Methods for production of vaccination site infiltrating lymphocytes and uses thereof

Abstract

The present invention relates to a method for producing a preparation comprising T cells recognizing at least one epitope of a disease antigen (disease epitope), the method comprising (a) incubating T cells from a sample of a site of vaccination of a subject with the disease epitope (vaccination site) under conditions suitable for proliferation of said T cells; and (b) thereby producing a preparation comprising T cells recognizing at least one disease epitope. The present invention also relates to a disease vaccine for use in improving an immune response of a subject to a disease epitope, to a preparation comprising T cells recognizing at least one cancer epitope, to a method of identifying a TCR binding to a disease epitope, a method of providing a T cell recognizing a cell presenting a disease epitope, and to a T cell recognizing a cell presenting a disease epitope related thereto.

Description

[0001] Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 1 DK17029PC___________________________________________________________________________ Methods for production of vaccination site infiltrating lymphocytes and uses thereof ___________________________________________________________________________ The present invention relates to a method for producing a preparation comprising T cells recognizing at least one epitope of a disease antigen (disease epitope), the method comprising (a) incubating T cells from a sample of a site of vaccination of a subject with the disease epitope(vaccination site) under conditions suitable for proliferation of said T cells; and (b) therebyproducing a preparation comprising T cells recognizing at least one disease epitope. The present invention also relates to a disease vaccine for use in improving an immune response of a subject to a disease epitope, to a preparation comprising T cells recognizing at least one disease epitope, to a method of identifying a TCR binding to a disease epitope, a method of providing a T cell recognizing a cell presenting a disease epitope, and to a T cell recognizing a cell presenting a disease epitope related thereto. T cell based immunotherapies have gained large interest in the last years due to the inventionof CAR-T cells and recently the generation of TCR transgenic T cell therapy [1, 2]. CAR Tcells have been successfully applied in hematological cancers, mainly targeting lineage-specific, cell-surface proteins like CD19 [3] or BCMA [4], at the cost of permanentlyeliminating all B cells or plasma cells [5]. The drawback of this approach is the availability of specific target proteins on the cell surface, which has been suggested to be overcome by vaccinating patients with tumor cells, optionally in combination with IL-2 producing fibroblasts

[0022] . Furthermore, transgenic T cells recognize a single tumor target, which can be downregulated by the tumor cells thus leading to immune escape. Another approach is aiming at the expansion of tumor-infiltrating lymphocytes (TIL) generated from tumor specimen of solid tumors. In a recent phase III trial in unresectable stage IIIC or IVmelanoma, TIL therapy resulted in longer progression-free survival compared to anti-CTLA4treatment [6]. The drawback of the TIL approach is that the specificity of the TIL is not knownand that TILs may be exhausted or suppressed and, in some cases, difficult to expand. Furthermore, it has been shown that the tumor microenvironment contains a large number ofbystander T cells [7] and that TILs do not always recognize neoepitopes eluted from autologoustumor cells [8]. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 2 DK17029PCTo enhance the number of tumor specific T cells, vaccination with appropriate antigens wasproposed. E.g. Tong et al.

[0021] performed intraperitoneal vaccination of mice with tumor-ganglioside glycomimetics and could detect tumor-specific T cells in the spleen with the abilityto become TILs. Highly mutated tumors contain so-called neoepitopes that are specific to the tumor and can berecognized by the patient’s immune cells to attack the tumor [8]. These patient-specific, somaticmutations can be identified by DNA sequencing of the tumor in comparison to control samples(e.g. blood) of the same patient [9]. Expression is confirmed by RNA sequencing and potential epitopes can be predicted by publicly available programs or alternatively be eluted from tumor cells and identified by mass spectrometry [8]. Peptides containing these putative epitopes canbe synthetically synthesized and administration of personalized tumor peptides as a therapeuticvaccine leads to stimulation of the body's own immune cells and sometimes to tumor control ortumor cell elimination [10, 11]. Analysis of subcutaneous vaccine-sites has revealedaccumulation and activation of T cells especially after repeated vaccination together with anincomplete Freund’s adjuvant [12, 13] or after vaccination with tumor lysate-loadedprofessional antigen-presenting cells

[0020] .Thus, there is still a need for improved methods for providing T-cells reactive to specific antigens, e.g. cancer antigens, and corresponding TCRs. This problem is solved by the embodiments characterized in the claims and described herein below. In view of the above, the present invention relates to a method for producing a preparation comprising T cells recognizing at least one epitope of a disease antigen (disease epitope), the method comprising (a) incubating T cells from a sample of a site of vaccination of a subject with the disease epitope (vaccination site) under conditions suitable for proliferation of said T cells; and (b) thereby producing a preparation comprising T cells recognizing at least one disease epitope.In general, terms used herein are to be given their ordinary and customary meaning to a personof ordinary skill in the art and, unless indicated otherwise, are not to be limited to a special or customized meaning. As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 3 DK17029PCmay both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e. are equivalent to "comprising at least one". In accordance, expressions relating to one item of a plurality, unless otherwise indicated, preferably relate to at least one such item, more preferably a plurality thereof; thus, e.g. identifying "a cell" relates to identifying at least one cell, preferably to identifying a multitude of cells. Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similarexpressions are intended to be optional features, without any restriction regarding furtherembodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention. The methods specified herein below, preferably, are in vitro methods. The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but preferably are performed in the indicated sequence; also, one or more, preferably all, of said steps may be assisted or performed by automated equipment. Moreover, the methods may comprise steps in addition to those explicitly mentioned above. As used herein, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ± 20%, more preferably ± 10%, most preferably ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 4 DK17029PCi.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, more preferably ± 10%, most preferably ± 5%. Thus, “consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of” encompasses any known acceptable additive, excipient, diluent, carrier, and the like. Preferably, a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of non-specified component(s). The term “polynucleotide", as used herein, refers to a linear or circular nucleic acid molecule. The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context) or in genetically modified form, preferably comprising at least one heterologous sequence. The term polynucleotide encompasses single- as well as, partially or completely, double-stranded polynucleotides. Preferably, the polynucleotide is a DNA polynucleotide, which may also be referred to as "DNA“. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificially modified derivatives such as biotinylated polynucleotides, locked nucleic acids, peptide nucleic acids, and the like. In view of the description herein, template DNA, e.g. for PCR, primers, e.g. sequencing or amplification primers, and probe oligonucleotides are included in the term polynucleotides. Unless specifically indicated otherwise, reference to specific polynucleotides herein preferably includes polynucleotide variants. The term “polynucleotide variant", as used herein, relates to a variant of a polynucleotide referred to herein comprising a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition and / or deletion, wherein the polynucleotide variant shall have the function and / or activity as specified for the specific polynucleotide. Thus, a variant of a polynucleotide may e.g. be an ortholog, a paralog, or another homolog of the specific polynucleotide; the polynucleotide variant may also be a mutant of the specific polynucleotide, preferably a naturally occurring mutant, e.g. identified in a cancer cell. Also preferably, said polynucleotide variant is or is derived from a non-naturally Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 5 DK17029PCoccurring allele of the specific polynucleotide. Further polynucleotide variants include polynucleotides comprising nucleic acid sequences which are at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%, identical to the specifically indicated nucleic acid sequences. The percent identity values are, preferably, calculated over the entire nucleic acid sequence region, preferably as specified herein elsewhere. The polynucleotides of the present invention either consist, essentially consist of, or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well. The term “polypeptide”, as used herein, refers to a molecule consisting of a multitude of amino acids that are covalently linked to each other by peptide bonds. Polypeptides consisting of less than 20 amino acids covalently linked by peptide bonds may also be referred to as "peptides". Preferably, the polypeptide comprises of from 4 to 1000, more preferably of from 5 to 1000, still more preferably of from 6 to 500, most preferably of from 7 to 400 amino acids. The polypeptide may also be comprised in a fusion polypeptide and / or in a polypeptide complex.Unless specifically indicated otherwise, reference to specific polypeptides herein preferablyincludes polypeptide variants. As used herein, the term "polypeptide variant" relates to anychemical molecule comprising at least one polypeptide as specified herein differing in structure from a specifically indicated polypeptide. Preferably, the polypeptide variant comprises a polypeptide having a contiguous amino acid sequence corresponding to at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%, of the amino acid sequence of the polypeptide specifically indicated. Moreover, it is to be understood that a polypeptide variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still, preferably, at least 70%, more preferably at least 80%, evenmore preferably at least 90%, even more preferably at least 95%, still more preferably at least98%, most preferably at least 99%, identical with the amino acid sequence of the specific polypeptide. The degree of identity between two amino acid sequences can be determined byalgorithms well known in the art and as described herein elsewhere. Polypeptide variantsreferred to above may be allelic variants or any other species specific homologs, paralogs, or orthologues. Moreover, the polypeptide variants referred to herein include fragments of the specific polypeptides or the aforementioned types of polypeptide variants, preferably as long Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 6 DK17029PCas these fragments and / or variants have the activity as specified, e.g. immunogenicity in the case of epitopes or polypeptides comprising them. Such fragments may be or may be derivedfrom, e.g., degradation products or splice variants of the polypeptides, or from or gene fusions,insertions / deletions (indels) and / or frameshift mutations. Further included are variants which differ due to posttranslational modifications such as phosphorylation, glycosylation, ubiquitinylation, sumoylation, or myristylation, by including non-natural amino acids, and / or by being peptidomimetics. The "activity" of a polypeptide as specified herein is, preferably, preserved in the polypeptide variant; as the skilled person will understand in view of the description herein, the activity of a polypeptide does not necessarily have to reflect its main natural function, but may be a further activity relevant in the context of the claimed invention.On a strictly exemplary basis, the p53 polypeptide has an activity in cell cycle regulation, butp53 and in particular mutants thereof also are immunogenic. Thus, a p53 mutant, including a fragment thereof, is a p53 variant as referred to herein, preferably having the activity of being immunogenic. The term "fragment" of a biological macromolecule, preferably of a polynucleotide or polypeptide, is used herein in a wide sense relating to any sub-part, preferably subdomain, of the respective biological macromolecule comprising the indicated sequence, structure and / or activity. Thus, the term includes sub-parts generated by actual fragmentation of a biological macromolecule, but also sub-parts derived from the respective biological macromolecule in anabstract manner, e.g. in silico. Thus, as used herein, an Fc or Fab fragment, but also e.g. a single-chain antibody, a bispecific antibody, and a nanobody may be referred to as fragments of an immunoglobulin. Also, a disease epitope may be a fragment of a disease antigen. Unless specifically indicated otherwise herein, the compounds specified, in particular the polynucleotides and polypeptides, may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further sequences, adjuvants, carrier molecules, retardants, and other excipients. In particular, polypeptides as specified may be comprised in fusion polypeptides comprising further polypeptides, which may serve e.g. as a tag for purification and / or detection, as a linker, or to extend the in vivo half-life of a compound. The term “detectable tag” refers to a stretch of amino acids which are added to or introduced into thefusion polypeptide; preferably, the tag is added C- or N- terminally to the fusion polypeptide.Said stretch of amino acids preferably allows for detection of the polypeptide by an antibody which specifically recognizes the tag; or it preferably allows for forming a functional Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 7 DK17029PCconformation, such as a chelator; or it preferably allows for visualization, e.g. in the case of fluorescent tags. Preferred detectable tags are the Myc-tag, FLAG-tag, 6-His-tag, HA-tag, GST- tag or a fluorescent protein tag, e.g. a GFP-tag. These tags are all well known in the art. Other further peptides preferably comprised in a fusion polypeptide comprise further amino acids or other modifications which may serve as mediators of secretion, as mediators of blood-brain- barrier passage, as cell-penetrating peptides, and / or as immune stimulants. Further polypeptides or peptides to which the polypeptides may be fused are signal and / or transport sequences, e.g. an IL-2 signal sequence, and linker sequences. The degree of identity (e.g. expressed as "%identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of sequence in the comparisonwindow may comprise additions or deletions (e.g., gaps or overhangs) as compared to thesequence it is compared to for optimal alignment. The percentage is calculated by determining, preferably over the whole length of the polynucleotide or polypeptide as specified herein, the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (e.g. BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. More preferably, the Basic LocalAlignment Search Tool (BLAST) implementation is used with default parameter values foralignment. In the context of biological sequences referred to herein, the term "essentially identical" indicates a %identity value of at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99%. As will be understood, the term essentially identical includes 100% identity. The aforesaid applies to the term "essentially complementary" mutatis mutandis. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 8 DK17029PCThe term "T cell" is understood by the skilled person to relate to a lymphocyte expressing at least one type of T cell receptor. Preferably, the T cell is a CD8+ T cell recognizing major histocompatibility (MHC) class I molecules on the surface of target cells, or is a CD4+ T cell recognizing MHC class II molecules on the surface of target cells. Preferably, the T cell is a cytotoxic T cell, more preferably a CD8+ cytotoxic T cell, which may also be referred to as "killer cell". Also preferably, the T cell is a helper T cell. Preferably, the T cell is reactive to a disease epitope, preferably a cancer epitope, i.e. is a cancer-reactive T cell. Thus, preferably, the T cell expresses a TCR recognizing a disease epitope. Preferably, the T cell is a recombinantcell expressing a chimeric antigen receptor (CAR), e.g. a TCR-like CAR

