Methods for producing vaccine site infiltrating lymphocytes and uses thereof

Abstract

This invention relates to a method for producing a formulation comprising T cells that recognize at least one epitope (disease epitope) of a disease antigen, the method comprising (a) incubating T cells from a sample taken at a site where the subject has been vaccinated with the disease epitope (vaccination site) under conditions suitable for the proliferation of the T cells; and (b) thereby producing a formulation comprising T cells that recognize at least one disease epitope. The invention also relates to disease vaccines for improving an immune response to a disease epitope, to formulations comprising T cells that recognize at least one cancer epitope, to methods for identifying TCRs that bind to a disease epitope, and to providing associated T cells that recognize cells presenting a disease epitope.

Description

[0001] This invention relates to a method for producing a formulation comprising T cells that recognize at least one epitope of a disease antigen (disease epitope), the method comprising (a) incubating T cells from a sample taken at a site of vaccination of the subject with the disease epitope (vaccination site) under conditions suitable for the proliferation of the T cells; and (b) thereby producing a formulation comprising T cells recognizing at least one disease epitope. The invention also relates to disease vaccines for improving an immune response to a disease epitope, to formulations comprising T cells recognizing at least one disease epitope, to methods for identifying TCRs that bind to a disease epitope, to methods for providing T cells that recognize cells presenting a disease epitope, and to T cells associated with recognizing cells presenting a disease epitope.

[0002] T-cell-based immunotherapy has garnered significant attention in recent years due to the invention of CAR-T cells and the recent development of TCR transgenic T-cell therapy [1,2]. CAR T-cells have been successfully used in hematologic malignancies, primarily targeting lineage-specific cell surface proteins such as CD19 [3] or BCMA [4], but at the cost of permanently eliminating all B cells or plasma cells [5]. A drawback of this approach is the requirement for the presence of specific target proteins on the cell surface, a drawback that has been proposed to be overcome by vaccinating patients with tumor cells (optionally combined with IL-2-producing fibroblasts)

[22] . Furthermore, transgenic T-cells recognize a single tumor target that can be downregulated by tumor cells, leading to immune escape.

[0003] Another approach aims to expand tumor-infiltrating lymphocytes (TILs) generated from tumor specimens of solid tumors. In a recent phase III trial for unresectable stage IIIC or IV melanoma, TIL therapy resulted in longer progression-free survival compared to anti-CTLA4 therapy [6]. The disadvantages of the TIL approach are that the specificity of TILs is unknown, and TILs may be depleted or suppressed and are difficult to expand in some cases. In addition, studies have shown that the tumor microenvironment contains a large number of bystander T cells [7], and TILs do not always recognize novel epitopes eluted from autologous tumor cells [8].

[0004] To increase the number of tumor-specific T cells, vaccination with appropriate antigens has been proposed. For example, Tong et al.

[21] administered intraperitoneal vaccination to mice with tumor-ganglioside glycosaminoglycans and were able to detect tumor-specific T cells that could become TILs in the spleen.

[0005] Highly mutated tumors contain so-called neoepitopes that are tumor-specific and can be recognized by the patient’s immune cells to attack the tumor[8]. These patient-specific somatic mutations can be identified by DNA sequencing of the tumor and control samples (e.g., blood) from the same patient[9]. Expression is confirmed by RNA sequencing and potential epitopes can be predicted by publicly available procedures or optionally eluted from tumor cells and identified by mass spectrometry[8]. Peptides containing these putative epitopes can be synthesized, and administration of personalized tumor peptides as therapeutic vaccines can lead to stimulation of the body’s own immune cells, sometimes resulting in tumor control or elimination of tumor cells[10,11]. Analysis of subcutaneous vaccine sites has shown accumulation and activation of T cells, particularly after repeated vaccination with incomplete Freund’s adjuvant[12,13] or after vaccination with specialized antigen-presenting cells loaded with tumor lysate

[20] .

[0006] Therefore, there remains a need for methods to provide T cells that are responsive to specific antigens (e.g., cancer antigens) and corresponding improvements to TCRs. This problem is solved by the embodiments defined in the claims and described below.

[0007] In view of the above, the present invention relates to a method for producing a formulation comprising T cells that recognize at least one epitope (disease epitope) of a disease antigen, the method comprising:

[0008] (a) Incubating T cells from a sample taken at the site of vaccination of the subject with the disease epitope (vaccination site) under conditions suitable for the proliferation of the T cells; and

[0009] (b) thereby producing a formulation containing T cells that recognize at least one disease epitope.

[0010] Generally, the terms used herein should have the conventional and common meanings understood by those skilled in the art, and should not be given special or custom meanings unless otherwise stated. As used below, the terms “having,” “comprising,” or “including,” or any of their grammatical variations, are used in a non-exclusive manner. Thus, these terms can refer both to the absence of any other feature in the entity described herein besides the feature introduced by that term, and to the presence of one or more other features. For example, the expressions “A has B,” “A contains B,” and “A includes B” can refer both to the absence of any other element in A besides B (i.e., A consists solely and exclusively of B), and to the presence of one or more other elements in entity A besides B, such as element C, element D, or even more elements. Furthermore, as understood by those skilled in the art, the expression “comprising / including” preferably means “comprising / including one or more / a kind or more,” that is, equivalent to “comprising / including at least one / at least one.” Accordingly, unless otherwise stated, a statement involving one of a plurality of terms preferably involves at least one such term, more preferably more than one such term; thus, for example, identifying a “cell” involves identifying at least one cell, preferably more than one cell.

[0011] Furthermore, as used below, the terms “preferred,” “more preferably,” “most preferably,” “particularly,” “more particularly,” “specifically,” “more specifically,” or similar terms are used in combination with optional features without limiting other possibilities. Therefore, features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As those skilled in the art will recognize, the invention can be implemented using alternative features. Similarly, features introduced by “in an embodiment” or similar expressions are intended to be optional features, without limiting other embodiments of the invention, without limiting the scope of the invention, and without limiting the possibility of combining features introduced in this way with other optional or non-optional features of the invention.

[0012] The methods described below are preferably in vitro methods. The method steps can, in principle, be performed in any arbitrary order that a person skilled in the art deems suitable, but are preferably performed in a specified order; furthermore, one or more of these steps, preferably all of them, can be assisted or performed by automated equipment. In addition to the steps explicitly mentioned above, the method may also include other steps.

[0013] As used herein, unless otherwise stated, the term "about" refers to a specified value having a technical precision generally accepted in the relevant field, preferably ±20%, more preferably ±10%, and most preferably ±5%. Furthermore, the term "substantially" means that there is no deviation affecting the indicated result or use, i.e., potential deviations will not cause the indicated result to deviate from the specified value by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, "substantially composed of..." means including the specified components but excluding other components, except for materials present as impurities, substances unavoidably present in the process of preparing the components, and components added for purposes other than achieving the technical effects of the invention. For example, compositions defined using the phrase "substantially composed of..." encompass any known and acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition substantially composed of one set of components will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of unspecified components.

[0014] As used herein, the term "polynucleotide" refers to a linear nucleic acid molecule or a circular nucleic acid molecule. The polynucleotides of the present invention are preferably provided as isolated polynucleotides (i.e., isolated from their naturally occurring state) or in a genetically modified form, and preferably contain at least one heterologous sequence. The term "polynucleotide" encompasses single-stranded as well as partially or fully double-stranded polynucleotides. Preferably, the polynucleotide is a DNA polynucleotide, which may also be referred to as "DNA". Furthermore, it includes 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, etc. In view of the description herein, template DNA (e.g., for PCR), primers (e.g., sequencing primers or amplification primers), and probe oligonucleotides are all included in the term polynucleotide.

[0015] Unless otherwise specified, references to specific polynucleotides herein preferably include polynucleotide variants. The term "polynucleotide variant" as used herein refers to a variant of the polynucleotide mentioned herein, comprising a nucleic acid sequence characterized in that the sequence can be derived from the specific nucleic acid sequence by at least one nucleotide substitution, addition, and / or deletion, wherein the polynucleotide variant should have the function and / or activity specified for the specific polynucleotide. Thus, a polynucleotide variant can be, for example, an ortholog, paralog, or other homolog of the specific polynucleotide; a polynucleotide variant can also be a mutant of the specific polynucleotide, preferably a naturally occurring mutant, such as those identified in cancer cells. Also preferably, the polynucleotide variant is or is derived from a non-naturally occurring allele of the specific polynucleotide. Other polynucleotide variants include polynucleotides comprising a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity with the specifically specified nucleic acid sequence. The percentage of sequence identity is preferably calculated over the entire nucleic acid sequence region, preferably as described in other parts of this document. The polynucleotides of the present invention are composed of, substantially composed of, or contain the above-described nucleic acid sequences. Therefore, they may also contain other nucleic acid sequences.

[0016] As used herein, the term "peptide" refers to a molecule composed of multiple amino acids covalently linked together by peptide bonds. A peptide consisting of fewer than 20 amino acids covalently linked by peptide bonds may also be called a "peptide". Preferably, the peptide comprises 4 to 1000, more preferably 5 to 1000, even more preferably 6 to 500, and most preferably 7 to 400 amino acids. The peptide may also be contained in fusion peptides and / or peptide complexes.

[0017] Unless otherwise specified, references to specific polypeptides herein preferably include polypeptide variants. As used herein, the term "polypeptide variant" refers to any chemical molecule containing at least one polypeptide as specified herein, whose structure differs from that of the specifically specified polypeptide. Preferably, the polypeptide variant contains a polypeptide whose sequential amino acid sequence corresponds 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%, and most preferably at least 99% of the amino acid sequence of the specifically specified polypeptide. Furthermore, it should be understood that the polypeptide variant according to the invention should have an amino acid sequence that 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%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identical to the amino acid sequence of the specific polypeptide. The degree of identity between the two amino acid sequences can be determined by algorithms known in the art and methods described elsewhere herein. The aforementioned polypeptide variants can be allelic variants or any other species-specific homologs, paralogs, or orthologs. Furthermore, the peptide variants mentioned herein include fragments of specific peptides or peptide variants of the aforementioned types, preferably provided that these fragments and / or variants possess the specified activity, such as an epitope or that the peptide containing it is immunogenic. Such fragments may be, or can be derived from, for example, degradation products of peptides or splice variants, or from gene fusions, insertions / deletions, and / or frameshift mutations. Variants also include those that differ due to post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, SUMOylation, or myristylation), by the inclusion of non-natural amino acids, and / or by functioning as peptide mimics. The “activity” of the peptides specified herein is preferably retained in the peptide variants; as those skilled in the art should understand from the description herein, the activity of a peptide does not necessarily have to reflect its primary natural function, but may be other activities relevant in the context of this invention. By strict example only, p53 peptides are active in cell cycle regulation, but p53, particularly its mutants, is also immunogenic. Therefore, p53 mutants (including fragments thereof) are p53 variants as referred to herein, preferably possessing immunogenic activity.

[0018] The term "fragment" of a biomolecule, preferably a fragment of a polynucleotide or polypeptide, is used in its broadest sense herein to refer to any sub-part, preferably a subdomain, of the respective biomolecule that contains the specified sequence, structure, and / or activity. Thus, the term includes sub-parts generated by actual fragmentation of a biomolecule, as well as sub-parts derived from the respective biomolecule in an abstract manner, such as through computer simulation. Therefore, as used herein, Fc fragments or Fab fragments, and, for example, single-chain antibodies, bispecific antibodies, and nanobodies, can all be referred to as fragments of immunoglobulins. Similarly, a disease epitope can be a fragment of a disease antigen.

[0019] Unless otherwise specified herein, the designated compounds, particularly polynucleotides and peptides, may be contained within larger structures, for example, covalently or non-covalently linked to other sequences, adjuvants, carrier molecules, sustained-release agents, and other excipients. In particular, the designated peptides may be contained within fusion peptides that include other peptides, which may serve, for example, as tags for purification and / or detection, as linkers, or for extending the in vivo half-life of the compound. The term "detectable tag" refers to a segment of amino acids added to or introduced into a fusion peptide; preferably, the tag is added at the C-terminus or N-terminus of the fusion peptide. This segment of amino acids is preferably detectable by antibodies that specifically recognize the tag; or preferably forms a functional conformation, such as a chelating agent; or preferably is visualized, for example, in the case of a fluorescent tag. Preferred detectable tags are Myc tags, FLAG tags, 6-His tags, HA tags, GST tags, or fluorescent protein tags, such as GFP tags. These tags are well known in the art. Preferably, other peptides included in the fusion polypeptide contain other amino acids or other modifications that can function as secretion mediators, as mediators for blood-brain barrier crossing, as cell-penetrating peptides, and / or as immunostimulants. Other polypeptides or peptides that the polypeptide may fuse to are signaling and / or transport sequences, such as IL-2 signaling sequences, and adapter sequences.

[0020] The degree of identity (e.g., expressed as "identity %") between two biological sequences (preferably DNA, RNA, or amino acid sequences) can be determined using algorithms known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the sequence fragment in the comparison window may contain additions or deletions (e.g., vacancies or single-stranded overhangs) compared to the sequences being compared for optimal alignment. This percentage is calculated by determining (preferably over the entire length of the polynucleotide or polypeptide specified herein) the number of positions in both sequences where the same residues occur to produce a number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the sequence identity percentage. The optimal alignment of sequences for comparison can be achieved through Smith and Waterman's (1981) local homology algorithm, Needleman and Wunsch's (1970) homology alignment algorithm, Pearson and Lipman's (1988) similarity search method, and computerized versions of these algorithms (e.g., BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection. Given that two sequences have been identified for alignment, GAP and BESTFIT are preferably used to determine their optimal alignment, thereby determining the degree of identity. Preferably, alignment is performed using default values ​​of 5.00 for gap weight and 0.30 for gap length. More preferably, the Basic Local Alignment Search Tool (BLAST) is used, and alignment is performed using the default parameter values. In the context of biological sequences mentioned herein, the term "substantially identical" means an identity value of at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%. It should be understood that the term "substantially identical" includes 100% identity. The foregoing content, after necessary modifications, applies to the term "substantially complementary".

