Methods and compositions for identifying epitopes

Abstract

To provide methods and reagents for identifying target antigens specific to T cells, for example cytotoxic T cells.SOLUTION: Provided are antigen presenting cells (APCs) comprising: a) an exogenous nucleic acid encoding one or more candidate antigens expressed and presented with MHC class I or MHC class II molecules, b) a molecular reporter of Granzyme B (GzB) activity, and c) an exogenous inhibitor of caspase-activated deoxyribonuclease (CAD)-mediated DNA degradation, a CAD knockout, or a caspase knockout.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claims This application is a continuation of U.S. Provisional Patent Application No. 62 / 516,977, filed June 8, 2017. The entire contents of the foregoing are incorporated herein by reference.

[0002] Government Rights This invention was made possible through grant number AI116833 awarded by the National Institutes of Health. This invention was made with government support under the terms of the present application. The government has certain rights in this invention.

[0003] Methods and assays for identifying target antigens specific for T cells, e.g., cytotoxic T cells The drugs are described herein. [Background technology]

[0004] Immunotherapeutic approaches based on cell-mediated immune responses have been used to treat, for example, cancer, autoimmune diseases, and sensitivities. It may be effective in treating diseases such as infectious diseases. The antigens that are expressed and play a role in modulating the immune response are difficult to identify. Therefore, specific targeting of T cells is essential for developing therapies and vaccines against diseases. There is a need for methods for identifying antigens. Summary of the Invention

[0005] In one embodiment, a) is expressed and presented by an MHC class I or MHC class II molecule; a) exogenous nucleic acids encoding one or more candidate antigens to be targeted; b) granzyme B (GzB) and c) molecular reporters of caspase-activated deoxyribonuclease (CA) activity. D) Mediated DNA degradation, CAD knockout, or caspase knockout (e.g., caspase Antigen-presenting cells (APCs) containing exogenous inhibitors of ribosomal kinase (RIK) and ribosomal kinase (RIK) are described herein. It will be published.

[0006] It may be applied to any aspect of the present invention and / or any other aspect described herein. Numerous further embodiments are provided that can be combined with the above embodiments. For example, various In various embodiments, the exogenous nucleic acid is optionally a lentiviral vector, a retroviral vector, or a It is stably introduced into the genome of APC via a vector or transposon. In various embodiments, the exogenous nucleic acid is flanked on both sides by predetermined primer recognition sequences. In various embodiments, the molecular reporter of GzB activity is a GzB cleavage molecule linked to a detection molecule. The fusion polypeptide contains a truncated portion (VGPD, SEQ ID NO: 1), e.g., a molecular reporter. The fusion proteins include engineered infrared fluorescent proteins, membrane-tethered CRE recombinase, and antibody-based Gz reporter of GzB activity, ER retention-based reporter of GzB activity, cell surface detectable-based reporter In various embodiments, the molecule comprises a reporter of GzB activity in a gene encoding the molecule. The reporter contains a membrane-tethered CRE recombinase, and the APC is flanked on both sides by LoxP sites. and optionally, the exogenous nucleic acid further comprises an inverted CRE reporter flanked by the exogenous nucleic acid. In various embodiments, the CAD-mediated DNA degradation sequence is located proximal to the CAD primer recognition sequence. The exogenous inhibitor is expressed in an expressible form as caspase-activating deoxyribonuclease inhibitor ( Nucleic acids encoding the ICAD gene; inhibitory nucleic acids targeting CAD or caspase 3 small molecule inhibitors of caspase-3; chemical DNAse inhibitors; or peptides of caspase-3 caspase-3 inhibitors or caspase knockouts. In various embodiments, the APCs are: i) do not express endogenous MHC molecules; and / or ii) have been engineered to express an exogenous MHC molecule, and / or cells, HEK 293 cells, HEK 293 T cells, U2OS cells, MelJuso cells Cells, MDA-MB231 cells, MCF7 cells, NTERA2 cells, LN229 cells, dendritic cells In various embodiments, the candidate antigen is selected from the group consisting of primary autologous B cells, primary autologous B cells, and primary autologous B cells. are 8, 9, 10, 11, 20, 30, 50, 100, 200, or 300 amino acids long In various embodiments, the candidate antigen is less than or equal to 300 amino acids in length. In various embodiments, the exogenous nucleic acid encoding the candidate antigen is from an infectious organism or Derived from human DNA. In various embodiments, the human DNA is obtained from cancer cells. In various embodiments, the infectious organism is a virus, a bacterium, a fungus, a protozoan, or a multicellular organism. Parasites are selected from the group consisting of:

[0007] In another embodiment, each APC encodes a candidate antigen, thereby encoding MHC class I and and / or a library of candidate antigens expressed and presented by MHC class II molecules. Libraries of APCs, such as those described above, containing a variety of exogenous nucleic acids are provided herein. It will be described.

[0008] As noted above, any aspect of the present invention and / or Numerous embodiments are further provided that can be combined with any other embodiment described. For example, in various embodiments, the exogenous nucleic acid is derived from an infectious agent or human DNA. In various embodiments, the library comprises about 10 2 from about 1014 Individual candidate antigens include.

[0009] In another embodiment, a fusion protein containing a GzB cleavage site (VGPD, SEQ ID NO: 1) linked to a detection molecule. Described herein are molecular reporters of Granzyme B activity, including synthetic polypeptides.

[0010] As noted above, any aspect of the present invention and / or Numerous embodiments are further provided that can be combined with any other embodiment described. For example, in various embodiments, the reporter is a pair of fluorescent dyes that form a FRET pair. or one of the fluorophores is quenched; not the intact fluorophore; For example, it is not an intact protein that is capable of fluorescing by itself; and / or or leuco dyes, e.g., between two chemical forms (one of which It does not contain a pair of fluorescent dyes that are not switchable dyes (one of which is colorless). In various embodiments, the detection molecule is an enzyme, a detectable label, an antibody binding antigen, or an affinity. In various embodiments, the detectable label is an infrared fluorescent protein (I FP), nucleic acid amplification target, composition recognized by antibody, composition released from ER, and and a composition present on a cell surface, detectable after GzB cleavage. In various embodiments, the IFP is functionally separated from the N by a GzB cleavage site. The N-IFP fragment and the C-IFP fragment are also included. The N-fragment of green fluorescent protein (N-GFP) located at the C-terminal end of N-IFP The resulting N-GFP fragment is flanked on both sides by the C-fragment of green fluorescent protein (C-GFP). and C-GFP are constitutively active. In various embodiments, the enzyme is a CRE recombinase. The fusion polypeptide is a GzB enzyme, and the plasma membrane-attached peptide is separated by a GzB cleavage site. In various embodiments, the affinity The -tag is a Flag epitope located C-terminal to the GzB cleavage site, resulting in This epitope is only recognized by the M1 Flag antibody when the GzB site is cleaved. Optionally, it further comprises GFP located C-terminal to the flag epitope. In one embodiment, the molecular reporter comprises an endoplasmic reticulum (ER) retention signal and an antibody-binding protein. It contains a membrane protein, and cleavage of the GzB site removes the ER retention signal, optionally The antigen of choice is CD40, CD4, CD19, CD20, or a tagged protein. Optionally, the tag is a Myc tag, a Flag tag, an HA tag, or a histidine tag. It is a tag.