[0019] , and / or arecombinant T cell receptor. Methods of producing appropriate recombinant T cells are knownin the art. As is known to the skilled person, human MHCs may also be referred to as "humanleukocyte antigens" (HLAs). The term "T cell receptor", abbreviated as "TCR", as used herein, relates to a polypeptide complex on the surface of T cells mediating recognition of antigenic peptides presented bytarget cells, preferably in the context of MHC molecules or MHC-related molecules such asMR1 or CD1, more preferably in the context of MHC molecules, still more preferably in the context of MHC class I or MHC class II molecules, most preferably in the context of MHC class I molecules. Typically, the TCR comprises one TCR-alpha chain and one TCR-beta chain, i.e. is an alpha / beta chain heterodimer. The TCR may, however, also comprise a TCR gamma and a TCR delta chain instead of the TCR alpha and beta chains. The TCR alpha and beta or gamma and delta chains mediate antigen recognition and each comprise a transmembrane region, a constant region, a joining region, and a variable region, the variable region of each TCR alpha, beta, gamma, or delta chain comprising three complementarity determining regions (CDRs), referred to as CDR1, CDR2, and CDR3, respectively. In accordance with usual nomenclature, the complex consisting of an alpha and a beta chain or a gamma and a delta chain is referred to as "T cell receptor" or "TCR" herein, the alpha and / or beta chain and the gamma and / or delta chains commonly or singly being referred to a "TCR polypeptide" or "TCR polypeptides", whereas the polypeptide complex comprising a TCR and accessory poly- peptides, such as CD3 and CD247, is referred to as "T cell receptor complex", abbreviated as "TCR complex". Preferably the T cell receptor binds to a major histocompatibility complex (MHC) molecule, preferably an MHC class I or class II, more preferably an MHC class Imolecule, presenting an epitope contributing to and / or associated with disease, preferably acancer epitope. Binding of a T cell receptor to an antigen can be determined by methods known Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 9 DK17029PCto the skilled person, e.g. by a tetramer assay. Preferably, binding of the TCR to an epitopepresented on an MHC activates the T cell. Activation biomarkers of various types of T cells areknown in the art and include in particular CD69, CD137, CD27, TRAP / CD40L, and CD134. The TCR may also be a soluble TCR. The term "soluble TCR" is, in principle, known to the skilled person to relate to a TCR as specified herein above lacking the transmembrane domains. Thus, preferably, the soluble TCR comprises the constant and the variable regions of the TCR polypeptides of a TCR. More preferably, the soluble TCR comprises the variable regions of the TCR polypeptides of a TCR, preferably in the form of a fusion polypeptide. The term "complementarity determining region", abbreviated as "CDR", is understood by the skilled person. As is known in the art, each TCR alpha, beta, gamma, and delta chain comprises three CDRs, which comprise the amino acids essentially providing the contacts of a TCR to a peptide presented by an MHC molecule as specified elsewhere herein. As is known to the skilled person, in TCRs, epitope binding specificity is essentially determined by the variabledomain and specifically by the CDR3 region of the variable region. Thus, to reproduce thebinding properties of a given TCR, it may be sufficient to transfer the CDR3s of said TCR to a suitable backbone, e.g. of another TCR. Preferably, in such case, all CDRs 1 to 3, or the variable regions or fragments thereof, are transferred. The term "preparation" is understood by the skilled person to relate to any composition of matter comprising at least the indicated components. Thus, the preparation may comprise further components as well, in particular components required to keep T cells at least viable, such as at least one solvent, preferably water, a buffer, and / or one or more salts, preferably sodium chloride, potassium chloride, or the like. Preferably, the preparation is a cell culture composition and / or a pharmaceutical composition. The preparation comprises T cells. In the context of the present description, the preparationpreferably is obtained within six weeks after starting step (a), more preferably within 4 weeks.Preferably, the preparation comprises at least 109T cells, more preferably comprises at least 1010T cells. Preferably, the aforesaid cell numbers are obtained within the aforesaid time frames; i.e., preferably, the preparation comprises at least 109T cells, more preferably at least 1010T cells, within six weeks, more preferably within 4 weeks, after starting step (a). Preferably the preparation comprises at least 50% T cells, preferably at least 75% T cells, preferably within six weeks, more preferably within 4 weeks, after starting step (a). Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 10 DK17029PCThe term "cell culture composition" is understood by the skilled person to relate to a composition comprising all compounds required for T cells to proliferate. Thus, the cell culturecomposition preferably comprises, preferably in addition to one or more additional componentsdescribed herein above for the preparation, at least one of nutrients required for T cell proliferation, one or more stimulant(s), such as one or more selected from the list consisting ofcytokines IL-2, IL-7, IL-15, IL-12, IL-21, and combinations of at least two thereof; an anti-CD3 antibody and / or an anti-CD28 antibody, and feeder cells. Preferably, the cell culturecomposition is a composition facilitating in vitro growth of T cells, and, more preferably is a Tcell culture medium. Appropriate culture media are known in the art and are described hereinin the Examples. The terms "medicament" and "pharmaceutical composition" are used essentially interchangeably herein and are, in principle, known to the skilled person. As referred to herein, the terms relate to any composition of matter comprising the specified active agent(s), in particular T cells, as pharmaceutically active compound(s) and one or more excipient. It will be appreciated that the form and character of the pharmaceutical acceptable excipient, e.g. carrier or diluent, is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. The excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. Thus, the excipient employed preferably is a liquid,preferably an aqueous solution. Exemplary of a liquid carrier is physiological salinesupplemented with a stabilizing agent like human serum albumin, optionally comprising furthersupplements. The carrier may also be an injectable freezing carrier, allowing the medicament to be frozen and injected directly after thawing. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington´s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The excipient(s) is / are selected so as not to compromise the biological activity of the combination and of the T cells comprised therein.Preferably, the medicament is administered systemically, preferably orally or parenterally, e.g.by intravenous administration, or is administered topically, preferably intra-tumorally, topicallyon a body surface, or by inhalation; in case of cancer treatment, topical administration may beintratumoral or peritumoral, and / or topical at a site of tumor excision. Administration may, however, also be into a blood vessel, typically an artery, afferent to an intended site of effect, Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 11 DK17029PCsuch as a tumor. However, depending on the nature of the formulation and the desired therapeutic application, the medicament may be administered by other routes as well. The medicament is, preferably, administered by a route as specified herein above. A therapeutically effective dose refers to an amount of T cells to be used in a medicament which prevents, ameliorates or cures the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of a drug can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, age, the particular formulation of the medicament to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The medicament referred to herein is, preferably, administered at least once, e.g. as a bolus. However, the medicament may be administered more than one time and, preferably, at least twice, e.g. permanently or periodically after defined time windows. Progress can be monitored by periodic assessment. Dosage recommendations may be indicated in the prescriber or userinstructions in order to anticipate dose adjustments depending on the considered recipient. Themedicament may comprise further active agents in addition to the aforementioned activeagent(s); e.g. in case of cancer treatment, said at least one further active agent preferably is a chemotherapeutic agent or a further immunotherapeutic agent, such as an immune checkpoint modulator. The term "disease" is known to the skilled person. Preferably, the disease is caused by at least one agent recognizable by a T cell. Thus, the disease preferably is cancer or is caused by a pathogenic microorganism, wherein the term "pathogenic microorganism" includes each and every microorganism causing, obligatorily or facultatively, disease in at least one subject. Thus, the pathogenic microorganism may in particular by a bacterium, a virus, a parasite, or a fungus. The term "cancer", as used herein, relates to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells (“cancer cells”). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue (invasion) and possibly spread of cancer cells to other locations in the body (metastasis). Preferably, also included by the term cancer is a recurrence of cancer after treatment (relapse). Thus, preferably, the cancer is a solid cancer, a metastasis, or a relapse thereof. Cancer may be Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 12 DK17029PCinduced by an infectious agent, preferably a virus, more preferably an oncogenic virus, more preferably Epstein-Barr virus, a hepatitis virus, Human T-lymphotropic virus 1, a papillomavirus, or Human herpesvirus 8. Cancer may, however, also be induced by chemical compounds, e.g. a carcinogen, or endogenously, e.g. caused by spontaneous mutation. Preferably, the cancer comprises at least one cancer epitope. Preferably, the cancer is selected from the list consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma (ACC), adrenocortical carcinoma, aids-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germcell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma,gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, throat cancer,thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor. More preferably, saidcancer is adenoid cystic carcinoma (ACC) or colorectal cancer (CRC).The term "antigen", in principle, includes each and every immunogenic chemical compound, i.e. each and every chemical compound having the activity to induce an immune response to its Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 13 DK17029PCown structure in a subject. As the skilled person understands, immunogenicity of a given chemical compound to a given subject depends on a variety of factors, including the health stateof the subject, the mode of administration of the antigen, and other factors. Also, in case of acancer antigen, tumor micro-environment and / or immune suppression may decrease or prevent immunogenicity of an antigen. Thus, as referred to herein, an antigen preferably is a chemical compound inducing an immune response to its own structure in a statistically significant portion of subjects to which the antigen is administered, wherein said subjects preferably are apparently healthy subjects. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student´s t-test, Mann-Whitneytest etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the antigen has the activity of inducing a T cell response in a subject. Preferably, the antigen has the activity of stimulating antigen specific T cells, the term "antigen specific T cells" relating to T cells presenting on their surface T cell receptor molecules specifically recognizing, i.e. binding to, an epitope of the antigen, i.e. preferably to a disease epitope of the present invention, presented in the context of an MHC molecule. Preferably, the antigen is a "disease antigen", i.e. an antigen comprised in an agent causing disease as described herein above. Thus, the antigen may in particular be a cancer antigen or an antigen of a pathogenic microorganism. Preferably, the antigen is a biological macromolecule, more preferably is a polypeptide. The term "cancer antigen" relates to an antigen, preferably a polypeptide, expressed by a cancer cell. Preferably, the cancer antigen is expressed at an at least 5fold, preferably at least 10fold, more preferably at least 25fold, lower rate in non-cancer cells compared to cancer cells. Preferably, the cancer antigen is not expressed in non-cancer cells of a subject, preferably of the same tissue, more preferably is not expressed in non-cancer cells of a subject; thus, thecancer antigen preferably is a cancer specific antigen. More preferably, the cancer antigen is aneoantigen and / or comprises a neoepitope, expressed by cancer cells. Preferably, one or more peptides of the cancer antigen are presented via MHC molecules, more preferably MHC class- I molecules, on the surface of cancer cells. As specified elsewhere herein, the cancer preferably is a solid cancer, i.e. a tumor-forming cancer; thus, the cancer antigen preferably is a tumorantigen, more preferably a tumor-specific antigen. Preferably, the cancer antigen is a Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 14 DK17029PCpolypeptide derived from a tumor-associated antigen, from a polypeptide encoded by a frame-shift mutation, from a polypeptide encoded by a fusion gene, or from a viral antigen. Viralcancer antigens are known in the art and include e.g. human papillomavirus E6 and E7, Epstein- Barr virus LMP-1, and the like. The term "epitope" is known to the skilled person to relate to a (sub-)structure of an antigen which is recognizable for an immune system of a subject. As referred to herein, the epitope is a sequence of amino acids, preferably a contiguous sequence of amino acids, i.e. a peptide. Preferably, the epitope has a length of at least three, more preferably at least four, more preferably at least five, most preferably at least six amino acids. Also preferably, the epitopehas a length of at most 50, more preferably at most 25, even more preferably at most 20, mostpreferably at most 15 amino acids. Thus, preferably, the epitope has a length of from 3 to 50,more preferably of from 4 to 25 amino acids. Preferably, the epitope is an MHC class-I epitopeand comprises of from 8 to 13 amino acids; also preferably, the epitope is an MHC class-IIepitope and comprises of from 13 to 25 amino acids. In accordance with the above, the term "disease epitope" is used herein to relate to an epitopeof a disease antigen. The disease epitope preferably is disease specific, i.e. preferably isexpressed and / or presented preferably only by cells contributing to disease, e.g. by cells infected with a pathogenic microorganism, or by cancer cells. Also preferably the disease epitope is an epitope derived from, in particular a sub-sequence of, a cancer antigen, i.e. is a cancer epitope. Thus, e.g. in case the disease antigen is a cancer neoantigen, the disease epitope preferably is or comprises the neoepitope, i.e. the subsequence comprising the modification constituting the neoepitope. A cancer epitope may be expressed by a cancer cell caused by any genetic modification compared to a non-cancer cell which is preferably expressed by a cancer cell. Such a genetic modification may be any type of mutation in a coding region of a gene, such as a point mutation, an insertion, a deletion, or a frameshift mutation. The genetic modification may,however, also be a mutation causing expression of a gene product not normally expressed bycells, in particular somatic cells, of a subject; such a modification may e.g. be a gene fusion, a translocation, a promoter mutation, a mutation causing alternative splicing, aberrant translationof non-coding open reading frames, and the like. The genetic modification may also be a non-mutation modification, such as a modification in chromatin structure and / or histonemodification, and the like. The genetic modification may also be a viral infection and thus give Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 15 DK17029PCrise to the expression of viral genes or to the reactivation of a retroviral gene giving rise to theexpression of retroviral proteins specifically in the cancer cells.The disease epitope, in particular the cancer epitope, may be an individual epitope, i.e. identified in a sample of the subject to be treated; it may, however, also be a shared disease epitope, i.e. a disease epitope known or assumed to be shared by a multitude of subjects,cancers, or other forms of disease. Shared cancer antigens and corresponding epitopes are well-known in the art. The disease epitope as referred to herein is recognized by T cells; thus, the disease epitope is a T cell epitope. The term "T cell epitope", as used herein, relates to a contiguous sequence of amino acids comprised in a polypeptide, which can be bound to a major histocompatibility complex (MHC) class I or class II molecule to be presented on the surface of a cell (MHC-I) or of a professional antigen presenting cell (MHC-II). The skilled artisan knows how to predict immunogenic peptides presented on MHC-I or MHC-II (

[0016] ,

[0017] ) and how to evaluate binding of specific peptides (e.g.