[0021] Those skilled in the art will understand that the term “T cell” refers to a lymphocyte expressing at least one T cell receptor. Preferably, the T cell is a CD8+ T cell that recognizes a major histocompatibility complex (MHC) class I molecule on the surface of a target cell, or a CD4+ T cell that recognizes a MHC class II molecule on the surface of a target cell. Preferably, the T cell is a cytotoxic T cell, more preferably a CD8+ cytotoxic T cell, which may also be referred to as a “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., a cancer-reactive T cell. Thus, preferably, the T cell expresses a TCR that recognizes a disease epitope. Preferably, the T cell is a recombinant cell that expresses a chimeric antigen receptor (CAR) (e.g., a TCR-like CAR

[19] ) and / or a recombinant T cell receptor. Methods for producing suitable recombinant T cells are known in the art. As is known to those skilled in the art, human MHC may also be referred to as “human leukocyte antigen” (HLA).

[0022] As used herein, the term "T-cell receptor" (abbreviated as "TCR") refers to a polypeptide complex on the surface of T cells that mediates the recognition of antigenic peptides presented by target cells, preferably in the presence of MHC molecules or MHC-related molecules (such as MR1 ​​or CD1), more preferably in the presence of MHC molecules, even more preferably in the presence of MHC class I or MHC class II molecules, and most preferably in the presence of MHC class I molecules. Typically, a TCR comprises one TCR-α chain and one TCR-β chain, i.e., it is an α / β chain heterodimer. However, a TCR may also comprise TCR-γ and TCR-δ chains instead of the TCR-α and TCR-β chains. TCR-α and TCR-β chains or TCR-γ and TCR-δ chains mediate antigen recognition and each contains a transmembrane region, a constant region, a linker region, and a variable region. The variable region of each TCR-α, TCR-β, TCR-γ, or TCR-δ chain contains three complementarity-determining regions (CDRs), referred to as CDR1, CDR2, and CDR3, respectively. According to common nomenclature, the complex composed of the α and β chains or the γ and δ chains is referred to herein as a “T cell receptor” or “TCR”. The α and / or β chains and the γ and / or δ chains are generally or individually referred to as “TCR polypeptides,” while polypeptide complexes containing TCR and helper polypeptides (such as CD3 and CD247) are referred to as “T cell receptor complexes,” abbreviated as “TCR complexes.” Preferably, the T cell receptor binds to major histocompatibility complex (MHC) molecules that present epitopes, preferably MHC class I or II, more preferably MHC class I molecules, said epitopes, preferably cancer epitopes, which contribute to and / or are associated with disease. The binding of T cell receptors to antigens can be determined by methods known to those skilled in the art, such as tetramer assays. Preferably, the binding of the TCR to an epitope presented on the MHC activates T cells. Activation biomarkers for various types of T cells are known in the art, particularly including CD69, CD137, CD27, TRAP / CD40L, and CD134. The TCR can also be a soluble TCR. The term "soluble TCR" is understood by those skilled in the art in principle to refer to a TCR lacking a transmembrane domain as described above. Therefore, preferably, a soluble TCR comprises both the constant and variable regions of the TCR polypeptide. More preferably, a soluble TCR comprises the variable region of the TCR polypeptide, preferably in the form of a fusion polypeptide.

[0023] Those skilled in the art will understand the term "complementarity-determining region," abbreviated as "CDR." As is known in the art, each TCR-α, TCR-β, TCR-γ, and TCR-δ chain contains three CDRs, the amino acids of which substantially provide contact sites for peptides presented by the MHC molecules described elsewhere in this document. As is known to those skilled in the art, in TCRs, epitope binding specificity is substantially determined by the variable domain, particularly by the CDR3 region of the variable region. Therefore, to reproduce the binding properties of a given TCR, it may be sufficient to transfer the CDR3 of the TCR to a suitable backbone, such as the backbone of another TCR. Preferably, in this case, all CDRs 1 to CDR 3, or the variable region or fragments thereof, are transferred.

[0024] Those skilled in the art will understand that the term "formulation" refers to any composition of substances containing at least the specified components. Therefore, the formulation may also contain other components, particularly those necessary to maintain the survival of T cells, such as at least one solvent, preferably water, a buffer solution, and / or one or more salts, preferably sodium chloride, potassium chloride, etc. Preferably, the formulation is a cell culture composition and / or a pharmaceutical composition.

[0025] The formulation contains T cells. In the context of this specification, the formulation is preferably obtained within six weeks after the start of step (a), more preferably within four weeks. Preferably, the formulation contains at least 10 9 T cells, more preferably containing at least 10 10 10 T cells. Preferably, the aforementioned cell number is obtained within the aforementioned time range; that is, preferably within six weeks after the start of step (a), more preferably within four weeks, the formulation contains at least 10 T cells. 9 T cells, more preferably containing at least 10 10 Each T cell. Preferably, within six weeks after the start of step (a), more preferably within four weeks, the formulation contains at least 50% T cells, and more preferably at least 75% T cells.

[0026] Those skilled in the art will understand that the term "cell culture composition" refers to a composition containing all the compounds required for T cell proliferation. Therefore, the cell culture composition preferably contains (preferably in addition to one or more additional components described above for the formulation) at least one nutrient required for T cell proliferation, one or more stimulants, such as one or more selected from the cytokines IL-2, IL-7, IL-15, IL-12, IL-21, and combinations of at least two thereof; anti-CD3 antibodies and / or anti-CD28 antibodies; and feeder cells. Preferably, the cell culture composition is a composition that promotes the in vitro growth of T cells, and more preferably, a T cell culture medium. Suitable culture media are known in the art and are described in the examples.

[0027] The terms “drug” and “pharmaceutical composition” are used interchangeably herein and are known in principle to those skilled in the art. As used herein, these terms refer to any composition of substances comprising a specified active agent (particularly T cells) as a pharmaceutically active compound and one or more excipients. It should be understood that the form and nature of a pharmaceutically acceptable excipient (e.g., a carrier or diluent) are determined by the amount of the active ingredient to be combined, the route of administration, and other well-known variables. The excipient must be acceptable, i.e., compatible with the other components of the formulation and harmless to its recipient. Therefore, the excipient used is preferably a liquid, preferably an aqueous solution. An example of a liquid carrier is physiological saline supplemented with a stabilizer (such as human serum albumin), optionally containing other supplements. The carrier may also be an injectable cryogenic carrier, allowing the drug to be frozen and injected directly after thawing. Suitable carriers include those described above and other carriers known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. Excipients are selected so as not to impair the bioactivity of the combination and the bioactivity of the T cells contained therein.

[0028] Preferably, the drug is administered systemically, preferably orally or parenterally, such as via intravenous administration, or locally, preferably intratumorally, on a body surface, or by inhalation; in the case of cancer treatment, local administration may be intratumoral or peritumoral, and / or at the site of tumor resection. However, administration may also be via blood vessels, typically arteries, flowing to the intended site of effect, such as the tumor. However, depending on the nature of the formulation and the desired therapeutic application, the drug may also be administered via other routes.

[0029] The drug is preferably administered via the route specified above. A therapeutically effective dose refers to the amount of T cells used in the drug that are capable of preventing, improving, or curing the disease or symptoms associated with the condition referred to in this specification. The therapeutic efficacy and toxicity of the drug can be determined through standard pharmaceutical procedures in cell culture or laboratory animals. The administration regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any single patient depends on many factors, including the patient's body size, age, the specific drug formulation to be administered, sex, time and route of administration, general health condition, and other concurrently administered drugs. The drug mentioned herein is preferably administered at least once, for example, as a bolus injection. However, the drug may be administered more than once, and preferably at least twice, for example, permanently or periodically after a defined time window. Progress can be monitored through periodic assessments. Dosage recommendations can be indicated in the prescriber's or user's instructions for dose adjustment based on the expected dose for the intended recipient. In addition to the active agents described above, the drug may contain other active agents; for example, in the case of cancer treatment, said at least one other active agent is preferably a chemotherapeutic agent or a further immunotherapeutic agent, such as an immune checkpoint modulator.

[0030] The term "disease" is known to those skilled in the art. Preferably, the disease is caused by at least one pathogen that can be recognized by T cells. Thus, the disease is preferably cancer or caused by a pathogenic microorganism, wherein the term "pathogenic microorganism" includes each and every microorganism that specifically or facultatively causes disease in at least one object. Thus, pathogenic microorganisms can be, in particular, bacteria, viruses, parasites, or fungi.

[0031] As used herein, the term "cancer" refers to a disease in animals (including humans) characterized by the uncontrolled growth of a group of body cells ("cancer cells"). This uncontrolled growth may be accompanied by invasion and destruction of surrounding tissues (invasion) and the potential spread of cancer cells to other locations in the body (metastasis). Preferably, the term cancer also includes the recurrence of cancer after treatment (relapse). Thus, preferably, cancer is a solid tumor, its metastasis, or its recurrence. Cancer can be induced by an infectious agent, preferably a virus, more preferably an oncogenic virus, and even more preferably EB virus, hepatitis virus, human T-lymphotropic virus-1, human papillomavirus, or human herpesvirus-8. However, cancer can also be induced by compounds, such as carcinogens, or endogenously induced, such as by spontaneous mutations. Preferably, cancer contains at least one cancer epitope.

[0032] Preferably, the cancers are selected from: acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma (ACC), adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, basal cell carcinoma, bile duct cancer, bladder cancer, brainstem glioma, breast cancer, Burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer. Colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, gonadal 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 carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and optic pathway glioma, intraocular melanoma, Kaposi's sarcoma, laryngeal cancer, medulloblastoma, medullary epithelial tumor, melanoma Merkel cell carcinoma, mesothelioma, oral cancer, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal carcinoma, osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade malignant potential ovarian tumor, pancreatic cancer, papilloma, paranasal sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma. Tumors, pituitary adenomas, pleural pulmonary blastomas, primary central nervous system lymphomas, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Cezari syndrome, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and nephroblastoma. More preferably, the cancer is adenoid cystic carcinoma (ACC) or colorectal cancer (CRC).

[0033] The term "antigen" in principle includes every and every immunogenic compound, that is, every and every compound that has the activity of inducing an immune response against its own structure in a subject. As those skilled in the art will understand, the immunogenicity of a given compound against a given subject depends on a variety of factors, including the health status of the subject, the manner of administration of the antigen, and others. Furthermore, in the case of cancer antigens, the tumor microenvironment and / or immunosuppression may reduce or prevent the immunogenicity of the antigen. Therefore, as used herein, an antigen is preferably a compound that induces an immune response against its own structure in a statistically significant proportion of subjects to which the antigen is administered, wherein the subject is preferably a surface-healthy subject. Whether a proportion is statistically significant can be determined by those skilled in the art using various well-known statistical assessment tools, such as the determination of confidence intervals, p-value determination, T-tests, Mann-Whitney tests, etc., without excessive effort. See Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 for details. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is 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 the subject. Preferably, the antigen has the activity of stimulating antigen-specific T cells, the term "antigen-specific T cell" referring to T cells that present T-cell receptor molecules on their surface, which specifically recognize (i.e. bind) epitopes of the antigen presented in the MHC molecular background, i.e., preferably the disease epitopes of the present invention. Preferably, the antigen is a "disease antigen," i.e., an antigen contained in pathogens that cause the diseases described above. Thus, the antigen can be, in particular, a cancer antigen or an antigen of a pathogenic microorganism. Preferably, the antigen is a biological macromolecule, more preferably a polypeptide.

[0034] The term "cancer antigen" refers to an antigen, preferably a polypeptide, expressed by cancer cells. Preferably, the cancer antigen is expressed in non-cancer cells at a rate of at least 5-fold, preferably at least 10-fold, and more preferably at least 25-fold, compared to cancer cells. Preferably, the cancer antigen is not expressed in non-cancer cells of the subject, more preferably not expressed in non-cancer cells of the same tissue, and even more preferably not expressed in non-cancer cells of the subject; therefore, the cancer antigen is preferably a cancer-specific antigen. More preferably, the cancer antigen is a novel antigen expressed by cancer cells and / or contains a novel epitope. Preferably, one or more peptides of the cancer antigen are presented on the surface of cancer cells via MHC molecules, more preferably MHC-I molecules. As described elsewhere herein, the cancer is preferably a solid cancer, i.e., cancer that forms a tumor; therefore, the cancer antigen is preferably a tumor antigen, more preferably a tumor-specific antigen. Preferably, the cancer antigen is a polypeptide derived from a tumor-associated antigen, a polypeptide derived from a polypeptide encoded by a frameshift mutation, a polypeptide derived from a polypeptide encoded by a fusion gene, or a polypeptide derived from a viral antigen. Viral cancer antigens are known in the field, including, for example, human papillomavirus E6 and human papillomavirus E7, Epstein-Barr virus LMP-1, etc.

[0035] Those skilled in the art understand that the term "epitope" refers to the (sub)structure of an antigen that can be recognized by the immune system of a subject. As used herein, an epitope is an amino acid sequence, preferably a continuous amino acid sequence, i.e., a peptide. Preferably, the length of an epitope is at least three, more preferably at least four, more preferably at least five, and most preferably at least six amino acids. Also preferably, the length of an epitope is at most 50, more preferably at most 25, even more preferably at most 20, and most preferably at most 15 amino acids. Therefore, preferably, the length of an epitope is 3 to 50 amino acids, more preferably 4 to 25 amino acids. Preferably, the epitope is an MHC-I epitope and contains 8 to 13 amino acids; also preferably, the epitope is an MHC-II epitope and contains 13 to 25 amino acids.