[0011] In another aspect, there is provided a nucleic acid encoding a molecular reporter described herein.

[0012] In another embodiment, a system for detecting granzyme B activity in antigen-presenting cells. a) a fusion polypeptide containing a CRE recombinase operably linked to a plasma membrane attachment peptide; The CRE recombinase and membrane-attached peptide are separated by the GzB cleavage site. a) a fusion polypeptide containing GFP and RFP in a head-to-head orientation; a reporter of CRE activity containing a nucleic acid sequence flanked on both sides by LoxP sites; and / or c) a CRE-activating primer containing an inactive primer flanked on both sides by LoxP sites. a nucleic acid sequence encoding an expressible form of the candidate antigen, located proximal to a functional primer recognition sequence; CRE-induced transposition of the LoxP site results in a functional primer recognition sequence. Described herein are systems that include nucleic acid sequences that

[0013] Similarly, in another embodiment, the antigen-presenting cells for cytotoxic lymphocytes or NK cells 1. A system for the detection of antigen presentation recognized by a cytotoxic lymphocyte, comprising: a) i) a cytotoxic lymphocyte; and / or NK cells, MHC class I and / or MHC class II molecules ii) an exogenous nucleic acid encoding a candidate antigen that is expressed and presented by the Molecular reporters of Granzyme B (GzB) or Granzyme B activity as described herein. and iii) a system for detecting antigen-presenting cells containing inhibitors of CAD-mediated degradation. and b) cytotoxic lymphocytes and / or NK cells. The system is described herein.

[0014] As noted above, any aspect of the present invention and / or Numerous embodiments are further provided that can be combined with any other embodiment described. For example, in various embodiments, the inhibitor of CAD-mediated degradation is an inhibitor of CAD-mediated DNA degradation. Exogenous inhibitors, CAD knockout, or caspase knockout (e.g., Caspase caspase-3 knockout), and optionally, the caspase knockout is a caspase-3 knockout. Either the CAD gene is a cDNA clone or an exogenous inhibitor of CAD-mediated DNA degradation is expressed in an expressible form. nucleic acid encoding the spase-activated deoxyribonuclease inhibitor (ICAD) gene; Inhibitory nucleic acids targeting CAD or caspase-3; small molecule inhibitors of caspase-3; and is a peptide or protein inhibitor of caspase 3. In various embodiments, the antigen Presented cells are K 562 cells, HEK 293 cells, HEK 293 T cells, and U2OS. cells, MelJuso cells, MDA-MB231 cells, MCF7 cells, NTERA2a cells In various embodiments, the cells are selected from the group consisting of primary autologous B cells, dendritic cells, and primary autologous B cells. Cytotoxic lymphocytes consist of cytotoxic CD4 T cells and cytotoxic CD8 T cells. In various embodiments, the cytotoxic lymphocytes and / or NK cells are selected from the group consisting of: The cells are modified to express an antigen receptor of interest. Cytotoxic lymphocytes and / or NK cells react with non-cytotoxic CD4 T cells to form T cell receptors The cytotoxic T cells and / or NK cells are modified to express

[0015] In another embodiment, the antigens recognized by cytotoxic T cells and / or NK cells are identified. a) administering to a subject an antigen-presenting cell (APC) or APCs described herein; The library of PCs is then subjected to the induction of one or more cytotoxic T cells under conditions appropriate for antigen recognition. (CTL) and / or NK cells; and b) contacting the APC with granulocytes. Identifying APCs expressing the recognized antigen by assaying for Zyme B activity step, wherein an increase in granzyme B activity compared to an appropriate control indicates that the APC is cytotoxic. stents, which show that they express antigens recognized by cytotoxic T cells and / or NK cells. and c) extracting nucleic acids encoding the recognized antigens from the APCs identified in step b). and isolating the same.

[0016] Similarly, in another embodiment, a cytotoxic T cell and / or NK cell-recognized antibody 1. A method for identifying an antigen, comprising: a) administering to a subject an antigen-presenting cell (APC) as described herein; Alternatively, a library of APCs can be combined with one or more CTLs under conditions appropriate for antigen recognition. contacting, wherein cleavage of the GzB site removes the ER retention signal. and releasing plasma membrane proteins from the ER for transport to the plasma membrane; b) contacting the APC with an antibody that binds to a plasma membrane protein recognized by the c) isolating APCs expressing the antigen and purifying the antibody-bound APCs; a step of isolating nucleic acids encoding the recognized antigen from the APCs isolated in step b); Described herein are methods that include:

[0017] As noted above, any aspect of the present invention and / or Numerous embodiments are further provided that can be combined with any other embodiment described. For example, in various embodiments, the method includes sequencing the isolated nucleic acid. In various embodiments, the cytotoxic T cells and / or NK cells further comprise: The biological sample is obtained from a subject. In various embodiments, the biological sample is blood, tumor, healthy tissue, or the like. tissue, ascites, autoimmune locations, tumor infiltration, viral infection sites, lesions, oral mucosa, and skin In various embodiments, the biological sample is selected from the group consisting of skin, a site of infection in a subject, or from autoimmune reactive sites. In various embodiments, the cytotoxic T cells are In various embodiments, the cytotoxic T cells and / or N The K cells are engineered to express an antigen receptor of interest. Cytotoxic T cells and / or NK cells transduce T cell receptors from non-cytotoxic CD4 T cells. In various embodiments, the identifying step b) is At least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more This is achieved by detecting a fluorescent signal in the APC that increases with increasing fluorescence intensity. The identifying step is carried out using flow cytometry or affinity purification. In various embodiments, the identifying step is performed using fluorescence activated cell sorting (FACS) or In various embodiments, the isolating step is performed using affinity purification. In various embodiments, sequencing is performed by PCR amplification. In various embodiments, the method is performed by sequencing or next generation sequencing. In this example, the library of APCs contains at least 5,000 different candidate antigens.