[0018] ). Preferably, the T cell epitope is an MHC-I epitope. Preferably, the T cell epitope is an epitope derived from a cancer antigen; thus, preferably, the T cell epitope is an epitope essentially not or not occurring on normal cells of a subject, i.e. on non-cancer cells of said subject. Preferably, the T cell epitope is a disease epitope. As will be understood by the skilled person, the disease vaccine described herein may comprise more than one T cell epitope, e.g. preferably of from 2 to 100, more preferably of from 2 to 50, even more preferably of from 2 to 25 T cell epitopes, wherein said T cell epitopes may be MHC-I and / or MHC-IIepitopes. In such case, the T cell epitopes preferably are MHC-I and MHC-II epitopes; morepreferably, the T cell epitopes are epitopes presented by MHC molecules encoded by different HLA-A, B, C, E and HLA-DR, DQ and DP loci. Also preferably, the T cell epitopes are epitopes presented by different MHC allotypes encoded by mentioned MHC class I and MHC class II loci. The term "vaccine" is understood by the skilled person to include each and every agent eliciting and / or improving an immune response in a subject. Preferably, said immune response is a cellular immune response, more preferably a T cell response. In accordance, the "disease vaccine", as related to herein, is a composition of matter comprising a at least one chemical compound, preferably a polypeptide, comprising at least one disease Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 16 DK17029PCepitope, more preferably comprising a multitude of disease epitopes. Thus, the disease vaccineis an agent for active vaccination. Preferably, the disease vaccine comprises at least onepolypeptide comprising at least one disease epitope; said at least one polypeptide may, however, also comprise a multitude of disease epitopes. More preferably, the disease vaccine comprisesa multitude of polypeptides, each comprising at least one disease epitope; each polypeptide ofsaid multitude may, however, also comprise a multitude of disease epitopes. Also preferably,at least one of said polypeptides comprises a disease epitope comprised in a long peptide asspecified herein elsewhere. Preferred amino acid sequences to be comprised in a disease vaccine are shown herein in Table 1 and in SEQ ID NOs:1 to 20, more preferably in SEQ ID NO:1 to 12 and 16 to 20. Preferably, the disease vaccine further comprises a carrier, preferably apharmaceutically acceptable carrier. In addition, the disease vaccine may also include otheradditional compounds, such as stabilizers and / or other compounds deemed appropriate by the skilled person, e.g. for galenic purposes. As referred to herein, the at least one disease epitope as specified herein above, is the "active compound" of the vaccine, although further active compounds may be present. Vaccine compositions are preferably prepared in a manner well known in the pharmaceutical art. Formulations are to be adapted to the mode of administration, i.e. in the forms of solutions, suspensions, tablets, capsules, suppositories, or the like. Preferably, the disease vaccine further comprises an adjuvant. More preferably, the disease epitope(s) and the adjuvant are comprised in a common mixture at administration. Thus, preferably, the disease epitope(s) and the adjuvant are mixed before administration. Preferred adjuvants in the context of the present description are mineral oil / surfactant mixtures (e.g., Montanide), muramyl dipeptide, saponins such as QS21 and Quil A, monophosphoryl lipid A, aluminum hydroxide, aluminum phosphate, hydroxyapatite, complete and / or incomplete Freund's adjuvant, or cytokines such as interleukins, macrophage derived chemokines, complement binding proteins and tumor necrosis factor (either free or fused to the scaffold protein) such as type I IFN or GMCSF, TLR agonists, universal helper peptides such as derived from tetanus toxin, and human use-approved live microbial carriers such as the live attenuated Salmonella enterica serovar Typhimurium strain. Preferably, the adjuvant comprises mineral oil; also preferably, the adjuvant comprises a surfactant, the term "surfactant", as used herein, relating to a compound or to a mixture of compounds having amphiphilic properties andlowering surface tension of a liquid comprising them. Preferably, the surfactant comprises along aliphatic chain mannitol conjugate, e.g. a mannide monooleate or a mannitol octodecenoate. Preferably, the adjuvant comprises, preferably consists of, a mineral oil and a Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 17 DK17029PCmannide oleate surfactant. Thus, the adjuvant comprising, preferably consisting of, a mineraloil and a mannide oleate surfactant, preferably is the only adjuvant comprised in the disease vaccine. Preferably, the aforesaid adjuvant is a Montanide adjuvant, more preferably is Montanide ISA™ 51. The disease vaccine comprises at least one disease epitope, preferably at least one cancerepitope. More preferably, the disease vaccine comprises a multitude of peptides comprisingdifferent disease epitopes, preferably cancer epitopes. Preferably, the disease vaccine comprises at least one synthetic long peptide comprising one or more disease epitopes from overlappingamino acid sequences, or comprises serial epitopes in case the disease antigen is derived froma frame shift mutation or from a gene fusion. Thus, the synthetic long peptide preferablycomprises disease epitopes from at least two non-identical disease antigens. In concurrence with its common use in the art, the expression "long peptide" does not relate to a relative length,but to the fact that the long peptide expands beyond the minimal disease epitope. Thus in apreferred embodiment, a long peptide comprises a sequence of from 15 amino acids to 35 amino acids, preferably of from 20 amino acids to 30 amino acids, more preferably about 25 aminoacids. In a preferred embodiment, the disease vaccine comprises, preferably consists of, asequence of any one of SEQ ID NO:1 to 61.In a preferred embodiment, the disease vaccine comprises at least one cancer neoepitope,wherein said cancer neoepitope is predicted or identified to be expressed by said cancer.Methods of predicting cancer neoepitopes are known in the art and include e.g. at least partiallysequencing the cancer genome and / or the cancer transcriptome, or identifying at least partially the cancer proteome. In a preferred embodiment, a cancer neoepitope is selected to match an MHC subtype of the subject to be treated. In a further preferred embodiment, the cancer neoepitope is comprised in the disease vaccine as a synthetic long peptide, preferably asdescribed herein above. In a further preferred embodiment, the disease vaccine comprises amultitude of cancer neoepitopes. Thus, in a preferred embodiment, the disease vaccine comprises at least one cancer neoepitope as synthetic long peptide, more preferably a multitude,i.e. preferably at least 2, more preferably at least 5, even more preferably at least 10, cancerneoepitopes as synthetic long peptides, wherein each of said synthetic long peptides preferablycomprises one of said cancer neoepitopes. In a further preferred embodiment, the diseasevaccine comprises at least one cancer neoepitope matched to an MHC subtype expressed by thesubject to be treated as synthetic long peptide, more preferably a multitude, i.e. preferably at Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 18 DK17029PCleast 2, more preferably at least 5, even more preferably at least 10, cancer neoepitopes assynthetic long peptides, wherein each of said synthetic long peptides preferably comprises oneof said cancer neoepitopes.Preferably, providing a disease vaccine comprises determining candidate disease epitopes in a disease-causing agent, such as a cancer cell, comprised in a subject. As the skilled person understands, determining candidate disease epitopes can be accomplished by any method deemed appropriate by the skilled person. To that end, in principle, the genome, transcriptome, proteome, metabolome, or parts thereof may be determined in a sample, preferably in an antigen sample as specified elsewhere herein. Preferably, the disease vaccine comprises at least one disease epitope in a water-in-oil emulsion, preferably in an adjuvant as described herein above. The term "vaccination" is well-known in the art. Preferably, the term includes administration of at least one disease epitope, preferably of a disease vaccine, to a subject at least once. The subject may be vaccinated a multitude of times, e.g. twice, three times, four times, or any number of times deemed appropriate by the skilled person. Preferably, administration of at least one disease epitope is repeated until signs of successful vaccination, in particular redness in the vicinity of the vaccination site, are observed. More preferably, administration of at least onedisease epitope is repeated until at least one discernible vaccination reaction forms at thevaccination site, e.g. discernible by means of local redness and / or swelling. Thus, a vaccinationsite sample is preferably obtained after the first, second, third, fourth, or fifth vaccination of the subject with the disease epitope, preferably the third or fourth vaccination of the subject with the disease epitope. Still more preferably, a vaccination site sample is obtained after the first, second, or third vaccination of the subject with the disease epitope causing a vaccinationreaction, preferably causing a discernible vaccination reaction, in particular an erythema or anerythematous swelling. The term "site of vaccination", which may also be referred to as "vaccination site", is understood by the skilled person. Preferably, the terms relate to the tissue of a subject directly contacted tothe disease epitope, preferably the disease vaccine. Preferably, in case the vaccine isadministered intradermally via a needle, the vaccination site includes the intradermal depositof disease vaccine. Thus, in a preferred embodiment, the vaccination comprises topicaladministration of said disease vaccine, preferably intradermally, subcutaneously, and / or intramuscularly. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 19 DK17029PCThe term "recognizing at least one disease epitope" is understood by the skilled person. Preferably, said recognizing is specific, i.e. more preferably, any other epitope is recognized less efficiently by a factor of at least 2, preferably at least 5, more preferably at least 10. Specificity of recognizing can be determined e.g. by determining activation of a T cell carrying a given TCR by a cognate disease epitope, e.g. when presented on a cell and comparing saidactivation to an activation caused by a non-cognate epitope. Specificity of recognizing can alsobe determined by binding of a MHC multimer incorporating the disease epitope.The term "sample" refers to a sample of biological material. The sample may, in principle, be of any type deemed appropriate by the skilled person. Preferably, the sample is a sample from a subject. The sample may be a sample of a bodily fluid, such as blood, urine, saliva, pleural effusions, ascites and the like, or a sample derived from a bodily fluid, such as e.g. plasma or serum. Preferably, the sample is a sample of separated cells or a sample from a tissue or anorgan, preferably from a vaccination site. As is known to the skilled person, tissue or organsamples may be obtained from any tissue or organ by, e.g., biopsy, surgery, or any other method deemed appropriate by the skilled person. Separated cells may be obtained from body fluid samples, such as lymph, blood, plasma, serum, pleural effusions, ascites, liquor and other, or from the tissues or organs by separating techniques such as centrifugation or cell sorting. Preferably, the sample is a tissue or body fluid sample which comprises cells. In the method for producing a preparation described herein, a sample of a site of vaccination of a subject with a disease epitope is used, said sample also being referred to as "vaccination site sample". Thus, the vaccination site sample preferably was obtained in the vicinity of a vaccination site and preferably comprises or is assumed to comprise lymphocytes, preferably T cells, more preferably cancer recognizing T cells. Preferably, the vaccination site sample isknown or assumed to be free of cancer cells; thus, the vaccination site sample is not a cancersample, i.e. preferably is a sample of non-cancer tissue. Preferably, the vaccine site sample was obtained within 50 mm, preferably within 40 mm, more preferably within 30 mm, of a vaccination site. Preferably, the vaccine site sample comprises biological material from the vicinity of a vaccination site showing signs of a successful vaccination, in particular rubor, dolor, and / or calor. Thus, the vaccine site sample may in particular be from a region in the vicinity of a vaccination showing redness after vaccination. More preferably, the vaccine site sample comprises a tissue showing a vaccination reaction or an aliquot thereof. The term "tissue Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 20 DK17029PCshowing a vaccination reaction" is known to the skilled person. As indicated herein above, thetissue showing a vaccination reaction, as referred to herein, is a tissue of a vaccination site. Methods of obtaining biological sample material, in particular cells, from a tissue showing avaccination reaction are known to the skilled person. The vaccine site sample is preferablyobtained within one week after the most recent vaccination with a disease vaccine at the vaccination site. For preparing a disease vaccine as described herein, an independent sample type may be used, which is indicative of the disease-causing agent and of disease antigen(s) said agent maycomprise, i.e. an "antigen sample", wherein said term is used herein in a broad sense relating toeach and every sample known or assumed to provide information on antigen(s) expressed by an agent causing disease. I.e. in case of cancer, the antigen sample may be a sample comprising cancer cells; in case of an infectious disease, the sample may be a sample from an infected site, e.g. a sample of pus from an infected site. As the skilled person understands in view of the description herein, the antigen sample does not necessarily have to comprise an antigen, such as a disease antigen. It may be sufficient that constituents of the antigen sample may allow the conclusion that a disease antigen is probable to be present. E.g. the presence of a certain metabolite in an antigen sample may allow to conclude on the presence of a specifically mutated gene causing production of said metabolite, or the presence of a certain gene product may allow to conclude on the presence of a pre-defined pathogenic microorganism, such as e.g. the presence of hepatitis virus polynucleotides and / or anti-hepatitis virus antibodies allows to identify hepatitis virus infection, and the presence of enterotoxin in a sample allows identification of the presence of at least one gram-negative bacterium. Preferably, the antigen sample is a cancer sample. From such a cancer sample, a cancer antigen may be identified from the proteome of cancer cells, e.g. by mass spectrometry, by transcriptome analysis, or by genome analysis. However, as indicated above, also metabolome analysis may provide anindication of which cancer antigen(s) are expressed by a cancer. It will be understood that ifgenome analysis is used, it may not be necessary in all cases to verify that an identified cancer antigen is indeed expressed by a cancer, in particular in case a multitude of cancer epitopes is comprised in the cancer vaccine. The term "subject", as referred to herein, relates to a vertebrate animal, preferably a mammal, in particular a livestock, companion, or laboratory animal. Most preferably, the subject is a human. Preferably, the subject is suspected or has been diagnosed to suffer from cancer. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 21 DK17029PCThe term "donor subject" is used herein in its common meaning to relate to a subject from which something is derived or obtained, in particular a vaccination site sample. Thus, the donor subject preferably was vaccinated against a disease epitope at least once. Correspondingly, an "acceptor subject" is a subject to which something is administered, e.g. a T cell immune product.The term "T cell immune product", as used herein, is comprising T cells recognizing a diseaseantigen, administered to a subject to improve an immune response to said disease antigen. Preferably, the T cell immune product is a preparation comprising T cells recognizing at leastone disease epitope as specified and / or produced as described herein elsewhere.The method comprises step (a) incubating T cells from a sample of a site of vaccination of a subject with the disease epitope (vaccination site sample) under conditions suitable for proliferation of said T cells. The term "conditions suitable for proliferation of T cells" is understood by the skilled person. Parameters included in said conditions are in particular temperature, water activity, presence and concentrations of nutrients, cell density, presence and concentration of stimulants, and the like. Preferably, said conditions are conditions for in vitro proliferation of T cells, such as cultured T cells. Appropriate conditions and protocols are known in the art. There may be cases in which a vaccination site sample does not comprise T cells or in which T cells comprised in such a sample are not viable or not cultivatable. In such case, the skilled person may envisage to take another vaccination site sample, and / or to repeat vaccination of the subject. The method further comprises step (b) thereby producing a preparation comprising T cells recognizing at least one disease epitope. As detailed herein elsewhere, the vaccination site sample is known or suspected to comprise T cells; preferably, said T cells in the vaccination site sample are less inhibited and / or exhausted compared to T cells comprised e.g. in a preparation of tumor infiltrating lymphocytes. Thus, the T cells comprised in a vaccination site sample preferably start proliferating as soon as they are incubated under conditions suitable for proliferation of T cells. Thus, the preparation as referred to herein may be produced within six weeks after starting step (a), preferably within 4 weeks, with acceptable T cell numbers. Preferably, no enrichment of T cells, of disease epitope specific T cells, and / or removal of inhibitory cells, such as regulatory T cells, is required in the method for producing a preparation. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 22 DK17029PCThus, the method preferably does not comprise binding said T cells recognizing at least one disease epitope to a solid surface; and / or does not comprise labelling said T cells recognizing at least one disease epitope and enriching said labelled T cells, e.g. by flow cytometry. Advantageously, it was found in the work underlying the present invention that vaccination site infiltrating lymphocytes (VIL) are a better starting material for producing preparations of disease epitope recognizing T cells than e.g. tumor infiltrating lymphocytes (TIL). As was found in the work underlying the present invention, VIL, in particular after Montanide adjuvanted vaccination, are highly viable and show higher proliferation compared to TIL fromthe same patient. Thus, T cells from VIL can be expanded more rapidly. Also, it was found thatafter vaccinating a subject with a multitude of disease epitopes, VIL specifically recognizing atleast some of these disease epitopes can be highly enriched already in VIL. Therefore, thevaccine site was found to be an ideal source for recently activated, tumor-specific T cells. In contrast to the TIL approach, cells are derived from the vaccination site with tumor-specific peptides (vaccination site-infiltrating lymphocytes, VIL), so that reinfusion of a large number of young, tumor-specific VIL with less depleted phenotype into the patient increases the chance of successful tumor defense by endogenous immune cells. Peptide epitope specificities of the VILs can be investigated in the VILs product and adoptively transferred cells can be monitoredwithin the patient’s blood. VILs have a number of advantages over TILs and other types of Tcell therapies: (1) due to the different source there is no risk of remaining tumor cells within the T cell product; (2) VILs are less repressed, more active and easier to expand; (3) specificities against neoepitopes can be selectively enhanced during vaccination and T cells with these specificities can be expanded and used for therapy; (4) the VILs product can have several specificities and thus target several epitopes from different antigens presented on different HLA alleles, which minimizes immune evasion; (5) due to the autologous nature of the T cells andthe selection process during maturation the risk for autoimmunity is minor. Also, in a preferredembodiment, in contrast to TIL preparation, where possible targets are not known, the proposedapproach of vaccination and preparation of T cells from VILs makes it possible to verifysuccessful vaccination and preparation, because the targets of resulting T cells are determinedby the disease epitopes in the vaccine, i.e. are known. The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 23 DK17029PCThe present invention also relates to a disease vaccine for use in improving an immune response of a subject to a disease epitope, said improving comprising (A) administering said disease vaccine to a donor subject at a vaccination site; (B) obtaining a sample from said vaccination site; (C) producing a preparation comprising T cells recognizing at least one disease epitope from the sample obtained in step (B) as a disease vaccine; and (D) administering the disease vaccine produced in step (C) to the subject. The term "immune response" is known to the skilled person. Preferably, the immune response is a T cell immune response. In accordance, improving an immune response preferably is increasing the number and / or persistence of T cells recognizing a disease antigen in a subject. It will be understood that increasing the number of T cells recognizing a disease antigen includes de novo establishing such T cells. Preferably, improving an immune response to a disease epitope is improving an immune response to a cancer. Improving an immune response comprises step (A) administering a disease vaccine to a donor subject at a vaccination site. Suitable disease vaccines have been described elsewhere herein, as have modes for their administration, which are known in the art. Improving an immune response further comprises step (B) obtaining a vaccination site sample from said vaccination site. Preferably, the vaccination site sample in step (B) is obtained in the vicinity of the vaccination site, as specified herein above. Preferably the vaccination site sample in step (B) is obtained within one week after the latest administration of said vaccine in step (A). As described herein above, the vaccination site sample preferably is a biopsy sample, preferably of a tissue showing a vaccination reaction. Improving an immune response further comprises step (C) producing a preparation comprising T cells recognizing at least one disease epitope from the sample obtained in step (B) as a T cellimmune product As detailed herein above, the methods described herein preferably do notrequire enrichment of T cells or of T cells specific for a disease antigen, so step (C) preferably does not comprise binding said T cells recognizing at least one disease epitope to a solid surface; and / or does not comprise labelling said T cells recognizing at least one disease epitope and enriching said labelled T cells, e.g. for FACS sorting. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 24 DK17029PCImproving an immune response further comprises step (D) administering the T cell immuneproduct produced in step (C) to the subject. Administering may be accomplished by any meansand methods deemed appropriate by the skilled person. Preferably, the T cells in the T cellimmune product are transferred to a suitable pharmaceutically acceptable carrier beforeadministration, such as physiological saline supplemented with human serum albumin.Preferably, the T cell immune product is administered intravenously; however, other routes ofadministration may be envisaged, such as intracranial, intraventricular and / or intratumoraladministration. Preferably, the T cell immune product is administered to a human leukocyteantigen (HLA)-matched subject, i.e. a subject expressing the same HLA-subtypes as the donorsubject. More preferably, the T cell immune product is administered to the donor subject, i.e.the donor subject and the acceptor subject preferably are identical, i.e. the T cell immune preferably is an autologous transplant. The present invention also relates to a preparation comprising T cells recognizing at least one cancer epitope produced or producible by a method for producing a preparation comprising Tcells recognizing at least one disease epitope as specified herein. In a preferred embodiment,the present invention also relates to a preparation comprising T cells recognizing at least onedisease epitope produced or producible by a method for producing a preparation comprising Tcells recognizing at least one disease epitope as specified herein.The present invention also relates to a preparation comprising T cells recognizing at least onedisease epitope (i) comprising at least 1%, preferably at least 2%, more preferably at least 3%, T cells specific to at least one cancer epitope and / or (ii) T cells specific to at least two, more preferably at least three non-identical disease epitopes, preferably at least three non-identicalcancer epitopes. Said preparation preferably was obtained by a method as specified hereinelsewhere.The present invention also relates to a preparation as specified herein for use in medicine; andto a use of a preparation as specified herein for the manufacture of a medicament. The present invention further relates to a preparation as specified herein for use in treating and / or preventing disease, preferably cancer, in a subject. The present invention also relates to a use of the preparation as specified herein for the manufacture of a medicament for treating and / or preventing cancer. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 25 DK17029PCThe terms "treating" and “treatment” refer to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of health with respect to the diseases or disorders referred to herein. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to hereincan be successfully treated. Whether a portion is statistically significant can be determinedwithout further ado by the person skilled in the art using various well-known statistic evaluation tools, e.g., as described herein above. Preferably, treating cancer is achieving stable disease, more preferably is reducing tumor burden in a subject. As will be understood by the skilled person, effectiveness of treatment of e.g. cancer is dependent on a variety of factors including, e.g. cancer stage and cancer type. Preferably, treating causes cancer cells, to be recognized by T cells administered to the subject or their progeny cells. Thus, preferably, treating has the effect of killing cancer cells, causing cancer cells to stop proliferating, in particular causing a tumor and / or metastasis to stop growing, more preferably causing regression of a tumor, more preferably causing a tumor to resolve. The term “preventing” refers to retaining health with respect to the diseases or disorders referred to herein for a certain period of time in a subject. It will be understood that the said period of time may be dependent on the amount of the preparation, e.g. the T cell immune product, which has been administered and individual factors of the subject discussed elsewhere in this specification. It is to be understood that prevention may not be effective in all subjects treated with a preparation according to the present invention. However, the term requires that, preferably, a statistically significant portion of subjects of a cohort or population are effectively prevented from suffering from a disease or disorder referred to herein or its accompanying symptoms. Preferably, a cohort or population of subjects is envisaged in this context whichnormally, i.e. without preventive measures according to the present invention, would developa disease or disorder as referred to herein. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well-known statistic evaluation tools discussed elsewhere in this specification. Thus, preferably, in case of cancer or disease caused by an infectious agent, preventing may be vaccination, in particular passive vaccination. Thus, preferably, the term preventing relates to administering a preparation as specified herein to elicit an immune response against a cancer and / or against at least one Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 26 DK17029PCinfectious agent. In the case of cancer preventing may in particular pertain to preventing relapse and / or metastasis. The present invention also relates to a kit comprising a preparation as described herein and a means of administration. The term “kit”, as used herein, refers to a collection of the indicated compounds, means or reagents which may or may not be packaged together. Components of the kit may be comprised by separate vials (i.e. as a kit of separate parts) or provided in a single vial. Moreover, it is to be understood that the kit of the present invention, preferably, is to be used for practicing the methods referred to herein elsewhere or steps thereof. It is, preferably, envisaged that all components are provided in a ready-to-use manner for practicing the methods referred to above. Further, the kit, preferably, contains instructions for carrying out said methods. The instructions can be provided by a user's manual in paper or electronic form. In addition, the manual may comprise instructions for administration and / or dosage instructions for carrying out the aforementioned methods using the kit of the present invention. Preferably, the components of the kit are comprised in a housing, the housing of the kit preferably allowing translocation of the compounds of the kit, in particular common translocation; thus, the housing may in particular be a transportable container comprising all specified components. "Means of administration", as referred to herein, are all means suitable for administering the preparation to a subject. Thus, preferably, a means of administration is any arbitrary means configured and / or suitable for bringing the preparation into the body of a subject. The means of administration may include a delivery unit for the administration of the preparation and a storage unit for storing said preparation until administration. However, it is also contemplatedthat the means may appear as separate devices and are, preferably, packaged together in saidkit. Preferred means for administration are those which can be applied without the particular knowledge of a specialized technician. Preferably, the means for administration is a syringe, more preferably with a needle, comprising the preparation of the invention. Also preferably, the means for administration is an intravenous infusion (i.v.) equipment comprising the preparation. Further preferably, the means for administration is an inhaler comprising the preparation of the present invention, wherein, more preferably, said preparation is formulated for administration as an aerosol. Appropriate means are known in the art. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 27 DK17029PCThe present invention also relates to a method for treating and / or preventing disease,preferably cancer, in a subject, said method comprising(i) administering preparation comprising T cells recognizing at least one disease epitopeproduced or producible by a method as described herein to said subject, and(ii) thereby treating and / or preventing disease in said subject.The present invention moreover relates to a method of treating disease, preferably cancer, in asubject, said method comprising (I) administering a disease vaccine comprising one or more disease epitopes to a donor subject at a vaccination site; (II) obtaining a sample from said vaccination site; (III) producing a preparation comprising T cells recognizing at least one disease epitope from the sample obtained in step (II); and(IV) administering the preparation comprising T cells produced in step (III) to a subject,preferably the subject of step (A). The present invention also relates to a use of a preparation as specified herein for eliminating cancer cells, wherein said use preferably is an in vitro use. Moreover, the present invention relates to a method of identifying a TCR binding to a disease epitope presented on a cell, preferably a cancer epitope, of a subject, said method comprising (AA) providing a preparation comprising T cells recognizing at least one disease epitope according to the method described herein; (BB) providing the amino acid sequences of at least the complementarity determining regions (CDR3s) of the TCRs of the T cells provided in step (AA); and, thereby,(CC) identifying a TCR binding to a disease epitope presented on a cell.The method of identifying comprises step (AA) providing a preparation comprising T cells recognizing at least one disease epitope according to the method described herein above.The method of identifying further comprises step (BB) providing the amino acid sequences ofat least the complementarity determining regions 3 (CDR3s) of the TCRs of the T cells provided in step (A). Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 28 DK17029PCThe term "providing a sequence", such as an amino acid sequence and / or a nucleic acid sequence, is used herein in a broad sense including any and all means and methods of providing information on said sequence or making said sequence information accessible. Thus, the sequence may be provided as a sequence information, preferably tangibly embedded on a data carrier. The sequence may, however, also be provided in the form of a molecule comprising said sequence, preferably as a TCR comprising TCR alpha and beta chains comprising said sequences, more preferably as a host cell comprising the same. As the skilled person understands, if the aforesaid host cell is provided, the sequence information can be provided by standard methods known to the skilled person, e.g. nucleic acid sequencing of the TCR ex- pressed by said host cell or of parts thereof. Preferably, the method of identifying a TCR recognizing a disease epitope further comprises step (BB1) expressing a TCR comprising at least the CDR3s determined in step (BB) in a host cell, preferably a T cell, i.e. preferably in a host T cell. More preferably, said method comprises further step (BB1) expressing a TCR comprising at least the CDR3s determined in step (BB) in a host cell, preferably a T cell, i.e. preferably comprises expressing a TCR comprising at least the CDR3s determined in step (BB) and at least one accessory TCR polypeptide in a host cell. The term "TCR comprising at least the CDR3s", as used herein, relates to a TCR in which at least the CDR3s are those as determined in step (BB), while the residual sequences of the TCR polypeptides may be sequences of one or more different alpha and beta or gamma and delta chains, e.g. heterologous sequences. More preferably, the CDRs 1 to 3 or the variable regions of the TCR molecules are provided in step (BB) and are expressed as parts of the TCR polypeptides in step (BB1). It is, however, also envisaged that sequences of further fragments of the TCR polypeptides or the complete TCR polypeptides are provided in step (BB), and areoptionally expressed in step (BB1). As the skilled person understands, it is also possible toprovide longer sequences in step (BB) than are expressed in step (BB1); e.g., preferably, the amino acid sequence of the variable regions of the TCR polypeptides may be provided in step (BB), while only the CDR3s thereof are expressed, in the context e.g. of heterologous TCR polypeptides, in step (BB1); or, the amino acid sequences of the variable regions of the TCR polypeptides may be provided in step (BB), and the amino acid sequence of the antigen binding region, including the CDRs, may be expressed, in the context e.g. of heterologous TCR polypeptides, in step (BB1). If not otherwise indicated, the TCR polypeptides preferably are expressed as complete molecules, i.e. each comprising a transmembrane region, a constant Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 29 DK17029PCregion, a joining region, and a variable region. In a preferred embodiment, the TCR comprisesat least the CRD3s of SEQ ID NO:62 and 63 and / or of SEQ ID NO:64 and 65. In a further preferred embodiment, the TCR further comprises the characteristics as shown in Table 5 herein below. Preferably, the method of identifying a TCR recognizing a disease epitope comprises further step (BB2) determining binding of the TCR expressed in step (BB1) to a cell presenting a disease epitope, preferably a cancer epitope, complexed in a major histocompatibility complex (MHC), preferably MHC class I, molecule. Methods of determining binding of a TCR, preferably comprised in a TCR complex, to a disease epitope complexed in a major histocompatibility complex (MHC) molecule are known in the art and include, preferably, determining binding of a disease epitope complexed in an MHC molecule carrying a detectable label to the TCR which may e.g. be expressed on the surface of a host cell. A well-known example of such a method is a tetramer assay, preferably using a soluble tetrameric MHC molecule complexed with a disease epitope. Preferably, the method identifying a TCR recognizing a disease epitope comprises further step (BB3) determining recognition of cells presenting said disease epitope by the TCR expressed in step (BB1). Assays for determining such recognition are known in the art and include in particular binding assays, activation assays, and cytotoxicity assays. In all of these assays, preferably cells presenting a disease epitope are co-incubated with host cells such as T cells expressing a TCR comprising at least the CDR3s as specified. In a binding assay, it is determined whether the presenting cells and the aforesaid host cells bind to each other, preferably to form an immunological synapse including at least an MHC molecule of the cell presenting a T cell activating antigen and the TCR. In an activation assay, the host cell, preferably the T cell, expressing a TCR comprising at least the CDR3s as specified, is tested after said co-incubation for biomarkers of immunological activation, e.g. interferon-gamma production. In a cytotoxicity assay, it is determined whether the host cells, preferably the T cells, expressing a TCR comprising at least the CDR3s as specified, lyse at least a fraction of the cells presenting the T cell activating antigen during said co-incubation. The method of identifying further comprises step (CC) identifying a TCR binding to a disease epitope presented on a cell. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 30 DK17029PCThe term "identifying a TCR" is used herein in a broad sense including any and all means and methods of providing information on a TCR allowing determination of at least its CDR3 sequences. In accordance, the TCR does not have to, but may, be provided in physical form. Thus, identifying a TCR may comprise providing at least the CDR3 sequences of the TCR or of a polynucleotide encoding at least said CDR3s. Preferably, said sequences are or were determined by single-cell determination of gene expression, preferably by single-cell RNAsequencing. Identifying a TCR may, however, also comprise physically providing said TCR,e.g. by providing a host cell, preferably a T cell, expressing said TCR, or by providing at least one polynucleotide encoding at least the variable regions of the TCR polypeptides identified. As will be understood, in case the TCR is provided in the context of a self-replicating entity such as a host cell, it may not be necessary to provide the amino acid sequences of at least the CDR3s of the TCR and / or the nucleic acid sequence of a polynucleotide encoding the same. The present invention also relates to a method of providing a host cell, preferably a T cell, recognizing a cell presenting a disease epitope, preferably a cancer epitope, said method comprising(aa) identifying a TCR binding to a disease epitope according to the method described hereinabove, (bb) expressing a TCR comprising at least the complementarity determining regions (CDR3s)of the TCR of step (aa) in a host cell, preferably a T cell, and, thereby,(cc) providing a host cell, preferably a T cell, recognizing a cell presenting a disease epitope,preferably a cancer epitope. As used herein, the term "host cell" relates to any cell capable of expressing, and preferably presenting on its surface, a TCR polypeptide as specified herein, preferably encoded by apolynucleotide and / or vector. Preferably, the host cell is a mammalian cell, in particular amouse or rat cell, more preferably, the host cell is a human cell. Preferably, the host cell is an immune effector cell, preferably a natural killer cell or a T cell. More preferably, the host cell is a T cell, more preferably a CD8+ T cell or a CD4+ T cell, more preferably a CD8+ T cell. As will be understood in view of the description herein, the host T cell preferably is a geneticallymodified cell, expressing a TCR polypeptide as specified herein, whereas a T cell in apreparation as described herein above preferably is non-recombinant. In view of the above, the following embodiments are particularly envisaged: Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 31 DK17029PCEmbodiment 1: A method for producing a preparation comprising T cells recognizing atleast one epitope of a disease antigen (disease epitope), the method comprising(a) incubating T cells from a sample of a site of vaccination of a subject with the diseaseepitope (vaccination site) under conditions suitable for proliferation of said T cells; and(b) thereby producing a preparation comprising T cells recognizing at least one diseaseepitope.Embodiment 2: The method of embodiment 1 wherein said sample was obtained within50 mm of the vaccination site, preferably wherein said sample comprises a tissue showing avaccination reaction or an aliquot thereof.Embodiment 3: The method of embodiment 1 or 2, wherein said sample was obtainedwithin one week after the most recent vaccination.Embodiment 4: The method of any one of embodiments 1 to 3, wherein said vaccinationwas the first, second, third, fourth, or fifth vaccination of the subject with the disease epitope, preferably the third or fourth vaccination of the subject with the disease epitope, and / or was the first, second, or third vaccination of the subject with the disease epitope causing a visible vaccination reaction, preferably causing a tissue showing a vaccination reaction.Embodiment 5: The method of any one of embodiments 1 to 4, wherein said preparationis produced within six weeks after starting step (a), preferably within 4 weeks.Embodiment 6: The method of any one of embodiments 1 to 5, wherein said method doesnot comprise binding said T cells recognizing at least one disease epitope to a solid surface.Embodiment 7: The method of any one of embodiments 1 to 6, wherein said method doesnot comprise labelling said T cells recognizing at least one disease epitope and enriching said labelled T cells.Embodiment 8: The method of any one of embodiments 1 to 7, wherein said vaccinationcomprised administration of at least one vaccine at the vaccination site, wherein said vaccine preferably comprises a peptide vaccine comprising at least one disease epitope, more preferably a plurality of disease epitopes.Embodiment 9: The method of any one of embodiments 1 to 8, wherein said vaccinationcomprised administration of disease vaccine in a water-in-oil emulsion, preferably comprisingmineral oil and anhydro mannitol ether octodecenoate, preferably as only adjuvant.Embodiment 10: A disease vaccine for use in improving an immune response of a subjectto a disease epitope, said improving comprising(A) administering said disease vaccine to a donor subject at a vaccination site; Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 32 DK17029PC(B) obtaining a sample from said vaccination site;(C) producing a preparation comprising T cells recognizing at least one disease epitope fromthe sample obtained in step (B) as a disease vaccine; and(D) administering the preparation comprising T cells produced in step (C) to the subject.Embodiment 11: The disease vaccine for use of embodiment 10, wherein said improvingan immune response to a disease epitope is improving an immune response to a cancer.Embodiment 12: The disease vaccine for use of embodiment 10 or 11, wherein said subjectin step (D) is a human leukocyte antigen (HLA)-matched subject, preferably is said donor subject of step (A).Embodiment 13: The disease vaccine for use of any one of embodiments 10 to 12, whereinsaid administering said disease vaccine was the first, second, third, fourth, or fifth vaccination of the subject, preferably the third or fourth vaccination of the subject with the vaccine.Embodiment 14: The disease vaccine for use of any one of embodiments 10 to 13, whereinsaid sample in step (B) is obtained within 50 mm of the vaccine injection site, preferablywherein said sample comprises a tissue showing a vaccination reaction or an aliquot thereof.Embodiment 15: The disease vaccine for use of any one of embodiments 10 to 14, whereinsaid sample in step (B) is obtained within one week after the latest administration of said vaccine in step (A).Embodiment 16: The disease vaccine for use of any one of embodiments 10 to 15, whereinstep (C) does not comprise binding said T cells recognizing at least one disease epitope to a solid surface.Embodiment 17: The disease vaccine for use of any one of embodiments 10 to 16, whereinstep (C) does not comprise labelling said T cells recognizing at least one disease epitope and enriching said labelled T cells.Embodiment 18: The disease vaccine for use of any one of embodiments 10 to 17, whereinsaid administration in step (A) comprises administration in a water-in-oil emulsion, preferably comprising mineral oil and a detergent, preferably anhydro mannitol ether octodecenoate.Embodiment 19: The disease vaccine for use of any one of embodiments 10 to 18, whereinsaid preparation is obtained within 4 weeks after start of incubation under conditions suitable for proliferation of said T cells.Embodiment 20: The subject matter of any one of embodiments 1 to 19, wherein saidsample is a biopsy sample, preferably of a tissue showing a vaccination reaction.Embodiment 21: The subject matter of any one of embodiments 1 to 20, wherein saidsample comprises T cells and is essentially free of cancer cells. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 33 DK17029PCEmbodiment 22: The subject matter of any one of embodiments 1 to 21, wherein saidsample is a tissue sample.Embodiment 23: The subject matter of any one of embodiments 1 to 22, wherein saidsample is a tissue sample comprising T cells.Embodiment 24: The subject matter of any one of embodiments 1 to 23, wherein saiddisease epitope is a cancer epitope derived from a cancer comprised in said subject.Embodiment 25: The subject matter of any one of embodiments 8 to 24, wherein saidvaccine comprises at least one disease epitope, preferably a cancer epitope, more preferably acancer neoepitope or an epitope derived from a shared cancer epitope.Embodiment 26: The subject matter of any one of embodiments 8 to 25, wherein saidvaccine comprises a multitude of disease epitopes.Embodiment 27: The subject matter of any one of embodiments 8 to 26, wherein saidvaccine comprises at least one synthetic long peptide.Embodiment 28: The subject matter of any one of embodiments 8 to 27, wherein at leastone disease epitope is human leukocyte antigen (HLA) class II restricted and, preferably, iscomprised in a synthetic long peptide.Embodiment 29: The subject matter of any one of embodiments 8 to 28, wherein saidpreparation comprises at least 109T cells.Embodiment 30: The subject matter of any one of embodiments 1 to 29, wherein saidpreparation comprises at least 1010T cells.Embodiment 31: The subject matter of any one of embodiments 1 to 30, wherein saidpreparation comprises at least 50% T cells, preferably at least 75% T cells.Embodiment 32: The subject matter of any one of embodiments 1 to 31, wherein at least30%, of said T cells are CD8+ T cells.Embodiment 33: A preparation comprising T cells recognizing at least one cancer epitopeproduced or producible by a method as specified in any one of embodiments 1 to 32.Embodiment 34: A preparation comprising T cells recognizing at least one cancer epitope(i) comprising at least 1%, preferably at least 2%, more preferably at least 3%, T cells specific to at least one disease epitope and / or (ii) T cells specific to at least one, preferably at least two, more preferably at least three non-identical disease epitopes, preferably at least three non- identical disease epitopes.Embodiment 35: The preparation of embodiment 33 or 34, wherein said T cells were notbound to a solid surface. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 34 DK17029PCEmbodiment 36: The preparation of any one of embodiments 33 to 35, wherein said T cellswere not labeled and were not enriched via said label.Embodiment 37: The preparation of any one of embodiments 33 to 36, for use in medicine.Embodiment 38: Use of a preparation according to any one of embodiments 33 to 36 forthe manufacture of a medicament.Embodiment 39: The preparation of any one of embodiments 33 to 36 for use in treatingand / or preventing disease, preferably cancer, in a subject.Embodiment 40: The preparation for use of embodiment 39, wherein said subject issuffering from cancer and wherein the preparation is for use in treating cancer.Embodiment 41: The preparation for use of embodiment 39 or 40, wherein said cancercomprises said cancer epitope.Embodiment 42: The preparation for use of any one of embodiments 39 to 41, whereinsaid treating and / or preventing comprises administering a disease vaccine comprising at least one epitope of said disease epitope to said subject.Embodiment 43: Use of the preparation of any one of embodiments 33 to 36, for themanufacture of a medicament for treating and / or preventing cancer.Embodiment 44: A kit comprising a preparation according to any one of embodiments 33to 36 and a means of administration.Embodiment 45: A method for treating and / or preventing cancer in a subject, said methodcomprising (i) administering preparation comprising T cells recognizing at least one cancer epitope produced or producible by a method according to any one of embodiments 1 to 9 to said subject, and (ii) thereby treating and / or preventing cancer in said subject.Embodiment 46: A method of treating cancer in a subject, said method comprising(I) administering a disease vaccine comprising one or more disease epitopes to a donor subject at a vaccination site;(II) obtaining a sample from said vaccination site;(III) producing a preparation comprising T cells recognizing at least one disease epitope from the sample obtained in step (II); and (IV) administering the preparation comprising T cells produced in step (III) to a subject, preferably the subject of step (A).Embodiment 47: Use of a preparation according to any one of embodiments 33 to 36 foreliminating cancer cells. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 35 DK17029PCEmbodiment 48: The use of embodiment 47, wherein said use is an in vitro use.Embodiment 49: A method of identifying a TCR binding to a disease epitope presented ona cell, preferably a cancer epitope, of a subject, said method comprising (AA) providing a preparation comprising T cells recognizing at least one disease epitope according to the method of any one of embodiments 1 to 9, (BB) providing the amino acid sequences of at least the complementarity determining regions (CDR3s) of the TCRs of the T cell provided in step (AA); and, thereby, (CC) identifying a TCR binding to a disease epitope presented on a cell.Embodiment 50: A method of providing a T cell recognizing a cell presenting a diseaseepitope, preferably a cancer epitope, said method comprising (aa) identifying a TCR binding to a disease epitope according to the method according to any one of embodiments 6 to 12, (bb) expressing a TCR comprising at least the complementarity determining regions (CDR3s) of the TCR of step (aa) in a T cell, and, thereby, (cc) providing a T cell recognizing a cell presenting a disease epitope, preferably a cancer epitope.Embodiment 51: A T cell recognizing a cell presenting a disease epitope produced orproducible according to the method according to embodiment 50, preferably for the use of any one of embodiments 37 to 42. All references cited in this specification are herewith incorporated by reference with respect totheir entire disclosure content and the disclosure content specifically mentioned in thisspecification. Figure LegendsFig. 1: (A) Concept of VIL treatment: patients are vaccinated with tumor-specific peptides.Infiltrating T cells are isolated from the vaccination site, ex vivo expanded and infused to thepatient. Concept is an adoptive cellular therapy (ACT) based on vaccine site infiltratinglymphocytes (VIL) shown as flow diagram: patients are vaccinated with suitable tumor antigen-derived peptides. VIL are isolated from the vaccination site, ex vivo expanded and infused tothe patient. (B) Exemplary schematic workflow for a VIL-based ACT development andtreatment shown with additional graphical illustration. IHC: Immunohistochemistry; WES:Whole exome sequencing, LS-MS: Liquid chromatography coupled with mass spectrometry Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 36 DK17029PCfor the identification of MHC-I or MHC-II presented peptide ligands (Immuno-peptidomics) that are eluted from tissue material such as a tumor biopsy. IL-2: Interleukin-2. TAA: Tumor- associated antigen.Fig. 2: Growth curves of VIL and TIL, shown is the change in total cell number over time forvarious preparations.Fig. 3: Number of days in culture needed to reach the target cell number of 4*107 cells in thegeneration phase normalized to 100 starting wells.Fig. 4: Growth properties of TIL and VIL. Time needed to grow 4*107 cells (y-axis) dependson the amount of starting material, which can be estimated by the number of wells filled withtissue pieces (x-axis).Fig. 5: VIL from a patient vaccinated with long peptides containing mutated epitopes werereactive against the mutated but not the wild-type peptide when tested for IFNγ secretion byELISpot assay (a, b) or for peptide-specific multimer staining (c); (d) Patient 001 VIL ACTdevelopment and CRC treatment schedule; (e) Functional T cell analysis towards vaccination- peptide (VAC)-specific and wild-type counterpart after VIL-based clinical-grade rapid expansion protocol (REP). Shown are VIL-derived T cell responses towards VAC, or wild-type (WT) variants and pools thereof measured by intracellular cytokine staining after 18h co-culture of isolated CD4+ and CD8+ T cells with peptide-pulsed B cells, that have been stimulated prior for 6 days with CD40L-multimer; (f) Serum level of soluble CEA (carcinoembryonic antigen) prior and after infusion of expanded VIL. (g,h) Tracking of mutation-specific CD8+ T cells in PBL ex vivo after infusion of a VIL ACT.Fig. 6: VIL of an adenoid cystic carcinoma patient vaccinated with a long peptide (AA 79-108)containing NY-ESO-1 epitopes were reactive against tested NY-ESO-1 peptides when measured for IFNγ secretion by ELISpot assay (a) or flow cytometry analysis of multimer- reactive CD8+ T cells (b); LP NY-ESO-1 long peptide; SPP: short peptide pool containing peptides that have been experimentally confirmed to bind the patient's HLA-A, B, C MHC-Ialleles and that are either part of NY-ESO-1 or of viral origin; (a) ELISPOT analysis of ex vivoVIL versus PBL isolated after the 8th vaccination with ISA51 emulsified SLP NY-ESO-179- 108 (SEQ ID: 16). CD4+ and CD8+ T cell were MACS isolated prior to 18h co-culture within Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 37 DK17029PCan ELISPOT plate with SLP, or short peptide pool (SPP)-pulsed autologous CD40L-stimulatedB cells (B cells CD40L); (b) Dual-color encoded NY-ESO-1 epitope-specific pMHC-Imultimer staining of VIL-derived and post vaccination derived peripheral blood (PBL) T cells;(c) NY-ESO-1-targeting VIL ACT shows immunogenicity and leads to clinical response in acase of metastatic adenoid cystic carcinoma (ACC); VIL ACT development and patient 002(ACC) treatment schedule; (d) schematic representation of ex vivo VIL preparation; (e)Functional vaccination-peptide (VAC)-specific T cell responses after VIL-based clinical-grade rapid expansion protocol (REP). Shown are VIL-derived T cell responses towards VAC, or shorter peptide variants and pools thereof (P01-P10) measured by intracellular cytokine staining after 18h co-culture of isolated CD4+ and CD8+ T cells with peptide-pulsed B cellsCD40L; Peptides are: VAC SLP: NY-ESO-179-108 (GARGPESRLLEFYLAMPFATPMEAELARRS,SEQ ID NO:16), P01: LEFYLAMPF (SEQ ID NO:20), P02: YLAMPFATPM (SEQ IDNO:17), P03: LAMPFATPM (SEQ ID NO:19), P04: FATPMEAEL (SEQ ID NO:18), P05:GARGPESRLLEFYLA (SEQ ID NO:39), P06: GPESRLLEFYLAMPF (SEQ ID NO:40), P07:SRLLEFYLAMPFATP (SEQ ID NO:41), P08: LEFYLAMPFATPMEA (SEQ ID NO:42),P09: YLAMPFATPMEAELA (SEQ ID NO:43), P10: MPFATPMEAELARRS (SEQ IDNO:44); (f) In-vitro killing measured by LDH-release of NY-ESO-1 P1-P4-peptide-pulsed HLA-C*03:04-expressing MCF-7 cells by isolated VIL-derived CD8+ T cells after REP; (g) Tracking of NY-ESO-1-specific T cells in PBL ex vivo after VIL infusion up to 470 days.Fig. 7: Detection of tumor epitope-specific or virus-specific CD8+ T cells in peripheral blood(PBL) after infusion of VIL for a patient with colorectal carcinoma (a) and a patient withadenoid cystic carcinoma (b).Fig. 8: Serum CEA levels of a CRC patient before and after VIL infusion.Fig.9: MRI images of different brain metastases from a patient suffering from adenoid cystic adenoma. A to D: Brain metastasis before (A: −57 days) and after VIL treatment (B: 41 days, C: 154 days, D: 174 days after VIL treatment) showing remarkable shrinkage of the metastases. E to H: a second brain metastasis of the same patient before (E) and after (F-H) VIL treatment.Fig. 10: Comparison of cytokine secretion from VIL, TIL and PBL in co-culture with autolo-gous tumor explants. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 38 DK17029PCFig. 11: Development and infusion of a personalized mutanome-targeting VIL ACT within 5months post vaccination start in a case of metastatic pancreatic ductal adenocarcinoma (PDAC). (a) Patient 003 VIL ACT development and treatment schedule. (b) ELISPOT analysis of ex vivo VIL versus peripheral blood (PBL) isolated after the 2nd vaccination with ISA51emulsified SLPs targeting in total 16 private gene alterations of patient 003 (NEO-VAC)including 9 SNV and 7 INDEL that have been identified after biopsy of the primary tumor. CD4+ and CD8+ T cell were MACS isolated prior to 18h co-culture within an ELISPOT platewith mutated SLP-pulsed autologous B cells that have been previously stimulated with CD40Lover 6 days.Fig. 12: Isolation and validation of patient 002 (ACC) VIL-derived NY-ESO-1-specific T cellreceptors (TCR). (a) DNA-barcoded pMHC-I multimer-guided single-cell TCR repertoire and cell surface protein expression analysis of VILs allows fast selection, cloning and subsequent validation of antigen-specific TCRs that may enable as part of a TCR warehouse off-the-shelfTCR-transgenic ACT. (b) pMHC-I multimer-based validation of three patient 002 VIL-derivedNY-ESO-1-specific TCRs recognizing in total three different NY-ESO-1 epitopes. Recombinant TCRs were stable expressed in CD8+ Jurkat lacking endogenous TCR expression (Jurkat-76 / CD8). The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention. Example 1: method overview (Fig.1) Highly mutated tumors contain so-called neoepitopes that are specific to the tumor and can be recognized by the patient’s immune cells to attack the tumor. These patient-specific peptide sequences are identified by tumor DNA sequencing and can be synthetically synthesized. Administration of personalized tumor peptides as a therapeutic vaccine leads to stimulation of the body's own immune cells and thus to tumor cell elimination. In addition to vaccination with tumor-specific peptides, cellular immunotherapies are another way to attack the tumor using the patient's own immune system. One well-known method is TIL therapy (tumor-infiltrating lymphocytes), where tumor-specific T cells are isolated from the tumor, expanded, and reinfused into the patient. Although TIL therapy has been successfully Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 39 DK17029PCused in certain tumor types such as melanoma, this type of cell harvesting has some disadvantages: antigen specificity of isolated T cells is very diverse and only a minor fraction of cells is tumor specific. In addition, isolated T cells originate from a highly immunosuppressive tumor milieu and are thus functionally impaired. A novel treatment procedure described here combines the approach of patient-specific peptide vaccination with T cell therapy in order to increase fitness of the infused T cells (Fig.1). In this approach, immune cells are expanded to high cell numbers according to a classical TIL protocol. However, in contrast to the TIL approach, cells are derived from the vaccination sitewith tumor-specific peptides (vaccination site-infiltrating lymphocytes, VIL), so that tumor-specific and recently activated T cells are used as starting material. Reinfusion of a large number of young, tumor-specific VIL with less depleted phenotype into the patient increases the chance of successful tumor defense by endogenous immune cells. Generally, the method may comprise the following steps:1) genome / exome / panel sequencing of tumor tissue and blood as control (not for panels)RNA sequencing of tumor tissue2) identification of genomic alterations (e.g. point mutations, INDELs, fusions,frameshifts) and prediction of neoepitopes, peptide selection3) peptide synthesis for vaccination, e.g. using Fmoc technology4) reconstitution of lyophilized peptides, addition of ISA-51 adjuvant5) repetitive vaccinations, optionally with preceding blood draws6) excision of vaccination site or parts thereof7) isolation and expansion of VIL8) detection of peptide-specific VIL using e.g. multimer technology, ELISpot9) i.v. infusion of cellular product (including preconditioning and subsequent IL-2application for T cell engraftment) Example 2: Epitope and peptide selection for vaccination The main criteria for the selection of shared antigen epitopes and vaccine peptides were shared antigen expression assessed e.g. by immunohistochemical or immunofluorescent staining oftissue sections, immunofluorescent staining of cells, amplification via polymerase chainreaction, and RNA sequencing. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 40 DK17029PCQuality of predicted epitopes with respect to parameters like the „eluted ligand rank“, andbinding affinity (nM) (current version: netMHCpan 4.1 algorithm,