[0036] Based on the foregoing, this document uses the term "disease epitope" to refer to an epitope of a disease antigen. Disease epitopes are preferably disease-specific, meaning they are preferably expressed and / or presented only by disease-causing cells, such as cells infected by pathogenic microorganisms or cancer cells. Also preferably, disease epitopes are epitopes derived from cancer antigens, particularly their subsequences, i.e., cancer epitopes. Thus, for example, if the disease antigen is a neocancer antigen, the disease epitope is preferably a novel epitope or contains a novel epitope, i.e., contains modified subsequences constituting a novel epitope. Cancer epitopes can be expressed by cancer cells resulting from any genetic modification compared to non-cancer cells, said genetic modification preferably being expressed by cancer cells. Such genetic modifications can be any type of mutation in the coding region of a gene, such as point mutations, insertions, deletions, or frameshift mutations. However, genetic modifications can also be mutations that cause the expression of gene products that are not normally expressed in the cells of the object, particularly somatic cells; such modifications can be, for example, gene fusions, translocations, promoter mutations, mutations leading to alternative splicing, aberrant translation of non-coding open reading frames, etc. Genetic modification can also be non-mutagenic, such as modifications to chromatin structure and / or histones. Genetic modification can also occur due to viral infection, leading to the expression of viral genes or the reactivation of retroviral genes, resulting in the specific expression of retroviral proteins in cancer cells.

[0037] Disease epitopes, particularly cancer epitopes, can be individual epitopes, i.e., epitopes identified in a sample of a subject to be treated; however, they can also be shared disease epitopes, i.e., disease epitopes known or presumed to be shared by multiple subjects, cancers, or other forms of disease. Shared cancer antigens and corresponding epitopes are well known in the art.

[0038] The disease epitopes described herein are recognized by T cells; therefore, disease epitopes are T-cell epitopes. As used herein, the term “T-cell epitope” refers to a continuous amino acid sequence contained in a polypeptide that can bind to major histocompatibility complex (MHC) class I or II molecules for presentation on the surface of cells (MHC-I) or specialized antigen-presenting cells (MHC-II). Those skilled in the art know how to predict immunogenic peptides presented on MHC-I or MHC-II (

[16] ,

[17] ) and how to assess the binding of specific peptides (e.g.,

[18] ). Preferably, the T-cell epitope is an MHC-I epitope. Preferably, the T-cell epitope is an epitope derived from a cancer antigen; therefore, preferably, the T-cell epitope is an epitope that is substantially absent or not present on normal cells of the subject (i.e., non-cancerous cells of the subject). Preferably, the T-cell epitope is a disease epitope. As those skilled in the art will understand, the disease vaccines described herein may contain multiple T-cell epitopes, such as preferably 2 to 100, more preferably 2 to 50, and even more preferably 2 to 25 T-cell epitopes, wherein the T-cell epitopes may be MHC-I epitopes and / or MHC-II epitopes. In this case, the T-cell epitopes are preferably MHC-I and MHC-II epitopes; more preferably, the T-cell epitopes are epitopes presented by MHC molecules encoded by different HLA-A, HLA-B, HLA-C, HLA-E, HLA-DR, HLA-DQ, and HLA-DP loci. Also preferably, the T-cell epitopes are epitopes presented by different MHC allotypes encoded by the mentioned MHC class I and MHC class II loci.

[0039] Those skilled in the art understand that the term "vaccine" includes every and every agent that induces and / or enhances an immune response in a subject. Preferably, the immune response is a cellular immune response, more preferably a T-cell response.

[0040] Accordingly, the “disease vaccine” referred to herein is a composition of substances comprising at least one compound, preferably a polypeptide, which comprises at least one disease epitope, more preferably multiple disease epitopes. Thus, a disease vaccine is a reagent for active vaccination. Preferably, the disease vaccine comprises at least one polypeptide containing at least one disease epitope; however, the at least one polypeptide may also contain multiple disease epitopes. More preferably, the disease vaccine comprises multiple polypeptides, each polypeptide containing at least one disease epitope; however, each of the multiple polypeptides may also contain multiple disease epitopes. Also preferably, at least one of the polypeptides comprises a disease epitope contained in a long peptide as described elsewhere herein. Preferred amino acid sequences to be included in the disease vaccine are shown in Table 1 and SEQ ID NO: 1 to SEQ ID NO: 20, more preferably SEQ ID NO: 1 to SEQ ID NO: 12 and SEQ ID NO: 16 to SEQ ID NO: 20. Preferably, the disease vaccine also comprises a carrier, preferably a pharmaceutically acceptable carrier. Furthermore, the disease vaccine may also include other additional compounds, such as stabilizers and / or other compounds deemed appropriate by the technician, for example, for pharmaceutical purposes. As used herein, at least one disease epitope specified above is the “active compound” of the vaccine, although other active compounds may be present. The vaccine composition is preferably prepared in a manner well known in the pharmaceutical field. The formulation is adapted to the method of administration, i.e., formulated as a solution, suspension, tablet, capsule, suppository, etc.

[0041] Preferably, the disease vaccine also contains an adjuvant. More preferably, the disease epitope and the adjuvant are contained in a common mixture at the time of administration. Therefore, preferably, the disease epitope and the adjuvant are mixed before administration. In the context of this specification, preferred adjuvants are mineral oil / surfactant mixtures (e.g., Montanide), muramyl dipeptides, saponins such as QS21 and Quil A, monophospholipid A, aluminum hydroxide, aluminum phosphate, hydroxyapatite, complete Freund's adjuvant and / or incomplete Freund's adjuvant, or cytokines such as interleukins, macrophage-derived chemokines, complement-binding proteins and tumor necrosis factors (free or fused to scaffold proteins), such as type I IFN or GMCSF, TLR agonists, universal helper peptides such as peptides derived from tetanus toxin, and live microbial vectors approved for human use such as attenuated Salmonella enterica serovar Typhimurium strains. Preferably, the adjuvant comprises mineral oil; equally preferably, the adjuvant comprises a surfactant, the term "surfactant" as used herein referring to a compound or mixture of compounds having amphiphilic properties and reducing the surface tension of liquids containing them. Preferably, the surfactant comprises a long-chain aliphatic mannitol conjugate, such as mannitol monooleate or mannitol octadecenoate. Preferably, the adjuvant comprises, and is composed of, mineral oil and mannitol oleate surfactant. Thus, an adjuvant comprising mineral oil and mannitol oleate surfactant is preferably the only adjuvant contained in the disease vaccine. Preferably, the aforementioned adjuvant is Montanide adjuvant, more preferably Montanide ISA™ 51.

[0042] The disease vaccine comprises at least one disease epitope, preferably at least one cancer epitope. More preferably, the disease vaccine comprises a plurality of peptides comprising different disease epitopes, preferably cancer epitopes. Preferably, the disease vaccine comprises at least one synthetic long peptide comprising one or more disease epitopes from overlapping amino acid sequences, or, in the case of disease antigens derived from frameshift mutations or gene fusions, tandem epitopes. Thus, the synthetic long peptide preferably comprises disease epitopes from at least two different disease antigens. Consistent with its common usage in the art, the term "long peptide" does not refer to relative length, but rather to the fact that the long peptide extends beyond the smallest disease epitope. Thus, in a preferred embodiment, the long peptide comprises a sequence of 15 to 35 amino acids in length, preferably 20 to 30 amino acids, more preferably about 25 amino acids. In a preferred embodiment, the disease vaccine comprises preferably any of the sequences in SEQ ID NO: 1 to 61.

[0043] In a preferred embodiment, the disease vaccine comprises at least one novel cancer epitope, wherein the novel cancer epitope is predicted or identified as being expressed by cancer. Methods for predicting novel cancer epitopes are known in the art, including, for example, at least partial sequencing of the cancer genome and / or cancer transcriptome, or at least partial identification of the cancer proteome. In a preferred embodiment, the novel cancer epitope is selected to match the MHC subtype of the subject to be treated. In another preferred embodiment, the novel cancer epitope is included in the disease vaccine as a synthetic long peptide, preferably as described above. In another preferred embodiment, the disease vaccine comprises multiple novel cancer epitopes. Therefore, in a preferred embodiment, the disease vaccine comprises at least one novel cancer epitope as a synthetic long peptide, more preferably more than one, i.e., preferably at least two, more preferably at least five, or even more preferably at least ten novel cancer epitopes as synthetic long peptides, wherein each synthetic long peptide preferably comprises one of the said novel cancer epitopes. In another preferred embodiment, the disease vaccine comprises at least one novel cancer epitope as a synthetic long peptide that matches the MHC subtype expressed by the subject to be treated, more preferably more than one, i.e., preferably at least two, more preferably at least five, or even more preferably at least ten, as novel cancer epitopes of the synthetic long peptide, wherein each of the synthetic long peptides preferably comprises one of the novel cancer epitopes.

[0044] Preferably, providing a disease vaccine includes identifying candidate disease epitopes in pathogenic factors (e.g., cancer cells) contained in the subject. As those skilled in the art will understand, the identification of candidate disease epitopes can be accomplished by any method that those skilled in the art deem appropriate. For this purpose, in principle, the genome, transcriptome, proteome, metabolome, or portions thereof can be identified in the sample (preferably in the antigen sample as specified elsewhere herein). Preferably, the disease vaccine contains at least one disease epitope in an oil-in-water emulsion, preferably in an adjuvant as described above.

[0045] The term "vaccination" is well known in the art. Preferably, this term includes administering at least one disease epitope (preferably a disease vaccine) to a subject at least once. The subject may be vaccinated multiple times, such as twice, three times, four times, or any number of times deemed appropriate by a technician. Preferably, at least one disease epitope is repeatedly administered until signs of successful vaccination are observed, particularly redness near the vaccination site. More preferably, at least one disease epitope is repeatedly administered until at least one identifiable vaccination reaction is formed at the vaccination site, for example, identifiable by local redness and / or swelling. Therefore, it is preferable to obtain a sample of the vaccination site after the subject has received the disease epitope for the first, second, third, fourth, or fifth time (preferably the subject has received the disease epitope for the third or fourth time). Even more preferably, a sample of the vaccination site is obtained after the subject has received the disease epitope for the first, second, or third time, which resulted in a vaccination reaction (preferably resulting in an identifiable vaccination reaction, particularly erythema or erythematous swelling).

[0046] Those skilled in the art should understand that the term "site of vaccination" can also be referred to as "vaccine site." Preferably, this term refers to the tissue in which the subject directly contacts the disease epitope, preferably the disease vaccine. Preferably, in the case of intradermal administration of the vaccine via a needle, the site of vaccination includes intradermal deposit of the disease vaccine. Therefore, in a preferred embodiment, vaccination includes local application of the disease vaccine, preferably intradermal, subcutaneous, and / or intramuscular administration.

[0047] Those skilled in the art understand the term "recognizing at least one disease epitope." Preferably, recognition is specific, i.e., more preferably, any other epitope is recognized with at least 2-fold less efficiency, preferably at least 5-fold, and more preferably at least 10-fold less efficiency. The specificity of recognition can be determined, for example, by determining the activation of T cells carrying a given TCR by a homologous disease epitope (e.g., when presented on the cell) and comparing said activation with activation caused by a non-homologous epitope. The specificity of recognition can also be determined by binding to an MHC multimer containing the disease epitope.

[0048] The term "sample" refers to a sample of biological material. A sample can, in principle, be of any type that a person skilled in the art would deem appropriate. Preferably, the sample is a sample from the subject. The sample can be a bodily fluid sample, such as blood, urine, saliva, pleural effusion, ascites, etc., or a sample derived from bodily fluids, such as plasma or serum. Preferably, the sample is an isolated cell sample or a sample from a tissue or organ, preferably from the site of vaccination. As known to a person skilled in the art, tissue or organ samples can be obtained from any tissue or organ by, for example, biopsy, surgery, or any other method that a person skilled in the art would deem appropriate. Isolated cells can be obtained from bodily fluid samples (e.g., lymph, blood, plasma, serum, pleural effusion, ascites, cerebrospinal fluid, etc.) or from a tissue or organ by separation techniques such as centrifugation or cell sorting. Preferably, the sample is a tissue or bodily fluid sample containing cells.

[0049] In the method of producing the formulation described herein, a sample from the site of vaccination of a subject with a disease epitope is used, also referred to as a "vaccination site sample". Therefore, the vaccination site sample is preferably obtained near the vaccination site and preferably contains or is presumed to contain lymphocytes, preferably T cells, and more preferably cancer-recognizing T cells. Preferably, the vaccination site sample is known or presumed to be free of cancer cells; therefore, the vaccination site sample is not a cancer sample, i.e., preferably a sample of non-cancerous tissue. Preferably, the vaccination site sample is obtained within 50 mm, preferably within 40 mm, and more preferably within 30 mm of the vaccination site. Preferably, the vaccination site sample comprises biological material from the vicinity of the vaccination site, which shows signs of successful vaccination, particularly redness, pain, and / or heat. Therefore, the vaccination site sample can particularly come from an area near the vaccination site showing post-vaccination redness. More preferably, the vaccination site sample comprises tissue showing a vaccination response or an aliquot thereof. The term "tissue showing a vaccination response" is known to those skilled in the art. As noted above, the tissue showing a vaccination response referred to herein is tissue from the vaccination site. Methods for obtaining biological sample material (particularly cells) from tissues that have shown a vaccine response are known to those skilled in the art. Preferably, the vaccine site sample is obtained within one week of the most recent administration of the disease vaccine at the vaccination site.