[0018] definition The articles "a" and "an" refer to one of the grammatical objects of this article. It is used to refer to one or more (i.e., at least one). For example, "one element" is used to refer to one or more (i.e., at least one). "Element" means one element or more than one element.

[0019] By "isolated" is meant the term "isolated" which refers to the components that normally accompany it when found in the native state. "Isolated" means material that is free, to varying degrees, from its original source or represents the degree of separation from the environment. For example, isolated cells are cells that have been removed from an animal and cultured. They can be placed in a dish or in another animal. Isolated means they are removed from all other cells. It is not necessarily required that the

[0020] The term "isolated population," as used herein, in reference to an isolated cell population, means: It is obtained from its natural environment (e.g., in the body) and is removed and removed from a mixed or heterogeneous population of cells. and isolated (e.g., during or following removal from the environment) In some embodiments, an isolated population refers to a population of cells (either isolated or isolated, or a combination of both). The population has substantially higher cell viability than the heterogeneous population from which the cells were isolated or enriched. In some embodiments, the isolated population is a substantially pure population of cells. Substantially pure cells compared to a heterogeneous population of cells including cytotoxic lymphocytes (e.g., cytotoxic lymphocytes) and contaminating cells. A population of isolated cells is a pure population of cells. Such cells are first isolated from adult cells. from a human or immature subject (e.g., from birth or from an embryo or fetus developing fetus) to 18 years of age or under 1 year of age or over 1 month of age It can be isolated from the oocyte (or oocytes) for up to 1 day.

[0021] The term "substantially pure" in reference to a particular cell population refers to the cells that make up the entire cell population. and at least about 50%, 60%, 70%, or 75%, preferably at least about 8%. 5%, more preferably at least about 90%, and most preferably at least about 95% pure cells. In other words, the term "substantially pure" or "essentially pure" refers to a population of cells. "Substantially purified" means less than about 20%, more preferably less than about 15%, 10%, 8%, or 7% , most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% contaminating cells. It refers to a cell population that does not contain

[0022] Antigen-presenting cells (APCs) are bound to MHC class I and / or MHC class II antigens. Any cell that can present an antigen to immune cells (e.g., cytotoxic immune cells). APCs are also referred to herein as APC targets, target cells, or target APCs. The APC used as described herein is a vector stably inserted into the genome of the APC. The cells are modified to present the candidate antigen by expression of an exogenous nucleic acid. In this embodiment, the APCs are used to prepare libraries encoding the candidate antigens described herein. Suitable cells for the production of erythrocytes (e.g., HEK293, HEK293T, U20S, K562, Me lJuso, MDA-MB231, MCF7, NTERA2a, dendritic cells and primary (autologous) (autologous) B cells.

[0023] The cells and subjects, as the term is used herein, are typically human. However, subjects and cells derived from non-human animals are also contemplated for use. The term "non-human animal" includes, but is not limited to, mammals (e.g., sheep, dogs, cattle, , horses, chickens, rodents (mice, rats, rabbits, guinea pigs), primates, canines mammals (equids, equids, bovines, felines, swine) and non-mammals The term "cell" refers to all vertebrates, including mammals, mammals, animals, mammals, reptiles, etc. The cells described herein are in this context or elsewhere herein, can be isolated from any such subject. Non-human primates are also a possible source. Experts believe that APCs and cytotoxic lymphocytes are the same. They will recognize that it should be derived from the object of the species.

[0024] As used herein, the term "antigen" refers to an antigen that induces an immune response in a host organism. In some embodiments, the term "antibody" refers to a molecule capable of being recognized by T cells, specifically a molecule that is recognized by T cells. , the antigen is a peptide.

[0025] As used herein, the term "candidate antigen" refers to a candidate antigen that is a target of the screening method described herein. A peptide encoded by an exogenous nucleic acid introduced into an APC target intended for use in The libraries described herein are designed to target cells containing introduced candidate antigens. Contains cells.

[0026] "Exogenous," as the term is used herein, refers to something that is outside or external to a cell. refers to material originating from a cell (e.g., nucleic acid originating from outside the cell that is inserted into the cell's genome) , considered exogenous nucleic acids).

[0027] The terms "nucleic acid," "nucleic acid sequence," "nucleic acid molecule," and "polynucleotide" are used herein and polymeric forms of nucleotides of any length, deoxyribonucleotides, It refers to either xylisothiazolinone or ribonucleotide, or their analogs. The polynucleotides may contain naturally occurring and / or modified nucleotides. Nucleotides may have any three-dimensional structure, known or unknown. Non-limiting examples of polynucleotides include: Genes, gene fragments, exons, introns, DNA, RNA, cDNA (complementary DNA) NA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNP A (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (small nuclear RNA) Small RNAs (small human RNAs), miRNAs (microRNAs), genomic DNA, synthetic DNA, synthetic R NA, and / or tRNA, recombinant polynucleotides, branched polynucleotides, Plasmids, vectors, isolated DNA of any sequence, control regions, isolated DNA of any sequence The nucleic acid molecule includes a linear RNA, a nucleic acid probe, and a primer. It may also be cyclic.

[0028] "Vector," "cloning vector," and "expression vector" are used herein. In this case, a polynucleotide sequence (e.g., a foreign gene) is introduced into a host cell to induce host and promote expression (e.g., transcription and translation) of the introduced sequence. Each refers to a nucleic acid that can transport another nucleic acid to which it is linked. Preferred vectors are molecules that are capable of autonomous replication and / or replication of nucleic acid molecules to which they are linked. They support the expression of genes to which they are operably linked. Vectors that can be used for this purpose are called "expression vectors." Examples include pages and viruses.

[0029] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein. The term "amino acid sequence" can be used to refer to polymeric forms of amino acids of any length, which are encoded by and non-encoded amino acids, chemically or biochemically modified or derivatives These may include polypeptides with modified amino acids, as well as polypeptides with modified peptide backbones. The term may include, but is not limited to, amino acids with or without an N-terminal methionine residue. Fusion proteins with heterologous amino acid sequences, heterologous and native leaders, regardless of -sequence fusions; immunologically tagged proteins; detectable fusion partners fusion proteins, e.g., fluorescent proteins, β-galactosidase, etc., as fusion partners; The present invention also includes fusion proteins, such as fusion proteins containing enzymes such as luciferase, luciferase, and the like.