[0014] , type and number ofHLA alleles predicted to bind the respective epitope were determined. Mutation specific epitopes were predicted based on point mutations („single nucleotide variations“, SNVs), insertions and deletions („Indels“) as well as gene fusions. For the selectionof mutation- specific epitopes, so called neoepitopes, and vaccine peptides all three categoriesof mutations were considered and the following parameters are applied: Ranking of epitopes and vaccine peptides based on the type / category of mutation: 1. Fusions, Indels and SNVs from oncogenic driver genes 2. Passenger SNVsExpression of the respective mutated geneQuality of predicted epitopes with respect to parameters like the „eluted ligand rank“, binding affinity, position of the mutation in the predicted epitope, allele frequency, type and number of HLA alleles predicted to bind the respective epitope, were evaluated. For gene fusions (identified by Arriba software,

[0015] ) in addition: „high confidence score“, distance of „breakpoints” > 1 Mio bases, number of “split reads” each > 0 and in sum > 10, epitopes spanning breakpoint, „eluted ligand rank“, binding affinity, position of the mutation in the predicted epitope, allele frequency, type and number of HLA alleles predicted to bind therespective epitope were evaluated. In case of out-of-frame mutation evens downstream epitopescould also be considered depending on the above listed parameters.Example 3: Preparation of peptide vaccineLyophilized peptides were dissolved in distilled water or a mixture of DMSO and water.Subsequently, peptides were mixed with an equal volume of mineral oil and anhydro mannitol ether octodecenoate (e.g. Montanide) in order to produce a water-in-oil emulsion. Example 4: Vaccination Peptide vaccine was injected intradermally. A second vaccination was applied 2 weeks after the first, followed by further vaccinations. Excision of the vaccine site was done after two or Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 41 DK17029PCmore vaccinations. The specimen was selected to include the injection site and the surroundingtissue. It was at least 1 cm in diameter.Example 5: Isolation and expansion of VIL A tissue biopsy from the vaccination site was chopped into small pieces and cultured in culturemedia (either T cell mix media: 80% RPMI 1640 + 20% AIM-V or X-Vivo 15) containing 10%human serum type AB, 6000 IU / ml IL-2 and 50 μg / ml gentamicin in 24-well suspension plates at 37°C, 5% CO2 and >90% humidity. The day after plating 50% of media was exchanged. Cells were observed daily under a microscope for outgrowth of T cells. If T cell outgrowth was visible, individual wells were resuspended and split 1:2 into new wells. Tissue debris wasdiscarded. T cells were combined in flasks at a starting cell concentration of 8*105 cells / ml andgrown until a sufficient number of cells (> 4*106cells) was reached, usually after approximately 14 days. Cells were monitored for cell composition using flow cytometry, and tested formicrobial and mycoplasma contamination prior to freezing. For the rapid expansion phase, cellswere thawed the day before stimulation. The next day, feeder cells from at least three donors were thawed, irradiated at an average dose of 30 Gy and prepared for culture at a ratio of 1:100 T cells : feeder cells. 2*107T cells were cultured in flasks containing 3 litres of T cell mix media or X-Vivo 15 with 10% human serum type AB, 30 ng / ml OKT3 anti-CD3 antibody, 3000 IU / ml IL 2 and feeder cells. A separate feeder cell control was cultured to ensuresuccessful irradiation. On day 4, 50% of the media was exchanged. After 5 days of culture thecells were transferred to the Xuri Cell Expansion System W25 and cultivated in 5 litres of mediacontaining 10% human serum type AB and 3000 IU / ml IL-2. Perfusion with media began twodays after cultivation in the Xuri system with 1 l / day on the third day of cultivation, increased to 2.5 l / day on day 4 and 5 and to 5 l / day on day 6 to 8. After 8 days of cell expansion in theXuri system cells were harvested, washed and resuspended in 0.9% NaCl with 2.5% humanserum albumin (HSA) for infusion. Cells were monitored for cell composition, microbial and mycoplasma contamination as well as endotoxin levels. Example 6: VIL are growing faster than TILGrowth properties of a T cell culture depend on a variety of parameters, including the amountof starting material. In general, vaccine-site infiltrating lymphocytes (VIL) grow faster than tumor infiltrating lymphocytes (TIL), as depicted in the corresponding growth curves (Fig.2). Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 42 DK17029PCThe number of days in culture needed to reach the target number of 4*107cells normalized to 100 starting wells after generation phase shows a significant difference between VIL and TIL (Fig.3). Example 7: VIL are growing faster than TIL As growth properties depend on the amount of starting material, we analyzed the time to reach the desired target cell number in correlation to the number of starting wells used for the culture of tissue pieces from the tumor or the vaccination site. In both cases, specimen are cut into small pieces and distributed to individual wells of a 24-well plate. Growth properties show that VIL are growing faster, even when starting with less material (Fig.4). Example 8: VIL do not contain tumor cellsTIL are generated from tumor tissue with the danger of tumor cell carry over. Therefore, carefulquality control measurements need to be implemented to detect residual tumor cells using tumor specific markers. In contrast, VIL, which are derived from a skin biopsy distant from the tumorsite, do not carry the risk of residual tumor cells in the VIL product.Example 9: VIL have increased tumor epitope-specific T cell frequencies compared to peripheral blood after vaccination A specimen from the vaccination site of a patient with colorectal cancer (CRC) vaccinated with synthetic peptides (Table 1) was used to generate VIL. VIL and lymphocytes from the peripheral blood (PBL) were analyzed for responses against wildtype and mutated peptides using ELISpot (Fig.5a + b) and multimer staining (Fig.5c). In contrast to PBL, which did not show any specific responses, VIL were reactive against mutated peptides. Reactivity againstthree predicted HLA class I epitopes could be detected by multimer staining in the CD8+ VILfraction, 4.4% of all CD8+ T cells were specific for one mutated 10-mer peptide in complex with HLA-A*11:01, whereas T cells did not recognize the corresponding wild-type peptides (Fig.5c). A second patient with an adenoid cystic carcinoma (ACC) was vaccinated with a 30-mer peptide from the known tumor antigen NY-ESO-1 Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 43 DK17029PC(GARGPESRLLEFYLAMPFATPMEAELARRS (SEQ ID NO:16), cancer epitopes:YLAMPFATPM (SEQ ID NO:17); FATPMEAEL (SEQ ID NO:18); LAMPFATPM (SEQ IDNO:19); LEFYLAMPF (SEQ ID NO:20). CD4+ and CD8+ T cells separated from VIL showedenhanced reactivity against NY-ESO-1 specific peptides as compared to isolated T cells from peripheral blood in an ELISpot assay (Fig.6a). Peptide-specific CD8+ T cells were detected by multimer staining in the VIL product before and after rapid expansion, with frequencies up to 6.8% whereas peptide-specific T cells in peripheral blood were below or close to detection limit (Fig.6b).In case of patient 001 (colorectal cancer, heavily pretreated CRC), a highly personalized mu-tanome-targeting SLP / ISA51 vaccine (NEO-VAC) was designed and applied based on our im- plemented automated tumor whole-exome and RNA-sequencing data analysis as well as in sil-ico T cell epitope prediction pipeline. Following mutanome-analysis of a liver CRC metastasis,patient 001 received 6 injections NEO-VAC prior to the vaccine site skin biopsy (Fig. 5d, Table1), which displayed enrichment for mutated as well as partial wild-type peptide cross-reactive CD4+T cell populations but exclusive mutation / neoepitope-specific CD8+T cell populations,while all these populations where not detectable ex vivo in peripheral blood (PBL) at the sametime as the skin biopsy was taken (Fig.6b). Following massive clinical-grade rapid expansion (REP) of ~ 40x106VILs till >1.0x1010cells,the final VIL product had a 1:1 CD4 / CD8 T cell ratio (data not shown). Post REP VILs fromCRC patient 001 maintained their functionality and ~ 48% of all CD4+ T cells were specificfor the mutated pool of NEO-VAC-derived SLPs and roughly 16% displayed in addition cross- reactive towards the wild-type peptide variants thereof. Among VIL CD8+ T cells roughly 25% displayed exquisite neoepitope-specificity after in vitro stimulation (Fig.5e) and pMHC-I mul-timer staining (Fig. 5c). Importantly, reactivity against three predicted HLA class I epitopescould be verified by multimer staining in the CD8+ VIL fraction, 4.4% of all CD8+ T cellswere specific for one mutated 10-mer peptide in complex with HLA-A*11:01, whereas T cells did not recognize the corresponding wild-type peptides (Fig.5c).In case of patient 002, a partial resection of a brain metastases derived from an adenoid cysticcarcinoma (ACC) showed NY-ESO-1 expression by immunohistochemistry staining providinga strong rationale for the start of a NY-ESO-179-108 SLP / ISA51 (NY-ESO-1 VAC) vaccinationand a subsequent VIL-based adoptive cell therapy (VIL ACT) (Fig. 6c, Table 2). Patient 002 Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 44 DK17029PCwas vaccinated with a 30-mer peptide from the known tumor antigen NY-ESO-1(GARGPESRLLEFYLAMPFATPMEAELARRS (SEQ ID NO:16), harboring know CD8+ Tcell epitopes such as: YLAMPFATPM (SEQ ID NO:17); FATPMEAEL (SEQ ID NO:18);LAMPFATPM (SEQ ID NO:19); LEFYLAMPF (SEQ ID NO:20). Following the 8th injectiona vaccination site biopsy was taken, that displayed a strong enrichment for NY-ESO-1179- 108-specific CD4+ as well as CD8+ T cell populations compared to peripheral blood-derivedT cells obtained at the same time as the skin biopsy (Fig. 6a). Here, CD4+ and CD8+ T cellsseparated from VIL or PBL were rested for NY-ESO-1-VAC reactivity by an ELISpot assayutilizing autologous CD40L-based expanded B cells as antigen-presenting cells (Fig.6a). In, addition peptide-specific CD8+ T cells were detected by multimer staining in the VIL product before and after rapid expansion, with frequencies up to 6.8% whereas peptide-specificT cells in peripheral blood were below or close to detection limit (Fig. 6a and 6b). Followingmassive clinical-grade rapid expansion (REP) of VILs until >1.0x109cells from a ~ 40x106VIL starting culture, these “post REP” VILs maintained their functionality as shown by effectormolecule expression upon NY-ESO-1-peptide based restimulation. Intracellular cytokinestaining (ICS) post restimulation combined with flow cytometry-based analysis indicated that ~23% and ~16% of the total CD8+ and CD4+ T cell population (overall 1:1 ratio post REP), respectively, were specific for multiple vaccine-derived epitopes that are accompanied by virus-specific bystander T cells (Fig. 6e and data not shown). In addition, post REP VIL-derivedCD8+ T cells maintained their capacity to specifically kill a NY-ESO-1 peptide-pulsed HLA-matched surrogate tumor cell line (Fig. 6f).Patient 003 suffering from pancreatic ductal adenocarcinoma (PDAC) received a highly per- sonalized neoepitope-specific SLP / ISA51 vaccine (NEO-VAC) based on mutanome analysis of a primary tumor biopsy (Fig.11a, Table 3). Here, the vaccination site biopsy was conducted already after the second NEO-VAC injection and surprisingly showed a vast enrichment of a single vaccination-peptide specific CD8+T cell population (Fig.11b) as well as various VAC peptide-specific CD4+T cell populations (data not shown). In the following, patient 3 received 3.2x1010rapid expanded VILs already 5 months after the first vaccine injection, which under- lines feasibility of rapid and effective production of a personalized VIL ACT. In terms of man- ufacturing time, VIL ACT may easily surpass personalized neoepitope-specific transgenic TCR therapy workflows, that require beforehand cumbersome extensive TCR screening, validation and cell product engineering steps, summing up to more than 250 days (>8 months) of devel- opment and manufacturing time