[0050] To prepare the disease vaccine described herein, a separate sample type can be used, indicating the causative agent and the disease antigen that said agent may contain, i.e., an "antigen sample," where the term is used broadly to refer to every and every sample that is known or presumed to provide information about the antigen expressed by the causative agent. That is, in the case of cancer, an antigen sample may be a sample containing cancer cells; in the case of an infectious disease, a sample may be a sample from an infection site (e.g., pus from an infection site). As understood by those skilled in the art based on the description herein, an antigen sample does not necessarily have to contain an antigen, such as a disease antigen. The requirement is satisfied if the components of the antigen sample lead to the conclusion that a disease antigen is likely present. For example, the presence of a specific metabolite in an antigen sample may allow the conclusion about the presence of a specific mutated gene that produces said metabolite, or the presence of a specific gene product may allow the conclusion about the presence of a predefined pathogenic microorganism; for example, the presence of hepatitis virus polynucleotides and / or anti-hepatitis virus antibodies allows the identification of hepatitis virus infection, and the presence of enterotoxins in the sample allows the identification of the presence of at least one Gram-negative bacterium. Preferably, the antigen sample is a cancer sample. From such cancer samples, cancer antigens can be identified from the proteome of cancer cells using methods such as mass spectrometry, transcriptomic analysis, or genomic analysis. However, as mentioned above, metabolomic analysis can also provide information about which cancer antigens are expressed by cancer. It should be understood that if genomic analysis is used, it is not always necessary to verify that the identified cancer antigens are indeed expressed by cancer, especially when cancer vaccines contain multiple cancer epitopes.

[0051] As used herein, the term "object" refers to vertebrates, preferably mammals, particularly livestock, companion animals, or laboratory animals. Most preferably, the object is a human being. Preferably, the object is suspected of or has been diagnosed with cancer.

[0052] The term "donor subject" is used herein in its common sense, referring to the subject from whom something is derived or obtained, particularly a sample from a vaccination site. Therefore, a donor subject is preferably someone who has received at least one dose of a vaccine against a disease epitope. Correspondingly, a "recipient subject" is the person to whom something, such as a T-cell immune product, is administered.

[0053] As used herein, the term "T-cell immune product" includes T cells that recognize disease antigens, which are administered to a subject to improve the immune response against said disease antigens. Preferably, a T-cell immune product is a T-cell preparation that recognizes at least one disease epitope as specified elsewhere herein and / or manufactured.

[0054] The method includes step (a): incubating T cells from a sample taken from a vaccination site (vaccination site) where the subject has been vaccinated with the disease epitope under conditions suitable for T cell proliferation. Those skilled in the art understand the term "conditions suitable for T cell proliferation." These conditions include parameters such as temperature, water activity, the presence and concentration of nutrients, cell density, the presence and concentration of stimulants, etc. Preferably, these conditions are for the in vitro proliferation of T cells, such as cultured T cells. Suitable conditions and protocols are known in the art. It is possible that the vaccination site sample may not contain T cells or that the T cells contained therein may be inactive or uncultureable. In such cases, those skilled in the art can envision obtaining another vaccination site sample and / or repeating the vaccination of the subject.

[0055] The method further includes step (b): thereby producing a formulation containing T cells that recognize at least one disease epitope. As detailed elsewhere herein, the vaccination site sample is known or suspected to contain T cells; preferably, the T cells in the vaccination site sample are less suppressed and / or exhausted compared to T cells contained in, for example, tumor-infiltrating lymphocyte formulations. Therefore, the T cells contained in the vaccination site sample will begin to proliferate once incubated under conditions suitable for T cell proliferation. Thus, the formulation described herein can be produced within six weeks, preferably four weeks, after initiating step (a) and has an acceptable number of T cells.

[0056] Preferably, the method for producing the formulation does not require the enrichment of T cells, disease epitope-specific T cells, and / or the removal of suppressor cells (such as regulatory T cells). Therefore, the method preferably does not include binding the T cells that recognize at least one disease epitope to a solid surface; and / or does not include labeling the T cells that recognize at least one disease epitope and enriching the labeled T cells, for example by flow cytometry.

[0057] Advantageously, in the work on which this invention is based, it was discovered that vaccine site infiltrating lymphocytes (VILs) are a better starting material for producing disease epitope-recognizing T-cell preparations, for example, superior to tumor infiltrating lymphocytes (TILs). In the work on which this invention is based, it was found that VILs, particularly after Montanide adjuvant vaccination, exhibit high viability and show higher proliferative capacity compared to TILs from the same patient. Therefore, T cells derived from VILs can expand more rapidly. Furthermore, it was found that after vaccination with multiple disease epitopes, VILs specifically recognizing at least some of these disease epitopes are highly enriched in the VILs. Therefore, the vaccine site is found to be an ideal source of recently activated tumor-specific T cells. Compared to the TIL approach, the cells are derived from the vaccine site (vaccine site infiltrating lymphocytes, VILs) containing tumor-specific peptides; therefore, reinfusing a large amount of newly generated, less phenotypically diminished tumor-specific VILs into the patient increases the chances of successful tumor defense by endogenous immune cells. Peptide epitope specificity can be studied in the VIL product, and cells after subtransfer can be monitored in the patient's blood. Compared to TILs and other types of T-cell therapies, VILs have several advantages: (1) due to their different origin, there is no risk of residual tumor cells in the T-cell products; (2) VILs are less inhibited, more active, and easier to expand; (3) during vaccination, specificity against novel epitopes can be selectively enhanced, and T cells with these specificities can be expanded for treatment; (4) VIL products can have multiple specificities, thereby targeting multiple epitopes from different antigens presented by different HLA alleles, which minimizes immune escape; (5) due to the autologous nature of T cells and the selection process during maturation, there is less risk of autoimmunity. Furthermore, in a preferred embodiment, unlike TIL preparation (where the possible targets are unknown), the proposed method for vaccination and T-cell preparation via VILs enables successful vaccination and preparation because the targets of the resulting T cells are determined by the disease epitopes in the vaccine, which are known.

[0058] The definitions made above, with necessary modifications, apply to the following. The additional definitions and explanations provided below, also with necessary modifications, apply to all embodiments described in this specification.

[0059] The present invention also relates to a disease vaccine for improving an immune response in a subject to a disease epitope, said improvement comprising...

[0060] (A) The disease vaccine is administered to the donor at the site of vaccination;

[0061] (B) Obtain a sample from the site of the vaccine administration;

[0062] (C) The sample obtained in step (B) is used to produce a formulation containing T cells that recognize at least one disease epitope as a disease vaccine; and

[0063] (D) Administer the disease vaccine produced in step (C) to the subject.

[0064] The term "immune response" is known to those skilled in the art. Preferably, the immune response is a T-cell immune response. Accordingly, improving the immune response preferably involves increasing the number and / or persistence of T cells in the subject that recognize disease antigens. It should be understood that increasing the number of T cells that recognize disease antigens includes de novo creation of such T cells. Preferably, improving the immune response to disease epitopes is improving the immune response to cancer.

[0065] Improving the immune response includes step (A): administering the disease vaccine to the donor subject at the site of vaccination. Suitable disease vaccines have been described elsewhere herein, as well as their administration methods, which are known in the art.

[0066] Improving the immune response further includes step (B): obtaining a sample from the vaccination site. Preferably, the sample from the vaccination site in step (B) is obtained near the vaccination site, as described above. Preferably, the sample from the vaccination site in step (B) is obtained within one week of the most recent administration of the vaccine in step (A). As mentioned above, the sample from the vaccination site is preferably a biopsy sample, preferably from tissue showing a response to vaccination.

[0067] Improving the immune response further includes step (C): producing a formulation containing T cells that recognize at least one disease epitope from the sample obtained in step (B) as a T cell immune product. As detailed above, the method described herein preferably does not require enrichment of T cells or disease antigen-specific T cells, therefore step (C) preferably does not include binding the T cells that recognize at least one disease epitope to a solid surface; and / or does not include labeling the T cells that recognize at least one disease epitope and enriching the labeled T cells, for example, by FACS sorting.

[0068] Improving the immune response further includes step (D): administering the T-cell immune product produced in step (C) to the subject. Administering may be done by any means and method deemed appropriate by a person skilled in the art. Preferably, the T cells in the T-cell immune product are transferred to a suitable pharmaceutically acceptable carrier, such as physiological saline supplemented with human serum albumin, prior to administration. Preferably, the T-cell immune product is administered intravenously; however, other routes of administration are also contemplated, such as intracranial, intraventricular, and / or intratumoral administration. Preferably, the T-cell immune product is administered to a human leukocyte antigen (HLA)-matched subject, i.e., a subject expressing the same HLA subtype as the donor subject. More preferably, the T-cell immune product is administered to a donor subject, i.e., the donor and recipient subjects are preferably identical, i.e., the T-cell immune product is preferably autologous transplantation.

[0069] The present invention also relates to T-cell-containing formulations produced by or through methods specified herein for producing formulations containing T cells, wherein the T cells recognize at least one cancer epitope. In a preferred embodiment, the present invention further relates to T-cell-containing formulations produced by or through methods specified herein for producing formulations containing T cells, wherein the T cells recognize at least one disease epitope.

[0070] The present invention also relates to formulations comprising T cells that recognize at least one disease epitope, wherein (i) at least 1%, preferably at least 2%, more preferably at least 3% of T cells specific to at least one cancer epitope, and / or (ii) T cells specific to at least two, more preferably at least three different disease epitopes, preferably at least three different cancer epitopes. The formulations are preferably obtained by methods specified elsewhere herein.

[0071] This invention also relates to formulations for use in pharmaceuticals as specified herein; and to the use of formulations as specified herein in the preparation of pharmaceuticals. This invention also relates to formulations as specified herein for the treatment and / or prevention of a target disease, preferably cancer. This invention further relates to the use of formulations as specified herein in the preparation of pharmaceuticals for the treatment and / or prevention of cancer.

[0072] The terms "treatment" and "management" refer to a significant improvement in the disease or condition mentioned herein or its accompanying symptoms. The treatment as used herein also includes complete recovery of health in relation to the disease or condition mentioned herein. It should be understood that the term "treatment" as used herein may not be effective for all subjects. However, preferably, the term requires that a statistically significant proportion of subjects suffering from the disease or condition mentioned herein be successfully treated. Whether a portion is statistically significant can be determined by those skilled in the art using various well-known statistical assessment tools (e.g., as described above) without excessive effort. Preferably, treating cancer aims to achieve disease stabilization, more preferably, to reduce the tumor burden of the subject. As those skilled in the art will understand, the effectiveness of treatment for cancer, for example, depends on a variety of factors, including, for example, the stage and type of cancer. Preferably, the treatment results in cancer cells being recognized by the subject's T cells or their progeny cells. Therefore, preferably, the treatment has the effect of killing cancer cells, stopping the proliferation of cancer cells (particularly stopping the growth of tumors and / or metastases), more preferably causing tumor regression, and even more preferably causing tumor remission.

[0073] The term "prevention" refers to maintaining health in respect of the diseases or conditions mentioned herein for a specific period of time in a subject. It should be understood that the period of time may depend on the amount of the administered preparation (e.g., T-cell immune products) and individual subject factors discussed elsewhere in this specification. It should be understood that prevention may not be effective for all subjects treated with the preparations of the present invention. However, the term requires, preferably, that a statistically significant proportion of subjects in a cohort or population be effectively prevented from suffering from the diseases or conditions mentioned herein or their accompanying symptoms. Preferably, in this context, a cohort or population of subjects would normally (i.e., without the preventive measures according to the invention) suffer from the diseases or conditions mentioned herein. Whether a portion is statistically significant can be determined by those skilled in the art without excessive effort using various well-known statistical assessment tools discussed elsewhere in this specification. Thus, preferably, in the case of cancer or diseases caused by infectious agents, prevention may be vaccination, particularly passive vaccination. Thus, preferably, the term prevention involves the administration of preparations as specified herein to elicit an immune response against cancer and / or against at least one infectious agent. In the case of cancer, prevention may particularly involve the prevention of recurrence and / or metastasis.

[0074] The present invention also relates to kits comprising the formulations and administration methods described herein.

[0075] As used herein, the term "kit" refers to a collection of indicated compounds, means, or reagents, which may or may not be packaged together. Kit components may be contained in individual vials (i.e., kits as individual components) or provided in a single vial. Furthermore, it should be understood that the kits of the present invention are preferably used to perform the methods or steps mentioned elsewhere herein. Preferably, all components are envisioned to be provided ready-to-use for performing the methods described above. Additionally, the kit preferably includes instructions for performing the methods. These instructions may be provided as a user manual in paper or electronic form. Furthermore, the manual may include instructions for administration and / or dosage of the methods performed using the kits of the present invention. Preferably, the kit components are contained in a housing, which preferably allows for the translocation of the kit's compounds, particularly co-translocation; thus, the housing may be a delivery container, particularly containing all specified components.

[0076] The term "means of administration" as used herein refers to all means suitable for administering the formulation to a subject. Therefore, preferably, the means of administration is any means configured and / or suitable for delivering the formulation into the subject's body. The means of administration may include a delivery unit for administering the formulation and a storage unit for storing the formulation prior to administration. However, it is also contemplated that the means may appear as a separate device and preferably packaged within the kit. Preferred means of administration are those that can be applied without the specific knowledge of a skilled technician. Preferably, the means of administration is a syringe containing the formulation of the present invention, more preferably with a needle. Also preferably, the means of administration is an intravenous infusion device containing the formulation of the present invention. Further preferably, the means of administration is an inhaler containing the formulation of the present invention, wherein, more preferably, the formulation is formulated for administration as an aerosol. Suitable means are known in the art.