[0030] As used herein, the term "library" refers to a collection of genetic material, As used herein, the term "library" refers to a collection of nucleic acids encoding candidate antigens. "Lee" describes how individual cells collectively contain and possibly express a library of nucleic acids. In some embodiments, the labeling of target APCs may also refer to a collection of target APCs. Libraries are used to identify, for example, pathogens, cells infected with pathogens, cancer cells, and cells involved in autoimmune diseases. either to cells to be transfected (e.g., targeted) and / or to cells derived from a healthy subject. The peptides comprise a plurality of peptides derived from MHC class I and / or M It is displayed on the surface of target cells as presented by HC class II molecules.

[0031] The terms "T cell" and "T lymphocyte" are used interchangeably and synonymously herein. Examples of such T cells include, but are not limited to, naive T cells, central memory T cells, and the like. These include immune-mediated T cells, effector memory T cells, or a combination thereof.

[0032] The term "transduction" as used herein refers to the transfer of foreign nucleic acid to a cell using a vector. Refers to introduction into cells.

[0033] The term "transfection," as used herein, refers to the process of transfection using recombinant DNA techniques. The term "transformation" refers to the introduction of exogenous nucleic acid into a cell, i.e. the introduction of a gene, DNA or RNA sequence (external, exogenous, or extracellular) into a host cell It means introduction, so that the host cell expresses the introduced gene or sequence and Produce a desired substance, such as a protein or enzyme, encoded by the gene or sequence One such method for transforming the cells described herein is to The introduced gene or sequence is "cloned" or or "foreign" genes or sequences, which are used by the cell's genetic machinery. the start, stop, promoter, signal, secretion, or other sequences that are A gene or sequence may contain a non-functional sequence or a sequence with a known function. A host cell that receives and expresses introduced DNA or RNA may contain sequences that do not have the The host cell has been "transformed" and is a "transformant" or "clone." The DNA or RNA introduced into cells of the same genus or species as the host cell, or of a different genus or species The cells may come from any source, including cells of any species.

[0034] The term "detector molecule" includes, but is not limited to, radioisotopes, fluorophores, chemiluminescent Chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal zones ligands (e.g., biotin, avidin, streptavidin, or heptavidin), etc. Exemplary molecules suitable for use as detection molecules include: Suitable detectable molecules include affinity tags and detectable labels. Examples of possible labels include, but are not limited to, fluorophores, chemiluminescent agents, and chromophores.

[0035] The term "chromophore" refers to a substance or portion thereof that is capable of exhibiting fluorescence in the detectable range. Refers to minutes.

[0036] The term "affinity tag" refers to a molecule that binds to an affinity tag and provides a detectable signal ( can be detected using molecules that result in fluorescent compounds (e.g., fluorescent compounds or proteins) It is used herein to refer to a peptide segment that is capable of binding to a target. In principle, any peptide or peptides for which an antibody or other specific binding agent is available. Alternatively, a protein can be used as an affinity tag.

[0037] The term "reaction mixture" as used herein refers to a library of target cells containing a candidate antigen. This refers to the liquid medium that comes into contact with the biological sample containing the cytotoxic lymphocytes. In this method, a library of target cells containing candidate antigens is first incubated with a biopsy sample containing cytotoxic lymphocytes. contacting the sample with the candidate antigens on the target cells and any subsequent washing steps to separate the candidate antigens from the cytotoxicity in the sample A reaction mixture designed to eliminate nonspecific or low-affinity binding between lymphocytes If desired, the stringency of the reaction mixture can be modified to allow for a better match between the candidate antigen and the cytotoxic activity in the sample. It can affect complex formation between lymphocytes.

[0038] As used herein, the terms "specific binding," "specifically bind," and the like refer to a reaction mixture. Preferentially binds to a second binding molecule or moiety relative to other molecules or moieties in the compound. Refers to the ability of a first binding molecule or moiety (by covalent or non-covalent bonding).

[0039] As used herein, the terms "determine," "measure," "assess," and "assess" "Assay" is used interchangeably and is defined as a quantitative determination unless the context clearly indicates otherwise. This includes both sexual determination.

[0040] As used herein, the term "sample" or "biological sample" refers to a sample isolated from its natural environment. It refers to a biological material that contains immune cells (e.g., including cytotoxic lymphocytes). The sample or biological sample may include a tissue sample or a biological fluid sample. Examples include, but are not limited to, blood, plasma, saliva, urine, cerebrospinal fluid, lavage fluid, and white blood cells. Examples include removal samples.

[0041] As used herein, the term "pathogen" refers to a pathogen that infects another organism (e.g., an animal or plant). infecting other organisms directly or causing disease in another organism They cause disease by producing agents that cause disease (e.g., bacteria that produce pathogenic toxins). As used herein, pathogens include, but are not limited to, organisms that cause disease, including microorganisms. Not limited to, but including, bacteria, protozoa, fungi, nematodes, viroids and viruses, or Any combination of these is included, each pathogen by itself or in combination with another pathogen. including, but not limited to, mammals (including, but not limited to, humans). As used herein, the term " A "pathogen" is a microorganism that may not normally be pathogenic in a non-immunocompromised host. It also includes things.

[0042] The term "immune cells" as used herein includes, but is not limited to, antigen-presenting cells. cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells , leukocytes, lymphocytes (e.g., cytotoxic lymphocytes), macrophages, mast cells, Includes memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells, and T cells Refers to cells of the mammalian immune system, including the immune system.

[0043] The term "immune response" as used herein includes, but is not limited to, innate immunity, Humoral immunity, cellular immunity, immunity, inflammatory response, acquired immunity, autoimmunity, and / or It refers to immunity, including overactive immunity.

[0044] The term "mammal," as used herein, includes, but is not limited to, human and non-human spirits. Long-legged animals, e.g., chimpanzees and other apes, human and other monkey species; domestic animals, e.g., cormorants domestic mammals, such as dogs and cats; mice, Any of the mammalian class, including rodent laboratory animals, including rats and guinea pigs The term does not denote a particular age or sex; therefore, males Adult and newborn subjects, as well as fetuses, whether male or female, are included within the scope of this term. It is intended to be included within

[0045] The term "tumor," as used herein, refers to any tumor, whether malignant or benign. The growth and proliferation of all neoplastic cells, as well as all precancerous and cancerous cells and Refers to organizations.