[0025] . Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 45 DK17029PCExample 10: VIL persist in peripheral blood and show clinical effects after infusion1*1010 VIL from the patient with colorectal cancer described in Fig. 5 were infusedintravenously into the patient. The frequencies of tumor-epitope specific CD8+ T cells wereenriched in PBL 7 and 37 days after infusion (Fig. 7a left panel), whereas the frequency ofcommon virus-specific T cells in PBL was not changed (Fig. 7a right panel). Similarly, NY-ESO-1 specific T cells could be detected in PBL of the patient with adenoid cystic carcinoma30 days after VIL infusion. After lymphodepletion of CRC patient 001 2*1010expanded VILs were re-infusedintravenously as adoptive cellular therapy (ACT) accompanied by systemic IL-2 injection (Fig.5d). The VIL-ACT was again overall well tolerated by patient 2 and no signs of potential autoimmunity were observed despite to the significant presence of wild-type peptide cross-reactive CD4+ T cells in the final ACT cell product in Fig.5e. The frequencies of tumor-epitopespecific CD8+ T cells were enriched in PBL 7 and were maintained at day 37 after infusion,whereas the frequency of common virus-specific T cells in PBL was not changed (Fig. 5g,h) .Similarly, following lymphodepletion, 1.1x109expanded VILs were re-infused as an ACT only 8-weeks after the 8thvaccination in case of patient 002 (last vaccination prior ACT) accompanied by systemic IL-2 treatment. The VIL-based ACT was overall well tolerated with clinical manageable side-effects mainly attributed to the IL-2 treatment. After reinfusion, inparticular NY-ESO-1-specific CD8+ T cell populations could be traced ex vivo up to 470 dayspost infusion indicating their long-term persistence in vivo, while they were not detectable exvivo prior VIL ACT (Fig. 6h).Example 11: VIL show clinical effects after infusion For the patient with colorectal cancer serum levels of the tumor marker carcinoembryonic- antigen (CEA) showed a sharp drop after infusion of VIL (Fig. 8). Brain metastases from the patient with adenoid cystic carcinoma (ACC) reduced markedly in size after treatment with VIL (Fig.9). Example 12: VIL show enhanced cytokine secretion in co-culture with autologous tumor cells Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 46 DK17029PCIn order to compare tumor-specific responses of VIL and TIL after the generation phase and peripheral blood T cells (CD3PB) before treatment, a co-culture experiment with autologoustumor explants from the patient with colorectal cancer was conducted. Cytokine secretion wasmeasured for tumor explants and T cells alone or in combination with T cells using the human MACSPlex Cytokine 12 Kit (Miltenyi). VIL co-cultured with tumor explants showedsignificantly increased cytokine secretion compared to TIL or T cells from peripheral blood.(Fig.10).Example 13: Isolation and validation of TCRs from patient 002-derived VIL targetingNY-ESO-1 Besides the use of VILs directly as an easy, customizable adoptive cellular therapy for an indi- vidual patient case as described in Fig.5 till 8, VILs also serve as a source of T cell receptors (TCRs) for off-the-shelf TCR-transgenic ACT. Similarly, TCRs isolated from patients that ex- perienced complete or partial responses upon TIL-based ACT have been further used to accel-erate off-the-shelf TCR-transgenic ACT manufacturing with great success