[0077] The present invention also relates to a method for treating and / or preventing a target disease, preferably cancer, said method comprising:

[0078] (i) administering to the subject a formulation comprising T cells that recognize at least one disease epitope, said formulation being produced or can be produced by the methods described herein and

[0079] (ii) thereby treating and / or preventing diseases of the subject.

[0080] The present invention also relates to a method for treating a target disease, preferably cancer, the method comprising:

[0081] (I) Administering a disease vaccine containing one or more disease epitopes to a donor at the site of vaccination;

[0082] (II) Obtain a sample from the site of the vaccine administration;

[0083] (III) Produce a formulation containing T cells that recognize at least one disease epitope from the sample obtained in step (II); and

[0084] (IV) The preparation containing T cells produced in step (III) is administered to the subject, preferably the subject of step (A).

[0085] The present invention also relates to the use of formulations as specified herein for eliminating cancer cells, wherein said use is preferably in vitro.

[0086] Furthermore, this invention relates to a method for identifying TCRs that bind to disease epitopes, preferably cancer epitopes, presented on the cells of a subject, the method comprising:

[0087] (AA) provides a formulation comprising T cells prepared by the methods described herein that recognize at least one disease epitope;

[0088] (BB) provides the amino acid sequence of at least the complementarity-determining region 3 (CDR3) of the T cell's TCR provided in step (AA); and, thereby,

[0089] (CC) Identification of TCRs that bind to disease epitopes presented on cells.

[0090] The identification method includes step (AA): providing a formulation comprising T cells prepared by the method described above that recognize at least one disease epitope.

[0091] The identification method also includes step (BB): providing the amino acid sequence of at least the complementarity-determining region 3 (CDR3) of the T cell's TCR provided in step (AA).

[0092] The term "providing a sequence," such as an amino acid sequence and / or a nucleic acid sequence, is used broadly herein to include any and all means and methods of providing information about the sequence or making the sequence information accessible. Thus, the sequence can be provided as sequence information, preferably tangibly embedded in a data carrier. However, the sequence can also be provided in molecular form containing the sequence, preferably as a TCR (containing the TCR α and β chains) containing the sequence, and more preferably as a host cell containing the sequence. As will be understood by those skilled in the art, if the aforementioned host cell is provided, the sequence information can be provided by standard methods known to those skilled in the art (e.g., nucleic acid sequencing of the TCR expressed by the host cell or a portion thereof).

[0093] Preferably, the method for identifying TCRs that recognize disease epitopes further includes the step (BB1): expressing a TCR comprising at least CDR3 as determined in step (BB) in host cells, preferably T cells, i.e., preferably in host T cells. More preferably, the method includes a further step (BB1): expressing a TCR comprising at least CDR3 as determined in step (BB) in host cells (preferably T cells), i.e., preferably including expressing a TCR comprising at least CDR3 as determined in step (BB) and at least one helper TCR polypeptide in host cells.

[0094] As used herein, the term "TCR containing at least CDR3" refers to a TCR in which at least CDR3 is identical to that identified in step (BB), while the remaining sequence of the TCR polypeptide can be one or more different sequences of α and β chains or γ and δ chains, such as heterologous sequences. More preferably, CDR1 to 3 or the variable region of the TCR molecule is provided in step (BB) and expressed as part of the TCR polypeptide in step (BB1). However, it is also contemplated that the sequence of a further fragment of the TCR polypeptide or the sequence of the complete TCR polypeptide is provided in step (BB) and optionally expressed in step (BB1). As will be understood by those skilled in the art, a longer sequence than that expressed in step (BB1) may also be provided in step (BB); for example, preferably, the amino acid sequence of the variable region of the TCR peptide may be provided in step (BB), while its CDR3 is expressed only in step (BB1), for example, in the context of a heterologous TCR peptide; or, the amino acid sequence of the variable region of the TCR peptide may be provided in step (BB), and the amino acid sequence of the antigen-binding region (including the CDR) may be expressed in step (BB1), for example, in the context of a heterologous TCR peptide. Unless otherwise stated, the TCR peptide is preferably expressed as a complete molecule, i.e., each comprising a transmembrane region, a constant region, a linker region, and a variable region. In a preferred embodiment, the TCR comprises at least the CDR3 of SEQ ID NO: 62 and 63 and / or the CDR3 of SEQ ID NO: 64 and 65. In another preferred embodiment, the TCR also comprises the features shown in Table 5 below.

[0095] Preferably, the method for identifying a TCR that recognizes a disease epitope further includes the step (BB2): determining the binding of the TCR expressed in step (BB1) to a cell that presents a disease epitope, preferably a cancer epitope, and is complexed in a major histocompatibility complex (MHC), preferably a class I MHC molecule. Methods for determining the binding of a TCR, preferably a disease epitope contained in a TCR complex and complexed in an MHC molecule, are known in the art, and preferably include determining the binding of a disease epitope carrying a detectable label and complexed in an MHC molecule to a TCR, which may, for example, be expressed on the surface of a host cell. A well-known example of this method is a tetramer assay, preferably using a soluble tetrameric MHC molecule complexed with a disease epitope.

[0096] Preferably, the method for identifying a TCR that recognizes a disease epitope further includes step (BB3): determining the recognition of the TCR expressed in step (BB1) on a cell presenting the disease epitope. Assays for determining such recognition are known in the art, and particularly include binding assays, activation assays, and cytotoxicity assays. In all these assays, it is preferable to co-incubate the cell presenting the disease epitope with a host cell (e.g., a T cell expressing a TCR containing at least a specified CDR3). In the binding assay, it is determined whether the presenting cell and the host cell bind to each other, preferably forming an immune synapse, which includes at least the MHC molecule and TCR of the cell presenting the T cell activation antigen. In the activation assay, after the co-incubation, an immune activation biomarker, such as interferon-γ production, of the host cell (preferably a T cell) expressing a TCR containing at least a specified CDR3 is tested. In the cytotoxicity assay, it is determined whether, during the co-incubation, the host cell (preferably a T cell) expressing a TCR containing at least a specified CDR3 lyses at least a portion of the cell presenting the T cell activation antigen.

[0097] The identification method also includes a step (CC): identifying TCRs that bind to disease epitopes presented on cells.

[0098] The term “identifying a TCR” is used broadly herein to include any and all means and methods relating to a TCR that allow for the determination of at least its CDR3 sequence. Accordingly, a TCR does not necessarily (but may) be provided in physical form. Thus, identifying a TCR may include providing at least the CDR3 sequence of the TCR or a sequence of a polynucleotide encoding at least said CDR3. Preferably, said sequence is determined or has been determined by single-cell gene expression assays (preferably by single-cell RNA sequencing). However, identifying a TCR may also include physically providing said TCR, for example by providing a host cell (preferably a T cell) expressing said TCR, or by providing at least one polynucleotide encoding at least the variable region of the identified TCR polypeptide. Those skilled in the art will understand that if the TCR is provided in the context of an autonomously replicating organism (such as a host cell), it may not be necessary to provide the amino acid sequence of at least the CDR3 of the TCR and / or the nucleic acid sequence of the polynucleotide encoding it.

[0099] The present invention also relates to a method for providing host cells, preferably T cells, that identify cells presenting disease epitopes, preferably cancer epitopes, the method comprising:

[0100] (aa) Identify TCRs that bind to disease epitopes using the methods described above.

[0101] (bb) In host cells, preferably T cells, a TCR containing at least the complementarity-determining region 3 (CDR3) of the TCR in step (aa) is expressed, and thus,

[0102] (cc) provides host cells, preferably T cells, that identify cells that present disease epitopes, preferably cancer epitopes.

[0103] As used herein, the term "host cell" refers to any cell capable of expressing (and preferably presenting on its surface) a TCR polypeptide as specified herein, which is preferably encoded by a polynucleotide and / or a vector. Preferably, the host cell is a mammalian cell, particularly mouse or rat cells, and more preferably, 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, and even more preferably a CD8+ T cell. As will be understood from the description herein, the host T cell is preferably a genetically modified cell expressing a TCR polypeptide as specified herein, and the T cells in the formulation described above are preferably non-recombinant.

[0104] In view of the above, the following implementation plan is specifically proposed:

[0105] Implementation Scheme 1: A method for producing a formulation of T cells comprising at least one epitope (disease epitope) that recognizes a disease antigen, the method comprising:

[0106] (a) Incubating T cells from a sample taken at the site of vaccination of the subject with the disease epitope (vaccination site) under conditions suitable for the proliferation of the T cells; and

[0107] (b) thereby producing a formulation containing T cells that recognize at least one disease epitope.

[0108] Implementation Scheme 2: The method of Implementation Scheme 1, wherein the sample is obtained within 50 mm of the vaccination site, preferably wherein the sample comprises tissue or an aliquot of tissue showing a vaccination response.

[0109] Implementation scheme 3: The method of implementation scheme 1 or 2, wherein the sample is obtained within one week after the most recent vaccination.

[0110] Implementation Scheme 4: The method of any one of Implementation Schemes 1 to 3, wherein the vaccination is the subject's first, second, third, fourth or fifth vaccination of the disease epitope, preferably the subject's third or fourth vaccination of the disease epitope, and / or the subject's first, second or third vaccination of the disease epitope resulting in a visible vaccination response (preferably resulting in tissue showing a vaccination response).

[0111] Implementation Scheme 5: The method of any one of Implementation Schemes 1 to 4, wherein the formulation is produced within six weeks, preferably four weeks, after the start of step (a).

[0112] Implementation Scheme 6: The method of any one of Implementation Schemes 1 to 5, wherein the method does not include binding the T cells that recognize at least one disease epitope to a solid surface.

[0113] Implementation Scheme 7: The method of any one of Implementation Schemes 1 to 6, wherein the method does not include labeling the T cells that recognize at least one disease epitope and enriching the labeled T cells.

[0114] Implementation Scheme 8: The method of any one of Implementation Schemes 1 to 7, wherein the vaccination includes administering at least one vaccine to the site of vaccination, wherein the vaccine preferably comprises a peptide vaccine containing at least one disease epitope, more preferably comprising multiple disease epitopes.

[0115] Implementation Scheme 9: The method of any one of Implementation Schemes 1 to 8, wherein the vaccination comprises administering the disease vaccine in the form of a water-in-oil emulsion, the water-in-oil emulsion preferably comprising mineral oil and anhydro mannitol ether octodecenoate, preferably as the sole adjuvant.

[0116] Implementation Scheme 10: A disease vaccine for improving an immune response in a subject to a disease epitope, said improvement comprising:

[0117] (A) The disease vaccine is administered to the donor at the site of vaccination;

[0118] (B) Obtain a sample from the site of the vaccine inoculation;

[0119] (C) Produce a formulation containing T cells that recognize at least one disease epitope from the sample obtained in step (B) as a disease vaccine; and

[0120] (D) The preparation containing T cells produced in step (C) is administered to the subject.

[0121] Implementation Scheme 11: A disease vaccine for use in Implementation Scheme 10, wherein the improvement in immune response to a disease epitope is an improvement in immune response to cancer.

[0122] Implementation Scheme 12: A disease vaccine for implementation scheme 10 or 11, wherein the object in step (D) is a human leukocyte antigen (HLA) matched object, preferably the donor object in step (A).

[0123] Implementation Scheme 13: A disease vaccine used in any one of Implementation Schemes 10 to 12, wherein the administration of the disease vaccine is the subject's first, second, third, fourth, or fifth vaccination, preferably the subject's third or fourth vaccination.

[0124] Implementation Scheme 14: A disease vaccine for any one of Implementation Schemes 10 to 13, wherein the sample in step (B) is obtained within 50 mm of the vaccine injection site, preferably wherein the sample comprises tissue or an aliquot of tissue showing a response to vaccination.

[0125] Implementation Scheme 15: A disease vaccine for any one of Implementation Schemes 10 to 14, wherein the sample in step (B) was obtained within one week after the most recent administration of the vaccine in step (A).

[0126] Implementation Scheme 16: A disease vaccine for use in any one of Implementation Schemes 10 to 15, wherein step (C) does not include binding the T cells that recognize at least one disease epitope to a solid surface.

[0127] Implementation Scheme 17: A disease vaccine for use in any one of Implementation Schemes 10 to 16, wherein step (C) does not include labeling the T cells that recognize at least one disease epitope and enriching the labeled T cells.

[0128] Implementation Scheme 18: A disease vaccine for use in any one of Implementation Schemes 10 to 17, wherein the application in step (A) comprises application in the form of a water-in-oil emulsion, the water-in-oil emulsion preferably comprising mineral oil and detergent, preferably dehydrated mannitol ether octadecenoate.

[0129] Implementation Scheme 19: A disease vaccine for use in any one of Implementation Schemes 10 to 18, wherein the formulation is obtained within 4 weeks after incubation conditions suitable for the proliferation of the T cells are initiated.

[0130] Implementation Scheme 20: The subject matter of any one of Implementation Schemes 1 to 19, wherein the sample is a biopsy sample, preferably from tissue showing a vaccine response.

[0131] Implementation Scheme 21: The subject matter of any one of Implementation Schemes 1 to 20, wherein the sample contains T cells and is substantially free of cancer cells.

[0132] Implementation Scheme 22: The subject matter of any one of Implementation Schemes 1 to 21, wherein the sample is an organization sample.

[0133] Implementation Scheme 23: The subject matter of any one of Implementation Schemes 1 to 22, wherein the sample is a tissue sample containing T cells.