[0046] The terms "cancer" and "cancerous," as used herein, typically refer to the development of unregulated cells. Refers to or describes the physiological condition in mammals characterized by cell growth. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin's lymphoma and and / or non-Hodgkin's lymphoma), brain tumors, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, Stomach cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary system cancer, thyroid cancer Adenocarcinoma, renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer (including but not limited to) Although not defined, androgen-dependent and androgen-independent prostate cancers (including)

[0047] "Appropriate controls," as that term is used herein, are controls that are designed to measure one or more important factors. Control refers to a control reaction that is otherwise treated identically to the experimental reaction, except for activation. cells that are identical except that they are not exposed to a molecule (e.g., activated cytotoxic lymphocytes) Alternatively, a control may be cells exposed to the activating molecule but lacking the reporter molecule. Suitable controls can be prepared by skilled technicians (or may be identical to the experimental cells). is determined by.

[0048] Exemplary embodiments are illustrated in the referenced drawings. It is intended that the embodiments and drawings be regarded as illustrative rather than restrictive. do. [Brief explanation of the drawings]

[0049] [Figure 1] FIG. 1 shows an overview of an exemplary approach for systematic identification of T cell antigens. [Figure 2]Figures 2A and 2B are graphs showing exemplary positive controls for CTL-target interactions. Figure 2A shows that CTLs recognize target cells and kill them when pulsed with IV9, but not with a control peptide, as determined by 7-AAD staining. Figure 2B shows that expression of a 56-amino acid peptide (also referred to as a 56-mer) containing the IV9 peptide from three different promoters leads to efficient antigen presentation and killing by IV9 CTLs, as measured by LDH release. [Figure 3] 1 shows a graph of exemplary fluorogenic reporter of GzB activity. GFP-labeled target cells expressing fluorogenic GzB reporter were pulsed with control peptide or homologous IV9 peptide before co-culture with IV9 CTL. GzB activity was detected by measuring infrared fluorescent protein signal in target cells. [Figure 4-1] Figures 4A and 4B show reconstitution experiments for enrichment of target cells presenting a cognate antigen. Figure 4A shows a schematic diagram of the reconstitution experiment. Figure 4B shows the fold enrichment of target cells presenting a cognate antigen when spiked into target cells presenting a control antigen at various ratios. [Figure 4-2] Same as Figure 4-1. [Figure 5-1] Figures 5A and 5B show the detection of CTL antigens in screening. Figure 5A shows a schematic diagram of the screening. Figure 5B shows the fold enrichment detected by qPCR of control (non-target) and cognate (target) peptides after screening relative to the input library. Rep1 and Rep2 are two independent biological replicates. [Figure 5-2] Same as Figure 5-1. [Figure 6-1]Figures 6A and 6B show the development of an exemplary Cre reporter for GzB activity. Figure 6A shows that antigen-presenting cells (APCs) expressing DNA-encoded peptides present epitopes derived from the peptide on MHC I molecules on their cell surface. When T cells recognize this complex via the T cell receptor (TCR), they form an immune synapse, secreting perforin and granzymes. Granzymes enter the APC and cleave the previously membrane-bound Cre recombinase. Cre recombinase reverses the orientation of the 3' primer site, allowing productive PCR amplification of the DNA-encoded peptide. Figure 6B shows the results of detecting Cre-mediated inversion (Cre activity) in target cells expressing membrane-tethered Cre and exposed to GzB delivered by NK cells. Cre activity was detected by qPCR using primers specific for the inversion reporter cassette. Genomic DNA from NK cell-treated and control untreated cells was purified, and inversion frequencies were quantified by qPCR and normalized to reporter abundance in each sample (quantified using inversion-independent qPCR primers). Target cells expressing only the reporter for Cre activity (no Cre control) demonstrated no detectable Cre activity upon GzB delivery. [Figure 6-2] Same as Figure 6-1. [Figure 7-1] Figures 7A and 7B show the development of an exemplary antibody-based reporter of GzB activity. Figure 7A shows a schematic diagram of the antibody-based reporter approach. Typically, the reporter substance contains a Flag epitope before the GzB cleavage site. After GzB cleavage, the Flag epitope is accessible for recognition by the M1 antibody, which recognizes a Flag epitope specific to the N-terminus of the protein. Figure 7B shows the results of Western blotting analysis using the M1 Flag antibody on cell lysates from target cells expressing the GzB reporter, with or without GzB delivery by NK cells. A dramatic increase in antibody targeting is observed in the presence of the reporter and subsequent GzB delivery. [Figure 7-2] Same as Figure 7-1. [Figure 8]1 is a graphical representation of the results of screening IV9 T cells. Each dot represents the fold enrichment in each of two biological replicates for one peptide from the library. Dots identified with numerical labels are all peptides containing known targets of IV9 T cells. [Figure 9] Figure 1 is a chart listing the motifs discovered by the analysis. The IV9 epitope was identified by this motif analysis. The top enriched motif identified by MEME analysis of the 100 most enriched peptides from the IV9 screen contains the exact IV9 epitope (ILKEPVHGV). [Figure 10] This table shows the percentage of target cells that activated the GzB reporter in experiments using the GzB reporter for the CD4 TCR. Primary CD8+ T cells were lentivirally modified to express either the Ob1A.12 TCR or a control TCR. The modified T cells were then mixed with target cells presenting the target antigen of the Ob1A.12 TCR or a mutant control antigen. Expression of the Ob1A.12 TCR resulted in specific recognition of the cognate MBP peptide in the context of MHC II, which could be detected using the GzB reporter. [Figure 11]