[0023] . Likewise, wehave implemented a robust workflow that allows us to interrogate several hundred predicted tumor (neo)epitopes per patient case for the presence of cognate CD8+ T cell populations using our highly sensitive and multiplex capable pMHC-I multimer-library based screening and sub-sequent compatible pMHC-I-guided single cell TCR sequencing approach

[0024] followed byTCR cloning and validation (Fig. 12a). Using this workflow, we have already identified three VIL-derived TCRs from patient 002. These three TCR recognized in total three different NY- ESO-1 epitopes with HLA-C*03:04 restriction (Fig.12b).Example 14: TCR sequencing and analysis from VILsA patient with a metastatic triple-negative breast cancer (TNBC) was enrolled in our institu- tional diagnostic and registry trial (NCT05652569) in February 2020. Longitudinal, molecularprofiling of tumor- and liquid biopsies where expanded by a translational program of immuno-monitoring as well as a research to identify and clone neoantigen‑specific T cells. Neoantigen calling from whole-genome and transcriptome sequencing of the first analyzed tu- mor lesion (Tumor 1) by neoepitope identification using NetMHCpan4.1 revealed 254 putative binding candidates to patient´s HLA-I alleles from 54 non-synonymous single-nucleotide vari-ants (SNV) considering a filter based on expression of the altered allele (FPKM > 0) and%RANK_EL < 2 (predicted binders). We selected 17 candidates based on gene expression, Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 47 DK17029PCallele frequency, and high affinity of the peptide-MHC, with the aim to select neoepitopes thatbest contribute to tumor immunogenicity. For the vaccination, extended peptide sequences(long peptides) flanking both sides of the predicted epitope were designed. The list of peptidesused in the vaccination can be found in Table 4. Intradermal application of the personalized vaccine started in February 2021 and after threerounds (every 4-6 weeks), the patient presented with a strong local inflammatory reaction at thevaccination site, manifesting induration, redness and swelling. Considering inflammation-re- lated adverse reactions as a consequence of a strong and antigen-specific immune response, thevaccinated lesion was referred to biopsy followed by an analysis of immune repertoire andantigen specificity. TCR sequencing (TCR-seq) was performed from cryopreserved VILs and PBMC at Genewiz(Azenta). Briefly, single cell TCR sequencing of VDJ regions was performed using theChromium Single Cell Immune Profiling kit (10x Genomics), followed by libraries sequencing on a MiSeq (Illumina). Analysis of full-length V(D)J sequences for paired T-cell receptors was performed using the Loupe VDJ 223 Browser v4.0.0 (10x Genomics). Analysis of single cell TCR sequencing (scTCR-seq) data revealed the presence of three common clonotypes at the original vaccination site (>5%), from which two exhibited significantexpansion after IVS (both in the bulk and the pure CD8 cultures) with the long peptideNCOR1L1475R (clonotypes 1 and 2, Table 5). We selected the TCRs from these two most prominent clonotypes (designated as TCR-1131 and TCR-1132), and we proceeded with their cloning and subsequent validation. References1. Zhao, L. and Y.J. Cao, Engineered T Cell Therapy for Cancer in the Clinic. FrontImmunol, 2019.10: p.2250.2. Kilian, M., et al., T-cell Receptor Therapy Targeting Mutant Capicua TranscriptionalRepressor in Experimental Gliomas. Clin Cancer Res, 2022. 28(2): p. 378-389.3. June, C.H. and M. Sadelain, Chimeric Antigen Receptor Therapy. N Engl J Med,2018.379(1): p.64-73.4. Susanibar Adaniya, S.P., A.D. Cohen, and A.L. Garfall, Chimeric antigen receptor Tcell immunotherapy for multiple myeloma: A review of current data and potential clinical applications. Am J Hematol, 2019. Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 48 DK17029PC5. Cordeiro, A., et al., Late Events after Treatment with CD19-Targeted ChimericAntigen Receptor Modified T Cells. Biol Blood Marrow Transplant, 2020. 26(1): p.26-33.6. Rohaan, M.W., et al., Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab inAdvanced Melanoma. N Engl J Med, 2022. 387(23): p. 2113-2125.7. Simoni, Y., et al., Bystander CD8 T cells are abundant and phenotypically distinct inhuman tumour infiltrates. Nature, 2018. 557(7706): p. 575-+.8. Bassani-Sternberg, M., et al., Direct identification of clinically relevant neoepitopespresented on native human melanoma tissue by mass spectrometry. NatureCommunications, 2016.7.9. Lang, F., et al., Identification of neoantigens for individualized therapeutic cancervaccines. Nat Rev Drug Discov, 2022. 21(4): p. 261-282.10. Patel, S.P., et al., Phase I / II trial of a long peptide vaccine (LPV7) plus toll-likereceptor (TLR) agonists with or without incomplete Freund's adjuvant (IFA) for resected high-risk melanoma. J Immunother Cancer, 2021. 9(8).11. Melssen, M.M., et al., Peptide emulsions in incomplete Freund's adjuvant createeffective nurseries promoting egress of systemic CD4(+) and CD8(+) T cells for immunotherapy of cancer. Journal for Immunotherapy of Cancer, 2022. 10(9).12. Chen, Q., et al., Characterization of antigen-specific CD8+ T lymphocyte responses inskin and peripheral blood following intradermal peptide vaccination. Cancer Immun,2005.5: p.5.13. Pollack, K.E., et al., Incomplete Freund's adjuvant reduces arginase and enhancesTh1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment. Journal for Immunotherapy of Cancer, 2020. 8(1).14. Reynisson, B., et al., NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions ofMHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res, 2020. 48(W1): p. W449-W454.15. Uhrig, S., et al., Accurate and efficient detection of gene fusions from RNA sequencingdata. Genome Res, 2021. 31(3): p. 448-460.16. Nielsen et al., (2004), Bioinformatics, 20 (9), 1388–1397) 17. Bordner (2010), PLoS ONE 5(12): e1438318. Bernardeau et al., (2011), J Immunol Methods, 371(1-2):97-10519. Sadelain et al. (2013) Cancer Discov 3(4):38820. Chakraborty et al., (1998) Cancer Immunol Immunother 47:58-6421. Tong et al. (2019) Cell Chem. Biol. 26(7):1013-102622. Mackensen et al. (1997), J. Mol. Med. 75(4):290-29623. Leidner, R. et al. Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer. NEngl J Med 386, 2112-2119 (2022). https: / / doi.org / 10.1056 / NEJMoa211966224. Meyer, M. et al. MediMer: a versatile do-it-yourself peptide-receptive MHC class Imultimer platform for tumor neoantigen-specific T cell detection. Front Immunol 14, 1294565 (2023). https: / / doi.org / 10.3389 / fimmu.2023.129456525. Foy, S. P. et al. Non-viral precision T cell receptor replacement for personalized celltherapy. Nature 615, 687-696 (2023). https: / / doi.org / 10.1038 / s41586-022-05531-1 Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 49 DK17029PCTable 1: Amino acid sequence, gene symbol and type of mutation of synthetic vaccine peptides, selected cancer epitopes and corresponding wildtype epitopes Amino acid sequence SEQ ID Gene symbol Type of cancer epitope SEQ ID wt epitope SEQ IDNO mutation NO NO KSGLSSRVQFQNQGSEPKYTQ 1 ITGA6 SNVSLAGIPAACDAFHPFIEALLP 2 NFIX SVN AACDAFHPF 10 AACDEFHPF 13LGYKASVKSLNTQVADLKLQL 3 OFD1 SNV SLNTQVADLK 11 SLTTQVADLK 14MAPAAVWAALAVGL 4 TNFRSF1B SNVSATPSSSLPKCTREEEEDSTI 5 TPR SNVRRRQALRGKSIPVLKKTPNKG 6 ZC3H3 SNVYPAGGGGGGGTSAA 7 NKX2-3 IndelDDNLKTPPECLLTPLPPSADDNLKKL 8 NPIPA5 IndelGLASSRTATVTFKHCLQPPSTSP 9 TYRO3 Indel ASSRTATVTFK 12 ASSRTATVHLQ 15

[0002] Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 50 DK17029PCTable 2: Amino acid sequence, gene symbol of the synthetic vaccine peptide NY-ESO-1 79-108 and identified CD4 and CD8 T cell epitopes ofpatient 002 Amino acid sequence SEQ Gene symbol CD8 T cell epitope SEQ CD4 T cell epitope SEQ IDID NOID NO NO GARGPESRLLEFYLAMPFATPMEAELARRS 16 NY-ESO-1 YLAMPFATPM 17 SRLLEFYLAMPFATP 21FATPMEAEL 18 LEFYLAMPFATPMEA 22LAMPFATPM 19LEFYLAMPF 20Table 3: Amino acid sequence, gene symbol and type of mutation of synthetic vaccine peptides of patient 003 Amino acid sequence SEQ IDGene Type ofAmino acid sequence SEQGene Type of NO symbol mutation ID NO symbol mutation LCNACGLYHKINGQNRPLIKP 23 GATA3 SNV ASTPIPPTPQAPSPAVDA 31 CDAN1 InDelTEYKLVVVGAVGVGKSALTIQ 24 KRAS SVN SPNTLILSHFVGRPMST 32 NES InDelLFQPFLKVIEQVGNREEKILN 25 PIK3CA SNV EDPPLPPTPMNSLVDEC 33 NPHP3 InDelTNHEIMPGGSFNITCVAVGSP 26 PTPRD SNV KMTQPQSKSASPLSRKNKGSG 34 PRUNE InDelRVGRIEKVWVLDGAAYFYGPI 27 SMIM4 SNV SKADTGSSNQGKASKMSSPET 35 NKIRAS2 SNVAEPNCADPATLTRPVHDAARE 28 CDKN2A InDel LYTLISKFFSFFCVSTLAYTK 36 MRPS18C SNVVFPSPPVPQTTQGFIG 29 FAM47C InDel AGRSKKETKYYLKAVEDMLET 37 BTBD10 SNVQSSRVLPQGPPTPAKTPGASA 30 C20orf85 InDel ASTPIPPTPQAPSPAVDA 38 EMC2 SNV

[0003] Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 51 DK17029PCTable 4: Long peptides used for vaccination in Example 14

[0004] Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 52 DK17029PCTable 5: Sequence characteristics of TCR-1131 and 1132 from Example 14

Claims

Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 53 DK17029PCClaims1. A preparation comprising T cells recognizing at least one epitope of a disease antigen(disease epitope) for use in treating and / or preventing disease in a human subject,wherein said preparation is produced or producible by a method comprising(a) incubating T cells from a sample of a site of vaccination of said human subject withthe disease epitope (vaccination site) under conditions suitable for proliferation of saidT cells; and (b) thereby producing a preparation comprising T cells recognizing at least one disease epitope.

2. The preparation for use of claim 1, wherein said disease is a cancer or is caused by apathogenic microorganism.

3. The preparation for use of claim 1 or 2, wherein said disease is a cancer.

4. The preparation for use of any one of claims 1 to 3, wherein said vaccination site is asite of topical administration of the disease epitope.

5. The preparation for use of any one of claims 1 to 4, wherein said vaccination site is asite of intradermal, subcutaneous, and / or intramuscular administration of the diseaseepitope.

6. The preparation for use of any one of claims 1 to 5, wherein said sample was obtainedwithin 50 mm of the vaccination site, preferably wherein said sample comprises a tissue showing a vaccination reaction or an aliquot thereof.

7. The preparation for use of any one of claims 1 to 6, wherein said sample comprises Tcells and is essentially free of cancer cells.

8. The preparation for use of any one of claims 1 to 7, wherein said vaccination was thefirst, second, or third vaccination of the subject with the disease epitope causing a visible vaccination reaction, preferably causing a tissue showing a vaccination reaction.Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 54 DK17029PC9. The preparation for use of any one of claims 1 to 8, wherein said vaccination comprisedadministration of at least one disease vaccine at the vaccination site.

10. The preparation for use of any one of claims 1 to 9, wherein said at least one diseaseepitope is comprised in a disease vaccine.

11. The preparation for use of claim 9 or 10, wherein said disease vaccine comprises at leastone cancer neoepitope.

12. The preparation for use of any one of claims 9 to 11, wherein said disease vaccinecomprises at least one cancer neoepitope as synthetic long peptide, preferably comprises a multitude of cancer neoepitopes as synthetic long peptides.

13. The preparation for use of any one of claims 9 to 12, wherein said disease vaccinecomprises at least one cancer neoepitope matched to an MHC subtype expressed by the subject to be treated as synthetic long peptide, preferably comprises a multitude of cancer neoepitopes matched to at least one MHC subtype expressed by the subject to betreated as synthetic long peptides.

14. The preparation for use of any one of claims 1 to 13, wherein said disease vaccinecomprises a plurality of disease epitopes.

15. The preparation for use of any one of claims 1 to 14, wherein said disease vaccinecomprises at least one synthetic long peptide.

16. The preparation for use of any one of claims 1 to 15, wherein said vaccination comprisedadministration of the disease vaccine in a water-in-oil emulsion, preferably comprisingmineral oil and anhydro mannitol ether octodecenoate, preferably as only adjuvant.

17. A disease vaccine for use in improving an immune response of a human subject to adisease epitope, said improving comprising(A) administering said disease vaccine to a donor subject at a vaccination site; (B) obtaining a sample from said vaccination site;Deutsches KrebsforschungszentrumStiftung des öffentlichen Rechts et al. 55 DK17029PC(C) producing a preparation comprising T cells recognizing at least one disease epitope from the sample obtained in step (B) as a disease vaccine; and (D) administering the preparation comprising T cells produced in step (C) to the humansubject.

18. The disease vaccine for use of claim 17, wherein said improving an immune responseto a disease epitope is improving an immune response to a cancer.

19. The disease vaccine for use of claim 17 or 18, wherein said improving comprises at leastone further feature of any one of claims 1 to 16.

20. A method of identifying a TCR binding to a disease epitope presented on a cell,preferably a cancer epitope, of a human subject, said method comprising (AA) providing a preparation comprising T cells recognizing at least one disease epitope according to the method specified in any one of claims 1 to 18,(BB) providing the amino acid sequences of at least the complementarity determiningregions 3 (CDR3s) of the TCRs of the T cell provided in step (AA); and, thereby,(CC) identifying a TCR binding to a disease epitope presented on a cell.

21. A method of providing a T cell recognizing a cell presenting a disease epitope,preferably a cancer epitope, said method comprising (aa) identifying a TCR binding to a disease epitope according to the method according to claim 20, (bb) expressing a TCR comprising at least the complementarity determining regions 3 (CDR3s) of the TCR of step (aa) in a T cell, and, thereby, (cc) providing a T cell recognizing a cell presenting a disease epitope, preferably a cancer epitope.

22. A T cell recognizing a cell presenting a disease epitope produced or producibleaccording to the method according to claim 21.

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