[0134] Implementation Scheme 24: The subject matter of any one of Implementation Schemes 1 to 23, wherein the disease epitope is a cancer epitope derived from cancer contained in the object.

[0135] Implementation Scheme 25: The subject matter of any one of Implementation Schemes 8 to 24, wherein the vaccine comprises at least one disease epitope, preferably a cancer epitope, more preferably a novel cancer epitope or an epitope derived from a shared cancer epitope.

[0136] Implementation Scheme 26: The subject matter of any one of Implementation Schemes 8 to 25, wherein the vaccine comprises multiple disease epitopes.

[0137] Implementation Scheme 27: The subject matter of any one of Implementation Schemes 8 to 26, wherein the vaccine comprises at least one synthetic long peptide.

[0138] Implementation Scheme 28: The subject matter of any one of Implementation Schemes 8 to 27, wherein at least one disease epitope is human leukocyte antigen (HLA) class II restricted, and is preferably contained in a synthetic long peptide.

[0139] Implementation Scheme 29: The subject matter of any one of Implementation Schemes 8 to 28, wherein the formulation comprises at least 10 9 T cells.

[0140] Implementation Scheme 30: The subject matter of any one of Implementation Schemes 1 to 29, wherein the formulation comprises at least 10 10 T cells.

[0141] Implementation Scheme 31: The subject matter of any one of Implementation Schemes 1 to 30, wherein the formulation comprises at least 50% T cells, preferably at least 75% T cells.

[0142] Implementation Scheme 32: The subject matter of any one of Implementation Schemes 1 to 31, wherein at least 30% of the T cells are CD8+ T cells.

[0143] Implementation Scheme 33: A formulation comprising T cells that recognize at least one cancer epitope, said formulation being manufactured or capable of being manufactured by a method defined in any one of Implementation Schemes 1 to 32.

[0144] Implementation Scheme 34: A formulation comprising T cells that recognize at least one cancer epitope, wherein (i) it comprises at least 1%, preferably at least 2%, more preferably at least 3% of T cells specific to at least one disease epitope, and / or (ii) it comprises T cells specific to at least one, preferably at least two, more preferably at least three different disease epitopes, preferably at least three different disease epitopes.

[0145] Implementation Scheme 35: The formulation of Implementation Scheme 33 or 34, wherein the T cells are not bound to a solid surface.

[0146] Implementation Scheme 36: The formulation of any one of Implementation Schemes 33 to 35, wherein the T cells are not labeled and are not enriched by the label.

[0147] Implementation Scheme 37: The formulation described in any one of Implementation Schemes 33 to 36, used in a pharmaceutical product.

[0148] Implementation Scheme 38: Use of the formulation described in any one of Implementation Schemes 33 to 36 in the preparation of a medicine.

[0149] Implementation Scheme 39: The formulation described in any one of Implementation Schemes 33 to 36 is used for the treatment and / or prevention of a target disease, preferably cancer.

[0150] Implementation Scheme 40: A formulation for use in Implementation Scheme 39, wherein the subject has cancer, and the formulation is used to treat cancer.

[0151] Implementation Scheme 41: A formulation for use in Implementation Scheme 39 or 40, wherein the cancer comprises the cancer epitope.

[0152] Implementation Scheme 42: A formulation for use in any one of Implementation Schemes 39 to 41, wherein the treatment and / or prevention comprises administering a disease vaccine to the subject, the disease vaccine comprising an epitope of at least one of the disease epitopes.

[0153] Implementation Scheme 43: Use of the formulation of any one of Implementation Schemes 33 to 36 in the preparation of a medicament for the treatment and / or prevention of cancer.

[0154] Implementation Scheme 44: A kit comprising the formulation and means of administration as described in any one of Implementation Schemes 33 to 36.

[0155] Implementation Scheme 45: A method for treating and / or preventing cancer in a target, the method comprising:

[0156] (i) Applying a preparation containing T cells that recognize at least one cancer epitope (produced or producible by the method described in any one of embodiments 1 to 9) to the subject, and

[0157] (ii) thereby treating and / or preventing cancer in the subject.

[0158] Implementation Scheme 46: A method for treating cancer, the method comprising:

[0159] (I) A disease vaccine containing one or more disease epitopes is administered to a donor at the site of vaccination;

[0160] (II) Obtain a sample from the site of the vaccine inoculation;

[0161] (III) Produce a formulation containing T cells that recognize at least one disease epitope from the sample obtained in step (II); and

[0162] (IV) The preparation containing T cells produced in step (III) is administered to the subject, preferably the subject of step (A).

[0163] Implementation Scheme 47: Use of the formulation described in any one of Implementation Schemes 33 to 36 for eliminating cancer cells.

[0164] Implementation Scheme 48: Use of Implementation Scheme 47, wherein the use is an in vitro use.

[0165] Implementation Scheme 49: A method for identifying TCRs that bind to disease epitopes, preferably cancer epitopes, presented on the cells of a subject, the method comprising:

[0166] (AA) Provides a formulation comprising T cells that recognize at least one disease epitope by any one of the methods described in embodiments 1 to 9;

[0167] (BB) Provides the amino acid sequence of at least the complementarity-determining region 3 (CDR3) of the T cell's TCR provided in step (AA); and, thereby,

[0168] (CC) Identification of TCRs that bind to disease epitopes presented on cells.

[0169] Implementation Scheme 50: A method for providing T cells that recognize cells presenting disease epitopes (preferably cancer epitopes), the method comprising:

[0170] (aa) Identifying TCRs that bind to disease epitopes by the method described in any one of embodiments 6 to 12;

[0171] (bb) T cells express TCRs containing at least complementarity-determining region 3 (CDR3) of the TCR in step (aa); and thus,

[0172] (cc) provides T cells that identify cells that present disease epitopes, with a preference for cancer epitopes.

[0173] Implementation Scheme 51: A T cell that recognizes cells that present disease epitopes, which is produced by or can be produced by the method described in Implementation Scheme 50, preferably for use in any one of Implementation Schemes 37 to 42.

[0174] All references cited in this specification are incorporated herein in their entirety, including their published content and any specific references to them. Attached Figure Description

[0175] Figure 1 (A) Concept of VIL therapy: Patients are inoculated with tumor-specific peptides. Infiltrating T cells are isolated from the vaccination site, expanded in vitro, and infused into the patient. This concept is based on adoptive cell therapy (ACT) using infiltrating lymphocytes (VILs) from the vaccination site, illustrated in the flowchart: Patients are inoculated with appropriate tumor antigen-derived peptides. VILs are isolated from the vaccination site, expanded in vitro, and infused into the patient. (B) Exemplary schematic flowchart of VIL-based ACT development and therapy, with additional graphic illustrations. IHC: Immunohistochemistry; WES: Whole exome sequencing; LS-MS: Liquid chromatography-mass spectrometry for identifying MHC-I or MHC-II presented peptide ligands eluted from tissue materials, such as tumor biopsies (immunopeptidomics); IL-2: Interleukin-2; TAA: Tumor-associated antigen.

[0176] Figure 2 The growth curves of VIL and TIL show the changes in total cell number over time for various formulations.

[0177] Figure 3During the expansion phase, the target cell count of 4*10⁴ cells was achieved. 7 The number of days required per cell was normalized to 100 starting wells.

[0178] Figure 4 Growth characteristics of TILs and VILs. Growing to 4*10 7 The time required to quantify a single cell (y-axis) depends on the amount of starting material, which can be estimated by the number of wells filled with the tissue slice (x-axis).

[0179] Figure 5 (a, b) VILs from patients inoculated with long peptides containing mutant epitopes were responsive to mutant peptides rather than wild-type peptides when IFNγ secretion was detected by ELISpot assay (a, b) or peptide-specific multimer staining (c); (d) Patient 001 VIL ACT development and CRC treatment timeline; (e) Functional T cell analysis against the inoculated peptide (VAC) and its wild-type counterpart following a VIL-based clinical-grade rapid expansion protocol (REP). The response of VIL-derived T cells to VAC or wild-type (WT) variants and their pools, as measured by intracellular cytokine staining, was shown after co-culturing isolated CD4+ T cells and CD8+ T cells with peptide-pulsed B cells (pre-stimulated with CD40L multimers for 6 days) for 18 hours; (f) Serum levels of soluble CEA (carcinoembryonic antigen) before and after infusion of expanded VILs; (g, h) In vitro tracking of mutation-specific CD8+ T cells in PBLs after VIL ACT infusion.

[0180] Figure 6VILs from adenoid cystic carcinoma patients inoculated with a long peptide (AA 79-108) containing the NY-ESO-1 epitope were responsive to the tested NY-ESO-1 peptide when measured by ELISPOT (a) or by flow cytometry analysis of multimeric reactive CD8+ T cells (b) to detect IFNγ secretion; LP NY-ESO-1 long peptide; SPP: short peptide pool containing peptides that are experimentally proven to bind to the patient's HLA-A, HLA-B, HLA-C MHC-I alleles and are part of NY-ESO-1 or derived from the virus; (a) ELISPOT analysis of isolated VILs and peripheral blood lymphocytes (PBLs) after the 8th inoculation with ISA51-emulsified SLP NY-ESO-1 79-108 (SEQ ID:16). (a) MACS-isolated CD4+ and CD8+ T cells co-cultured with autologous CD40L-stimulated B cells (B cell CD40L) pulsed with SLP or short peptide pool (SPP) for 18 hours in ELISPOT plates; (b) staining of VIL-derived and post-vaccination peripheral blood (PBL) T cells with two-color encoded NY-ESO-1 epitope-specific pMHC-I multimers; (c) VIL ACT targeting NY-ESO-1 demonstrating immunogenicity and leading to a clinical response in a case of metastatic adenoid cystic carcinoma (ACC); VILACT development and patient 002 (ACC) treatment timeline; (d) schematic diagram of ex vivo VIL preparation; (e) functional T cell response to the vaccine peptide (VAC) following a VIL-based clinical-grade rapid expansion protocol (REP). The figure shows the response of VIL-derived T cells to VAC or its short peptide variants and pools (P01-P10) as measured by intracellular cytokine staining, after co-culturing isolated CD4+ and CD8+ T cells with peptide-pulsed B cells CD40L for 18 hours; the peptide was: VAC SLP: NY-ESO-1 79-108(GARGPESRLLEFYLAMPFATPMEAELARRS, SEQ ID NO: 16), P01: LEFYLAMPF (SEQ ID NO: 20), P02: YLAMPFATPM (SEQ ID NO: 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 ID NO: 44); (f) In vitro killing 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, measured by LDH release; (g) NY-ESO-1 specific T cells were tracked in vitro in PBL up to 470 days after VIL infusion.

[0181] Figure 7 Tumor epitope-specific or virus-specific CD8+ T cells detected in peripheral blood (PBL) of colorectal cancer patients (a) and adenoid cystic carcinoma patients (b) after VIL infusion.

[0182] Figure 8 Serum CEA levels in CRC patients before and after VIL infusion.

[0183] Figure 9 MRI images of different brain metastases in a patient with adenoid cystic adenoma. A to D: Brain metastases before VIL treatment (A: -57 days) and after VIL treatment (B: 41 days, C: 154 days, D: 174 days), showing significant shrinkage of the metastases. E to H: Images of a second brain metastasis in the same patient before (E) and after (FH) VIL treatment.

[0184] Figure 10 Comparison of cytokine secretion when VIL, TIL, and PBL are co-cultured with autologous tumor explants.

[0185] Figure 11In cases of metastatic pancreatic ductal adenocarcinoma (PDAC), personalized mutant-targeted VIL ACTs were developed and infused within 5 months of vaccination initiation; (a) a timeline of VIL ACT development and treatment in patient 003; (b) ELISPOT analysis of ex vivo VILs isolated from peripheral blood (PBL) after a second inoculation with ISA51-emulsified SLPs (targeting a total of 16 proprietary gene alterations (NEO-VAC) in patient 003, including 9 SNVs and 7 INDELs identified after primary tumor biopsy). CD4+ T cells and CD8+ T cells isolated from MACS were co-cultured in ELISPOT plates for 18 hours with autologous B cells pulsed with mutant SLPs (pre-stimulated with CD40L for more than 6 days).

[0186] Figure 12 : Isolation and validation of NY-ESO-1 specific T-cell receptors (TCRs) from patient 002 (ACC) VILs. (a) DNA barcoded pMHC-I multimer-guided VIL single-cell TCR library and cell surface protein expression analysis allowed for rapid selection, cloning, and subsequent validation of antigen-specific TCRs, potentially enabling off-the-shelf TCR transgenic ACTs as part of a TCR repository. (b) Validation of three patient 002 VIL-derived NY-ESO-1 specific TCRs based on pMHC-I multimers, recognizing three distinct NY-ESO-1 epitopes. The recombinant TCRs were stably expressed in CD8+ Jurkat cells (Jurkat-76 / CD8) lacking endogenous TCR expression.

[0187] The following examples are for illustrative purposes only. They should not in any way be construed as limiting the scope of the invention.

[0188] Example 1: Method Overview ( Figure 1 )

[0189] Highly mutated tumors contain so-called neoepitopes, which are tumor-specific and can be recognized by the patient's immune cells to attack the tumor. These patient-specific peptide sequences are identified through tumor DNA sequencing and can be synthesized. Administering personalized tumor peptides as a therapeutic vaccine stimulates the body's own immune cells, thereby eliminating tumor cells.