[0033] Figure 1 shows a linear schematic of an IFP-based Gzb reporter. The reporter contains two halves of an IFP separated by a linker containing a GzB cleavage sequence. The entire IFP cassette is flanked on both sides by split GFP. [Figure 12] Figure 1. Schematic of the reporter activation mechanism. Prior to activation, the reporter contains two IFP halves whose maturation is inhibited by a linker sequence. Upon GzB cleavage, the linker is released and the two IFP halves join together to form the active, fluorescent IFP. Split GFP at the N- and C-termini of the construct provides constitutive GFP fluorescence and helps stabilize the entire protein. [Figure 13-1]FIG. 1 shows the nucleotide (SEQ ID NO: 2) and amino acid (SEQ ID NO: 33) sequences of an exemplary GzB IFP reporter. [Figure 13-2] Same as Figure 13-1. [Figure 13-3] Same as Figure 13-1. [Figure 14] FIG. 1 shows qPCR primers designed to facilitate detection of reporter cassette inversion events for the presence of Cre by qPCR rather than by fluorescent detection via activation of GFP and loss of RFP. [Figure 15-1] FIG. 1 shows the nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 34) sequences of an exemplary reporter cassette for the presence of Cre, which allows fluorescent detection of Cre activity by activation of GFP and loss of RFP. [Figure 15-2] Same as Figure 15-1. [Figure 15-3] Same as Figure 15-1. [Figure 16] 1 is a graph showing the results of a genome-wide screen of CMV for the NLV2 TCR. Two distinct DNA barcodes encoded 2,882 56-amino acid peptides, arranged in 28-amino acid increments across the CMV genome. Each dot in the scatter plot represents the performance of two barcodes corresponding to one peptide sequence. The two dots in the upper right corner, identified in association with the numerical labels, are the only two epitopes in the library containing known markers of the NLV2 TCR (NLVPMVATV (SEQ ID NO: 4)). [Figure 17]18 shows the results of a viromes-wide screening of patient T cells. 93,904 56-amino acid peptides, arranged by 28 amino acids across the genomes of 206 viral species, were screened on patient T cells grown in the presence of NLV peptides. Each dot in the scatter plot represents the performance of one peptide in each of two biological replicates of the screen. The two dots identified with the numerical labels "24741" and "24742" represent the only two epitopes in the library containing the NLV epitope (NLVPMVATV (SEQ ID NO: 4)). The two dots identified with the numerical labels "32255" and "32256" encode overlapping 56-mers from the UL123 (IE1) protein, which is targeted by 2% of the sample T cells shown in FIG. 18. [Figure 18] Figure 1 shows the validation of novel epitopes discovered in the virome-wide screening. HLA-A2 tetramers loaded with a negative control peptide, a known pp65 NLV peptide, or the newly discovered IE1 (UL123) peptide were used to stain the T cell population used in the virome-wide screening. 27.7% of CD8-positive T cells in the input population recognized the NLV peptide, while 2.1% recognized the IE1 epitope. [Figure 19] This graph shows the results of library screening using a CMV genome-wide library versus polyclonal memory T cells. Memory T cells from a CMV-positive, HLA-A2-positive donor were used to screen 2,882 56-amino acid peptides aligned at 28 amino acids across the CMV genome. Each dot represents the performance of two independent DNA barcodes encoding a specific 56-amino acid peptide. Peptides with two overlapping 56-amino acid peptides enriched are identified by the associated numerical label. [Figure 20]1 is a graph showing the results of side-by-side mutagenesis characterization of TCR binding. T cells grown against the NLV epitope (NLVPMVATV (SEQ ID NO: 4)) were screened against an exhaustive mutagenesis library of the NLV epitope and its two flanking amino acids. Each box in the heatmap represents one mutant, with shading and values ​​indicating how this mutant performed compared to the wild-type version of the epitope. [Figure 21] Graphs showing that detection of TCR markers improves with multiple rounds of screening. Each dot represents the performance of two DNA barcodes for a given peptide after the first (left panel) or second (right panel) round of screening with an IV9-specific TCR. The six dots identified in association with numerical labels represent six peptides in the antigen library that are known markers of the TCR. [Figure 22] Figure 1 shows the results of a signal-to-noise analysis of tumor-specific TCRs. NLV-specific CMV TCR (viral TCR) and MAGE-A3 tumor-specific TCR (tumor TCR, not affinity-enhanced) were introduced into donor CD8 T cells. Recognition of MHC-matched cells in the absence (control) or presence (+antigen) of cognate antigen was measured, demonstrating comparable TCR performance. [Figure 23] (Figure 1) Overexpression of mutant ICAD prevents DNA degradation during apoptosis. Target cells without (control) or with (ICAD) mutant ICAD overexpression were treated with the apoptosis inducers camptothecin and staurosporine. Purified genomic DNA showed the characteristic DNA smear and laddering when apoptosis was induced in control cells but not in the presence of mutant ICAD. [Figure 24]1 is a graph showing that expression of mutant ICAD enhances antigen cassette recovery in a screening setting. The number of antigen cassettes recovered by sequencing was compared to the number of cells sorted to calculate the efficiency of antigen recovery. Each bar represents the percent antigen recovery in one screening replicate performed in the absence (No ICAD) or presence (ICAD) of overexpressed mutant ICAD. [Figure 25] Figure 1 shows the results of designing and testing an engineered protease reporter. The reporter protein (CD4) was fused to GFP and retained in the ER by the addition of a C-terminal KKXX motif. Expression of TEV protease (right panel) leads to increased surface expression of CD4 by cleaving the ER retention motif, as detected by staining with an anti-CD4 APC antibody. DETAILED DESCRIPTION OF THE INVENTION

[0050] The immune response is a complex process that involves several "molecular players." However, one of the fundamental parts of the immune response is the recognition of epitopes / antigens by CTLs. Epitopes are proteins that are presented on cell membranes by major histocompatibility complexes (MHC). or fragments of proteins. Large proteins are broken down into hundreds of short proteins by specific enzymes. It is broken down into large peptide fragments, only a few of which induce an immune response. It will be issued.

[0051] Interaction of T cells (e.g., cytotoxic T cells) with antigens, such as antigens presented by APCs Productive interactions between target cells are extremely rare, occurring in fewer than one in a million target cells. The antigen recognized by a given T cell is typically very low For example, they occur at a frequency of 1 in 100,000 or less. In other words, all target cells presenting a given antigen will be targeted by their cognate T cells, especially in mixed T cell populations. Therefore, the T-cell interaction with the antigen and its epitope is not necessarily a given specificity of the group. Efforts to identify cell receptor interactions have focused on the complexities between T cell receptors and epitopes / antigens. The inability to detect such rare events in a diverse mixture can lead to a lack of predictive power for T cell responses. have focused on individual or small pairs of interactions based on direct measurements (e.g., physiological Furthermore, existing methods do not provide a comprehensive approach to the analysis of antigen presentation. This results in an inability to allow endogenous processing and loading of peptides onto MHC by the presenting cells. We focus on cell-based platforms that can provide effective therapeutic effects. The target antigens identified by this platform are indeed functional in vivo. There is little tendency for this to be presented objectively.

[0052] To solve this problem, we have developed a method for producing high-resolution immunoglobulin (HIL) antibodies specific to T cells, e.g., cytotoxic T cells. Regenerative backups are performed using a combination of elements that allow for high throughput discovery. A method to identify interaction signals above background noise and correlate them with the APCs presented To detect such rare interactions in a manner that allows for the recovery of the antigens that drive the interaction, Compositions and methods for this purpose are provided herein. These are low-throughput problems as well as high-throughput (e.g., genome-wide) ) Scaled and modular complex antigen libraries are used to target T cell receptor- By enabling sensitive and robust detection of epitope / antigen interactions, It is particularly useful because it overcomes the problem of T cell receptor-epitope / antigen discovery.

[0053] Generally, the compositions and methods provided herein involve the use of recognized APC markers. By isolating DNA from target APCs presenting candidate antigens using a CARD / readout This allows the identification of antigens recognized by cytotoxic lymphocytes, thereby preventing the proliferation of a wide variety of It is possible to stimulate an immune response from a library of possible antigen fragments (e.g., a library of test antigens). This is useful for enabling the identification of antigens that can be targeted to the target cells. viral vectors) in MHC class I and / or MHC class II molecules encodes one or more candidate antigens for expression and presentation to multiple APCs. The resulting library of genetic material results in a library of target APCs. The libraries are screened for cytotoxic lymphocyte-mediated cytotoxicity by the methods described herein. This allows the identification of antigens that are more efficiently recognized by APCs or APCs. The library is expressed and presented by MHC class I or MHC class II molecules. The exogenous nucleic acid includes an exogenous nucleic acid encoding one or more candidate antigens to be detected by the APC. into the genome (e.g., via a vector, e.g., a viral vector, or a transposon) In some embodiments, the inserted exogenous nucleic acid can be stably introduced into a mammalian cell line. Both the upstream and downstream primers contain predetermined primer recognition sequences (referred to herein as flanking primers). These sequences allow the APC to recognize the antigen. This makes it easier to identify the problem later.

[0054] APCs contain molecular reporters that indicate antigen recognition by cytotoxic lymphocytes Productive antigen recognition may be further modified, for example, by directly measuring responding T cells. They are identified by detecting the activity resulting from antigen recognition rather than by detecting the activity resulting from antigen recognition. For example, cytotoxic T Surrogate measures of cellular activity, e.g., IFN-γ secretion measured by ELISPOT, are typically used in the art to investigate T cell interactions. However, measures of these aspects have uncertain implications for the in vivo function of CTLs. (Sekaly, JEM 205(1): 7(2008)) and TCR-epitope / antigen binding. In contrast, the modified APCs described herein do not directly identify proteases. The APC's cellular responses, such as the release of cytotoxic granules containing the enzyme granzyme B (GzB), By detecting reporters of productive antigen recognition caused by cytotoxic lymphocyte-mediated alterations This allows the identification of the binding of cytotoxic lymphocytes by APCs. and cytotoxic lymphocytes expressing T cell receptors capable of binding to antigens presented on the surface. This occurs when the APC contacts the antigen and under conditions suitable for antigen recognition. The detected T cell modifications are involved in the induction of cytolysis and therefore functionally relevant T cells. For example, productive antigen recognition by cytotoxic lymphocytes results in the production of antigens in APCs. This results in the activation of GzB, a serine protease involved in cell lysis in Even a 1% false signal level is sufficient to mask true positives, so background The objective of this invention is to minimize background noise and enable identification of true positive signals. In some embodiments, the GzB reporter is a fluorogenic reporter of GzB. In some embodiments, the GzB reporter produces an optically detectable signal. However, it is possible to express antigens that are recognized by CTLs, for example, by affinity purification. In some embodiments, GzB produces a cell surface signal that allows for the isolation of APCs that respond to GzB. The detectable signal generated by the reporter is used to identify the expressed antigen on the cell. Enrich APCs that are productively recognized by cytotoxic lymphocytes.

[0055] Markers of productive antigen recognition enhance signal-to-noise and increase the complexity of candidate antigens. This increases the number of T cell targets included in the library (i.e., the signal is corrected). (This can be useful in identifying a number of candidate antigens that can be successfully identified.) For example, T cell receptor-antigen Unlike traditional methods of analyzing interactions, the assay can be performed on one million target cells. The complexity of the candidate antigens that can be identified is greater than 5k (i.e., 5,000), or is less than 10k, 15k, or 20k, 25k, 30k, 35k, 40k, 45k, 50k, 55k, 60k, 65k, 70k, 75k, 80k, 85k, 90k, 95k, 100k, 105k, 110k, 1 15k, 120k, 125k, 130k, 135k, 140k, 145k, 150k, 1 55k, 160k, 165k, 170k, 175k, 180k, 185k, 190k, 1 95k, 200k, 210k, 220k, 230k, 240k, 250k, 260k, 2 70k, 280k, 290k, 300k, 310k, 320k, 330k, 340k, 3 50k, 360k, 370k, 380k, 390k, 400k, 410k, 420k, 4 30k, 440k, 450k, 460k, 470k, 480k, 490k, 500k, 6 00k, 700k, 800k, 900k, 1000k, 1100k, 1200k, 130 0k, 1400k, 1500k, 1600k, 1700k, 1800k, 1900k, 2 000k or more, or any range therebetween, inclusive (e.g., 1 The target cells can be any size (00K to 2000K), each of which exhibits a unique peptide. Some antigen library formats, such as libraries of unlabeled peptides, require multiple screening steps. The antigen that can be used is 1 × 10 8 (i.e., hundreds of millions) to 1×10 9 or it It is an order of magnitude higher.

[0056] An increase in antigens that can be screened according to the compositions and methods described herein In addition to the increased complexity, the methods and compositions preferably increase the efficiency of antigen recovery. To achieve this, DNA degradation (e.g., caspase-activated deoxyribonuclease (CAD) The APC may also contain an inhibitor of cytotoxicity (cytotoxicity-mediated DNA degradation). The antigens recognized by the antigens are then circulated from the modified APCs, which have been marked by productive antigen recognition. If the target gene can be recovered (e.g., a gene encoding the cognate antigen bound by the T cell receptor), However, the exogenous nucleic acid sequence can be determined by the CTL. The resulting cell lysis initiates DNA degradation that prevents efficient recovery of antigen identity. 100 modifications marked by productive antigen recognition without the inclusion of inhibitors of NA degradation Approximately one single antigen (i.e., 100 modified APCs) was isolated from the modified APCs. The antigen presented by one of the antibodies can be identified (with an efficiency of 1% or less). As described in, for example, an inhibitor of DNA degradation, e.g., an inhibitor of CAD-mediated DNA degradation. By using the antibody, antigen recovery was increased by at least 5-fold (i.e., 5% efficiency), At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of , 50%, or more, or any range therebetween, inclusive (e.g., 5% Thus, using the method of the present invention, it is possible to obtain a densification rate of more than 5%, e.g., 50 % or higher recoveries (100% is the theoretical limit) can be achieved.