[0190] Besides vaccination with tumor-specific peptides, cellular immunotherapy is another way to use a patient's own immune system to attack tumors. One well-known method is TIL therapy (tumor-infiltrating lymphocyte therapy), in which tumor-specific T cells are isolated from the tumor, expanded, and then reinfused into the patient. Although TIL therapy has been successful for certain tumor types such as melanoma, this cell collection method has some drawbacks: the antigen specificity of the isolated T cells is highly diverse, with only a small fraction being tumor-specific. Furthermore, the isolated T cells originate from a highly immunosuppressed tumor microenvironment, and are therefore functionally impaired.

[0191] This article describes a novel treatment approach that combines patient-specific peptide vaccination with T-cell therapy to increase the fitness of infused T cells. Figure 1 In this approach, immune cells are expanded to a high cell count according to the classic TIL protocol. However, compared to the TIL method, the cells are derived from the vaccination site (vaccination site infiltrating lymphocytes, VILs) containing tumor-specific peptides, thus using tumor-specific and recently activated T cells as starting material. Reinfusing a large number of newly generated, less depleted tumor-specific VILs into the patient increases the chances of endogenous immune cells successfully defending against the tumor.

[0192] Typically, the method may include the following steps:

[0193] 1) Genome / exome / targeted gene sequencing of tumor tissue and blood as a control (not required for targeted sequencing panels), and RNA sequencing of tumor tissue;

[0194] 2) Identification of genomic alterations (e.g., point mutations, insertions / deletions, fusions, frameshifts) and prediction of new epitopes, peptide selection;

[0195] 3) Peptide synthesis for vaccination, for example, using Fmoc technology;

[0196] 4) Reconstruction of lyophilized peptides by adding ISA-51 adjuvant;

[0197] 5) For repeat vaccinations, blood can be drawn at any previous time;

[0198] 6) Remove the vaccination site or part thereof;

[0199] 7) Isolation and amplification of VILs;

[0200] 8) Use techniques such as multimer technology and ELISpot to detect peptide-specific VILs;

[0201] 9) Intravenous infusion of cell products (including pretreatment and subsequent IL-2 administration to promote T cell engraftment).

[0202] Example 2: Epitope and peptide selection for vaccination

[0203] The primary criterion for selecting shared antigenic epitopes and vaccine peptides is shared antigen expression, which is assessed through methods such as immunohistochemistry or immunofluorescence staining of tissue sections, immunofluorescence staining of cells, amplification via polymerase chain reaction, and RNA sequencing.

[0204] The quality of predicted epitopes was evaluated in parameters such as “elution ligand grade” and binding affinity (nM) (current version: netMHCpan4.1 algorithm,

[14] ), the type and number of HLA alleles predicted to bind the corresponding epitopes.

[0205] Mutation-specific epitopes were predicted based on point mutations (single nucleotide variants, SNVs), insertions / deletions (“Indels”), and gene fusions. For the selection of mutation-specific epitopes (so-called novel epitopes) and vaccine peptides, all three types of mutations were considered, and the following parameters were applied:

[0206] Epitopes and vaccine peptides are sorted based on mutation type / category:

[0207] 1. Fusions, indels, and SNVs from oncogenic driver genes;

[0208] 2. Passenger SNV.

[0209] Expression of the corresponding mutated gene.

[0210] The quality of predicted epitopes was evaluated on parameters such as "elution ligand rank", binding affinity, location of mutations in predicted epitopes, allele frequency, and the type and number of HLA alleles predicted to bind to the corresponding epitopes.

[0211] For gene fusions (identified using Arriba software,

[15] ): In addition, the following parameters were evaluated: high confidence score, breakpoint distance > 1 million bases, number of each split sequencing read > 0 and total > 10, epitopes crossing breakpoints, elution ligand rank, binding affinity, position of the mutation in the predicted epitope, allele frequency, and the type and number of HLA alleles predicted to bind to the corresponding epitope. In the case of frameshift mutations, downstream epitopes may also be considered based on the above parameter list.

[0212] Example 3: Preparation of peptide vaccine

[0213] The lyophilized peptides are dissolved in distilled water or a mixture of DMSO and water. Subsequently, the peptides are mixed with an equal volume of mineral oil and dehydrated mannitol ether octadecenoate (e.g., Montanide) to produce a water-in-oil emulsion.

[0214] Example 4: Vaccination

[0215] The peptide vaccine is administered intradermally. A second dose is given two weeks after the first, followed by multiple doses. The vaccine site is excised after two or more doses. A specimen including the injection site and surrounding tissue is selected. The specimen should be at least 1 cm in diameter.

[0216] Example 5: Isolation and Amplification of VIL

[0217] Tissue biopsy samples from the vaccination site were cut into small pieces and cultured in 24-well suspension plates at 37°C, 5% CO2, and >90% humidity in a medium containing 10% human AB serum, 6000 IU / ml IL-2, and 50 μg / ml gentamicin (T cell mixed medium: 80% RPMI 1640 + 20% AIM-V or X-Vivo 15). The medium was replaced with 50% medium the day after plating. Cells were observed daily under a microscope for T cell growth. If T cell growth was observed, the individual wells were resuspended and aliquoted into new wells at a 1:2 ratio. T tissue fragments were discarded. T cells were cultured at 8*10-10... 5 A starting cell concentration of 10 cells / ml was added to the culture flask and cultured until a sufficient number of cells (>4*10) was reached. 6 (100 cells), typically after approximately 14 days. Cell composition is monitored using flow cytometry, and microbial and mycoplasma contamination is tested before freezing. For the rapid expansion phase, cells are thawed one day before stimulation. The next day, feeder cells from at least three donors are thawed, irradiated at an average dose of 30 Gy, and prepared for culture at a T cell:feeder cell ratio of 1:100. 2*10 7 T cells were cultured in 3 liters of mixed T cell culture medium or in X-Vivo 15 flasks containing 10% human AB serum, 30 ng / ml OKT3 anti-CD3 antibody, 3000 IU / ml IL-2, and feeder cells. A control group of feeder cells was cultured separately to ensure successful irradiation. On day 4, the culture medium was replaced with 50% medium. After 5 days of culture, cells were transferred to the Xuri cell expansion system W25 and cultured in 5 liters of medium containing 10% human AB serum and 3000 IU / ml IL-2. Culture medium perfusion began two days after culture in the Xuri system, increasing to 1 liter / day on day 3, 2.5 liters / day on days 4 and 5, and 5 liters / day from days 6 to 8. After 8 days of cell expansion in the Xuri system, cells were harvested, washed, and resuspended in 0.9% NaCl containing 2.5% human serum albumin (HSA) for infusion. Cell composition, microbial and mycoplasma contamination, and endotoxin levels were monitored.

[0218] Example 6: VIL grows faster than TIL

[0219] The growth characteristics of T cell cultures depend on several parameters, including the amount of starting material. Generally, vaccine site infiltrating lymphocytes (VILs) grow faster than tumor infiltrating lymphocytes (TILs), as shown in the corresponding growth curves. Figure 2 After the expansion phase, the target quantity of 4*10 was reached. 7 The number of culture days required per cell (normalized to 100 starting wells) showed a significant difference between VIL and TIL. Figure 3 ).

[0220] Example 7: VIL grows faster than TIL

[0221] Since growth characteristics depend on the amount of starting material, we analyzed the correlation between the time to reach the desired target cell number and the number of starting wells used to culture tissue slices from tumor or vaccination sites. In both cases, specimens were cut into small pieces and allocated to the individual wells of a 24-well plate. Growth characteristics showed that VILs grew faster even with less starting material. Figure 4 ).

[0222] Example 8: VIL does not contain tumor cells

[0223] Tumor-induced endothelial cells (TILs) are produced by tumor tissue and carry the risk of residual tumor cells. Therefore, careful quality control measures are required to detect residual tumor cells using tumor-specific markers. In contrast, venous endothelial cells (VILs) are derived from skin biopsies located far from the tumor site, eliminating the risk of residual tumor cells in the VIL product.

[0224] Example 9: Compared with peripheral blood after vaccination, VIL has an increased frequency of tumor epitope-specific T cells.

[0225] VILs were generated using specimens from the vaccination site of a patient with colorectal cancer (CRC) who received a synthetic peptide (Table 1) . ELISpot ( Figure 5 a+b) and multimer staining ( Figure 5 c) The responses of VILs and peripheral blood lymphocytes (PBLs) to wild-type and mutant peptides were analyzed. VILs were reactive to the mutant peptides compared to PBLs, which showed no specific response. Reactivity to three predicted HLA class I epitopes was detected in the CD8+ VIL fraction by multimer staining; 4.4% of all CD8+ T cells were specific for a mutant 10-mer peptide complexed with HLA-A*11:01, while T cells did not recognize the corresponding wild-type peptide (…). Figure 5 c).

[0226] A second patient with adenoid cystic carcinoma (ACC) was inoculated with 30-mer peptides derived from the known tumor antigen NY-ESO-1 (GARGPESRLLEFYLAMPFATPMEAELARRS (SEQ ID NO: 16), cancer epitopes: YLAMPFATPM (SEQ ID NO: 17); FATPMEAEL (SEQ ID NO: 18); LAMPFATPM (SEQ ID NO: 19); LEFYLAMPF (SEQ ID NO: 20)). In an ELISpot assay, CD4+ and CD8+ T cells isolated from VIL showed enhanced responsiveness to NY-ESO-1-specific peptides compared to T cells isolated from peripheral blood. Figure 6 a). In VIL products before and after rapid amplification, peptide-specific CD8+ T cells were detected by multimer staining, with a frequency as high as 6.8%, while peptide-specific T cells in peripheral blood were below or close to the detection limit. Figure 6 b).

[0227] For patient 001 (colorectal cancer, CRC with extensive pretreatment), a highly personalized mutant-targeted SLP / ISA51 vaccine (NEO-VAC) was designed and applied based on our automated whole-exome and RNA sequencing data analysis and computer-simulated T-cell epitope prediction workflow. Following mutant analysis of liver CRC metastases, patient 001 received six NEO-VAC injections before skin biopsy at the vaccine site. Figure 5 d, Table 1), which shows enriched mutations and partial wild-type peptide cross-reactivity with CD4. + T cell population, but only specific to mutations / novel epitopes. CD8+ T Cell populations that are undetectable in peripheral blood (PBL) during skin biopsy. Figure 6 b).

[0228] In approximately 40x10 6 The number of VILs increased significantly to >1.0x10 10 Following rapid clinical-grade expansion (REP) of 1,000 cells, the final VIL product exhibited a 1:1 CD4 / CD8 T cell ratio (data not shown). In CRC patient 001, the VIL retained its function after REP, with approximately 48% of all CD4+ T cells showing specificity for the NEO-VAC-derived SLP mutant library, and approximately 16% also showing cross-reactivity to its wild-type peptide variants. In VILCD8+ T cells, approximately 25% showed strong neoepitope specificity upon in vitro stimulation (…). Figure 5 e) and pMHC-I polymer staining ( Figure 5c). Importantly, reactivity to three predicted HLA class I epitopes was validated in the CD8+ VIL fraction by multimer staining, with 4.4% of all CD8+ T cells showing specificity for a mutant 10-mer peptide complexed with HLA-A*11:01, while T cells did not recognize the corresponding wild-type peptide. Figure 5 c).

[0229] For patient 002, partial resection of brain metastases originating from adenoid cystic carcinoma (ACC) was performed, and immunohistochemical staining showed NY-ESO-1 expression, which is initiation of NY-ESO-1... 79-108 SLP / ISA51 (NY-ESO-1 VAC) vaccination and subsequent VIL-based adoptive cell therapy (VIL ACT) provide a strong justification. Figure 6 c, Table 2). Patient 002 was inoculated with a 30-mer peptide (GARGPESRLLEFYLAMPFATPMEAELARRS (SEQ ID NO: 16)) derived from the known tumor antigen NY-ESO-1, which contains known CD8+ T cell 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 injection, a biopsy of the inoculation site showed a strong enrichment of NY-ESO-1 79-108-specific CD4+ T cells and CD8+ T cell populations compared to peripheral blood-derived T cells obtained during skin biopsy. Figure 6 a). Here, using autologous CD40L-amplified B cells as antigen-presenting cells, the responsiveness of CD4+ T and CD8+ T cells isolated from VIL or PBL to NY-ESO-1-VAC was detected by ELISpot assay. Figure 6 a).

[0230] Furthermore, peptide-specific CD8 was detected by multimer staining in the VIL products before and after rapid amplification. + T cells, with a frequency as high as 6.8%, while peptide-specific T cells in peripheral blood were below or close to the detection limit. Figure 6 a and 6b). In the approximately 40x10 6 The VILs in the VIL-initiated culture were significantly amplified to >1.0 x 10⁻⁶. 9Following rapid clinical-grade expansion (REP) of individual cells, these "post-REP" VILs retained their function, as evidenced by effector molecule expression following restimulation with the NY-ESO-1 peptide. Intracellular cytokine staining (ICS) combined with flow cytometry-based analysis after restimulation revealed that approximately 23% and 16% of the total CD8+ T and CD4+ T cell populations (a 1:1 ratio overall after REP), respectively, were specific for multiple vaccine-derived epitopes accompanied by virus-specific bystander T cells (VTS). Figure 6 e and data not shown). Additionally, CD8 derived from VIL after REP. + T cells maintained their ability to specifically kill HLA-matched replacement tumor cell lines of the NY-ESO-1 peptide pulse. Figure 6 f).

[0231] Patient 003, suffering from pancreatic ductal adenocarcinoma (PDAC), received a highly personalized neoepitope-specific SLP / ISA51 vaccine (NEO-VAC) based on primary tumor biopsy mutationome analysis. Figure 11 a, Table 3). Here, a biopsy of the vaccination site was performed immediately after the second NEO-VAC injection, and surprisingly, a significant enrichment of CD8+ T cell populations specific to the single vaccination peptide was observed (a, Table 3). Figure 11 b) and multiple VAC peptide-specific CD4+ T cell populations (data not shown). Subsequently, patient 3 received 3.2*10^3 vaccines only 5 months after the first vaccination. 10 The rapid expansion of VILs underscores the feasibility of rapidly and efficiently producing personalized VIL ACTs. In terms of preparation time, VIL ACTs may easily surpass the workflow of personalized novel epitope-specific transgenic TCR therapies, which require extensive and cumbersome TCR screening, validation, and cell product engineering steps, totaling more than 250 days (>8 months) of development and preparation time

[25] .

[0232] Example 10: VIL persists in peripheral blood and shows clinical efficacy after infusion.

[0233] will come from Figure 5 The colorectal cancer patient 1*10 10 One venous intravenous infusion of VIL was administered to the patient. At 7 and 37 days post-infusion, the frequency of tumor epitope-specific CD8+ T cells in the PBL was enriched (…). Figure 7 (a left figure), while the frequency of common virus-specific T cells in PBL did not change ( Figure 7 (a right figure). Similarly, NY-ESO-1 specific T cells could be detected in the PBL of patients with adenoid cystic carcinoma 30 days after VIL infusion.

[0234] After lymph node dissection of CRC patient 001, 2*10 10 One expanded VIL was infused intravenously as adoptive cell therapy (ACT), accompanied by systemic IL-2 injection. Figure 5 d). Patient 2 tolerated VIL-ACT well overall, and although significant wild-type peptide cross-reactive CD4+ T cells were present in the final ACT cell product, no potential signs of autoimmunity were observed. Figure 5 e). The frequency of tumor epitope-specific CD8+ T cells was enriched in the PBL at 7 days post-infusion and maintained at 37 days post-infusion, while the frequency of common virus-specific T cells in the PBL remained unchanged. Figure 5 g、 Figure 5 h).

[0235] Similarly, for patient 002, only 8 weeks after lymph node clearance and the 8th vaccination (the last vaccination before ACT), the level was 1.1*10. 9 Expanded VILs were reinfused as ACT, accompanied by systemic IL-2 therapy. VIL-based ACT was generally well-tolerated, with clinically manageable side effects primarily attributed to IL-2 therapy. Following infusion, particularly the NY-ESO-1-specific CD8+ T cell population, could be tracked ex vivo for up to 470 days, indicating their long-term persistence in vivo, whereas they were undetectable ex vivo prior to VIL-ACT. Figure 6 h).

[0236] Example 11: VIL showed clinical efficacy after infusion

[0237] In patients with colorectal cancer, serum levels of the tumor marker carcinoembryonic antigen (CEA) drop sharply after VIL infusion. Figure 8 Brain metastases from patients with adenoid cystic carcinoma (ACC) showed a significant reduction in size after VIL treatment. Figure 9 ).

[0238] Example 12: VILs exhibit enhanced cytokine secretion when co-cultured with autologous tumor cells.

[0239] To compare tumor-specific responses of VILs and TILs after the expansion phase and of peripheral blood T cells (CD3PB) before treatment, co-culture experiments were performed using autologous tumor explants from colorectal cancer patients. Cytokine secretion was measured using the human MACSPlex cytokine 12 kit (Miltenyi) with tumor explants alone and in combination with T cells. VILs co-cultured with tumor explants showed significantly increased cytokine secretion compared to TILs or peripheral blood T cells. Figure 10 ).

[0240] Example 13: Isolation and validation of TCR from VILs targeting NY-ESO-1 derived from patient 002

[0241] In addition to Figures 5 to 8 In addition to using VILs directly as a convenient, customizable adoptive cell therapy for individual patient cases, VILs also serve as a source of T-cell receptors (TCRs) for off-the-shelf TCR-transgenic ACTs. Similarly, TCRs isolated from patients who achieved complete or partial remission from TIL-based ACTs have been further used to accelerate the production of off-the-shelf TCR-transgenic ACTs with great success

[23] . Likewise, we implemented a robust workflow that uses our highly sensitive and multifunctional pMHC-I-based multimer library screening followed by a compatible pMHC-I-guided single-cell TCR sequencing method

[24] , followed by TCR cloning and validation, enabling us to query hundreds of predicted tumor (neo)epitopes for each patient case to determine the presence of homologous CD8+ T cell populations (CD8+ T cells). Figure 12 a). Using this workflow, we have identified three VIL-derived TCRs from patient 002. These three TCRs collectively identify three distinct NY-ESO-1 epitopes with HLA-C*03:04 restriction ( Figure 12 b).

[0242] Example 14: TCR sequencing and analysis from VIL

[0243] A patient with metastatic triple-negative breast cancer (TNBC) was enrolled in our diagnostic and registry trial (NCT05652569) in February 2020. Longitudinal molecular analyses of tumors and liquid biopsies were expanded through a transformative procedure for immune surveillance and studies identifying and cloning neoantigen-specific T cells.

[0244] Neoantigen mobilization was performed using NetMHCpan 4.1. Whole-genome and transcriptome sequencing was conducted on the first analyzed tumor lesion (tumor 1). Novel epitope identification predicted 254 putative candidates binding to the patient's HLA-I allele from 54 non-synonymous single nucleotide variants (SNVs), considering filtering based on altered allele expression (FPKM>0) and %RANK_EL<2 (predicted binders). We selected 17 candidates based on gene expression, allele frequency, and peptide-MHC high affinity, aiming to select novel epitopes that would most contribute to tumor immunogenicity. For vaccination, peptide sequences extending flanking the predicted epitopes (long peptides) were designed. A list of peptides used in vaccination can be found in Table 4.

[0245] Intradermal administration of the personalized vaccine began in February 2021. After three rounds (every 4 to 6 weeks), patients experienced a strong local inflammatory reaction at the vaccination site, manifested as induration, redness, and swelling. Considering that inflammation-related adverse reactions are the result of a strong and antigen-specific immune response, biopsies were performed on the vaccinated lesions, followed by immune repertoire analysis and antigen-specific analysis.

[0246] TCR sequencing (TCR-seq) was performed on cryopreserved VILs and PBMCs using Genewiz (Azenta). Briefly, single-cell TCR sequencing of the VDJ region was performed using the Chromium Single-Cell Immunoassay Kit (10x Genomics), followed by library sequencing on MiSeq (Illumina). The full-length V(D)J sequence of the paired T cell receptor was analyzed using the Loupe VDJ 223 browser v4.0.0 (10xGenomics).

[0247] Single-cell TCR sequencing (scTCR-seq) data analysis revealed three common clonal types (>5%) at the site of the original vaccination, two of which showed significant amplification after IVS (in both bulk and pure CD8 cultures), carrying the long peptide NCORR1L1475R (clone 1 and clone 2, Table 5). We selected TCRs from these two most prominent clonal types (named TCR-1131 and TCR-1132) and performed cloning and subsequent validation.

[0248] References

[0249] 1. Zhao, L. and YJ Cao, Engineered T Cell Therapy for Cancer in theClinic. Front Immunol, 2019. 10: p. 2250. 2. Kilian, M. et al., T-cell Receptor Therapy Targeting Mutant CapicuaTranscriptional Repressor in Experimental Gliomas. Clin Cancer Res, 2022. 28(2): p. 378-389. 3. June, CH and M. Sadelain, Chimeric Antigen Receptor Therapy. NEngl J Med, 2018. 379(1): p. 64-73. 4. Susanibar Adaniya, S.P., A.D. Cohen, and A.L. Garfall, Chimeric antigen receptor T cell immunotherapy for multiple myeloma: A review of current data and potential clinical applications. Am J Hematol, 2019. 5. Cordeiro, A. et al., Late Events after Treatment with CD19-Targeted Chimeric Antigen 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 in Advanced 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 in human tumour infiltrates. Nature, 2018. 557(7706): p. 575-+. 8. Bassani-Sternberg, M. et al., Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nature Communications, 2016. 7. 9. Lang, F. et al., Identification of neoantigens for individualized therapeutic cancer vaccines. 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-like receptor (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 create effective 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 in skin and peripheral blood following intradermal peptide vaccination. Cancer Immun, 2005. 5: p. 5.

[0250] 13. Pollack, K.E. et al., Incomplete Freund's adjuvant reduces arginase and enhances Th1 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 of MHC 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 sequencing data. 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): e14383. 18. Bernardeau et al (2011), J Immunol Methods, 371(1-2):97-105. 19. Sadelain et al (2013) Cancer Discov 3(4):388. 20. Chakraborty et al (1998) Cancer Immunol Immunother 47:58-64. 21. Tong et al (2019) Cell Chem. Biol. twenty-six(7):1013-1026. 22. Mackensen et al. (1997), J. Mol. Med. 75(4):290-296. 23. Leidner, R. et al. Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer. N Engl J Med 386, 2112-2119 (2022). https: / / doi.org / 10.1056 / NEJMoa2119662. 24. Meyer, M. et al. MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection. Front Immunol 14, 1294565 (2023). https: / / doi.org / 10.3389 / fimmu.2023.1294565. 25. Foy, S. P. et al. Non-viral precision T cell receptor replacement for personalized cell therapy. Nature 615, 687-696 (2023). https: / / doi.org / 10.1038 / s41586-022-05531-1.

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Claims

1. A formulation comprising at least one epitope (disease epitope) of a T cell that recognizes a disease antigen, for the treatment and / or prevention of a disease in a human subject, wherein said formulation is produced by or can be produced by a method comprising the following steps: (a) Incubating T cells from a sample taken at the site of vaccination of the human subject with the disease epitope (vaccination site) under conditions suitable for the proliferation of the T cells; and (b) thereby producing a formulation containing T cells that recognize at least one disease epitope.

2. The formulation for the use of claim 1, wherein the disease is cancer or caused by a pathogenic microorganism.

3. The formulation for the use according to claim 1 or 2, wherein the disease is cancer.

4. The formulation for use according to any one of claims 1 to 3, wherein the vaccination site is the site of topical application of the disease epitope.

5. The formulation for use according to any one of claims 1 to 4, wherein the vaccination site is the site of intradermal, subcutaneous and / or intramuscular administration of the disease epitope.

6. The formulation for use according to any one of claims 1 to 5, wherein the sample is obtained within 50 mm of the vaccination site, preferably wherein the sample comprises tissue or an aliquot of tissue showing a vaccination response.

7. The formulation for use according to any one of claims 1 to 6, wherein the sample comprises T cells and is substantially free of cancer cells.

8. The formulation for use according to any one of claims 1 to 7, wherein the vaccination is a first, second, or third vaccination of the subject with the disease epitope, which results in a visible vaccination response, preferably resulting in a tissue-displayed vaccination response.

9. A formulation for use according to any one of claims 1 to 8, wherein the vaccination comprises administering at least one disease vaccine at the vaccination site.

10. A formulation for use according to any one of claims 1 to 9, wherein the at least one disease epitope is contained in a disease vaccine.

11. The formulation for use according to claim 9 or 10, wherein the disease vaccine comprises at least one novel cancer epitope.

12. The formulation for use according to any one of claims 9 to 11, wherein the disease vaccine comprises at least one novel cancer epitope as a synthetic long peptide, preferably comprising multiple novel cancer epitopes as synthetic long peptides.

13. The formulation for use according to any one of claims 9 to 12, wherein the disease vaccine comprises at least one novel cancer epitope as a synthetic long peptide that matches an MHC subtype expressed by the subject of treatment, preferably comprising multiple novel cancer epitopes as synthetic long peptides that match at least one MHC subtype expressed by the subject of treatment.

14. A formulation for use according to any one of claims 1 to 13, wherein the disease vaccine comprises multiple disease epitopes.

15. A formulation for use according to any one of claims 1 to 14, wherein the disease vaccine comprises at least one synthetic long peptide.

16. A formulation for use according to any one of claims 1 to 15, wherein the vaccination comprises administering the disease vaccine in the form of a water-in-oil emulsion, the water-in-oil emulsion preferably comprising mineral oil and dehydrated mannitol ether octadecenoate, and preferably as the sole adjuvant.

17. A disease vaccine for improving the immune response of a human subject to a disease epitope, said improvement comprising: (A) Administering the disease vaccine to a donor at the vaccination site; (B) Obtain a sample from the said vaccination site; (C) Produce a formulation containing T cells that recognize at least one disease epitope from the sample obtained in step (B) as a disease vaccine; and (D) The preparation containing T cells produced in step (C) is administered to the human subject.

18. The disease vaccine for the use of claim 17, wherein the improvement in immune response to a disease epitope is an improvement in immune response to cancer.

19. A disease vaccine for use according to claim 17 or 18, wherein the improvement includes at least one other feature according to any one of claims 1 to 16.

20. A method for identifying TCRs that bind to disease epitopes, preferably cancer epitopes, presented on cells of a human subject, the method comprising: (AA) Provides a formulation comprising T cells that recognize at least one disease epitope by the method defined in any one of claims 1 to 18. (BB) Provides the amino acid sequence of at least the complementarity-determining region 3 (CDR3) of the T cell's TCR provided in step (AA); and, thereby, (CC) Identification of TCRs that bind to disease epitopes presented on cells.

21. A method of providing T cells, said T cells recognizing cells presenting disease epitopes, preferably cancer epitopes, said method comprising: (aa) Identification of TCRs that bind to disease epitopes by the method according to claim 20. (bb) TCRs containing at least the complementarity-determining region 3 (CDR3) of the TCRs in step (aa) are expressed in T cells, and thus, (cc) provides T cells that identify cells that present disease epitopes, with a preference for cancer epitopes.

22. A T cell that recognizes cells presenting disease epitopes, which is produced by or can be produced by the method according to claim 21.

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