[0057] The large number of antigens that can be screened and the efficiency of antigen recovery in individual experiments Therefore, the methods described herein require only a small number of T cells, and therefore ex A limited number of T cells can be applied to a sample directly in vivo.

[0058] Also provided herein are a plurality of APCs modified as described herein, Here, the APC contains various exogenous nucleic acids encoding candidate antigens, such that the APC In some embodiments, each APC collectively presents a library of candidate antigens. The nucleic acid is contained and expressed, possibly in multiple copies, thereby forming an MHC class. A single candidate antigen is presented by MHC class I and / or MHC class II molecules. In an embodiment, each APC carries a handful of different nucleic acids, possibly multiple, that express different candidate antigens. and expressing it in multiple copies, thereby inhibiting MHC class I and / or MHC class II molecules are able to recognize several candidate antigens (e.g., 2, 3, 4, 5, 6, or (or even more)

[0059] Preferably, the APCs for the library are of the same cell type (e.g., cloned prior to modification). In various embodiments described herein, the library is derived from: An isolated and / or substantially pure cell population, comprising a plurality of APCs. Examples of suitable cells include, but are not limited to, K562 cells, HEK 293 cells, and the like. cells, HEK 293 T cells, U2OS cells, MelJuso cells, MDA-MB23 1 cells, MCF7 cells, NTERA2a cells, dendritic cells, and primary autologous B cells. can be.

[0060] In the methods described herein, the APC, or a plurality thereof, is generally a cell suitable for antigen recognition. In some embodiments, the APC target is contacted by a cytotoxic lymphocyte under conditions. The reaction sample containing the target cytotoxic lymphocytes is mixed with a biological sample containing the target cytotoxic lymphocytes, and the mixture is incubated. This allows for the recognition by cytotoxic lymphocytes of any target cell that displays the cognate antigen. Upon recognition, the cytotoxic lymphocytes modify the target cell in a detectable manner (e.g., by causing death). To initiate the destruction process, the serine protease granzyme B (GzB) The cells thus modified are identified and then treated with the same type of antibody. An exogenous nucleic acid encoding the gene is isolated therefrom. The isolated exogenous nucleic acid is sequenced. The antigens recognized by the antibody are identified by the antibody binding. Using this method, specific targets for a given cytotoxic lymphocyte can be used with PCs. Antigens can be generically identified. Additional details and representative embodiments are further provided below. Write it down.

[0061] Use of the Compositions and Methods CTLs have long been understood to recognize cells infected by intracellular pathogens. , which are essential for the control of many infectious diseases, including HIV. Recognition by the immune system can lead to autoimmune diseases, including type 1 diabetes. Recent advances have highlighted another important function of CTLs: their ability to recognize and eliminate tumors. This function supports promising immune strategies such as adoptive T cell transfer and immune checkpoint blockade. It serves as a foundation for therapeutic approaches and has demonstrated durable cure in a subset of patients with previously refractory cancers. The major ongoing challenge in each of these situations is the antigen-driven The purpose of this study is to characterize the activity of T cells.

[0062] Understanding protective and pathogenic T cell responses will help improve immunity to help a wider range of patients. Important for informing the discovery of biomarkers or co-interventions that can improve immunotherapy The techniques disclosed herein can be used to provide protective measures for fair profiling. or characterizing pathogenic T cell responses, as well as identifying target antigens for T cells of interest. This can be done.

[0063] Identifying the target antigen of a TCR of interest As demonstrated herein, this method was used to identify targets for isolated T cell clones. The technology can be applied directly to the identification of genes by DNA sequencing. This technique can be used to identify the target of interest for the TCR. These TCR sequences can be applied to TCRs arising from either CD8 or CD8 T cells. synthesized and introduced into primary T cells, then screened on our platform Remarkably, the technology for sequencing TCRs has improved dramatically. This has been improved upon and is expected to reveal many other applications for this platform. There is.

[0064] autoimmune disease Advances in high-throughput sequencing have enabled the identification of potentially pathogenic T cell clones. These cells are grown in the body or in patients with type 1 diabetes, multiple sclerosis, ankylosing spondylitis, recurrent Diseases such as aplastic anemia, large granular lymphocyte leukemia, polymyositis, thyroiditis, and cardiomyopathy These T cells allowed the identification of TCRs that are conserved across patients with the disease. Existing methods for identifying antigens recognized by TCRs are based on the use of TCR sequences, even when the TCR sequence is known. However, these T cells and / or antigens lack the throughput to allow for unbiased antigen discovery. or using TCRs in the methods described herein to identify their target antigens. This can provide insight into the etiology, provide biomarkers, and identify pathogenicity. This opens the door to targeted therapies that specifically suppress autoimmune responses.

[0065] Cancer immunotherapy A major and outstanding challenge in the field of cancer immunotherapy is to mediate productive antitumor immunity. The goal is to identify tumor antigens that are involved in tumor infiltration. TCR sequencing demonstrated oligoclonal expansion of tumor-specific T cells. Patients' tumors containing novel protein fragments arising from somatic mutations in the patient's tumor A specific neoantigen library can then be generated. Tumor-specific T cells can then be immunized with these neoantigens. Systematically screened for recognition of native epitopes and for recognition of non-mutated tumor antigens. This allows genome-wide screening to understand productive anti-tumor immunity. This could guide the development of biomarkers and co-interventions that enhance the success of immunotherapy. Cut.

[0066] Unbiased profiling of protective or pathogenic T cell responses The techniques described herein can be applied to identify specificities of mixed populations of T cells. This allows for the identification of specific clones or TCRs of interest even if they have not yet been identified. , allowing the characterization of protective or pathogenic T cell responses.

[0067] Applying this platform to T cell populations in each of the above situations For example, this platform can be used to treat bulk T cells isolated from patients with type 1 diabetes. to identify the complete set of pancreatic autoantigens recognized by patient T cells. Similarly, it can be used to identify polyclonal tumors infiltrating T cells. Screening for mutated and non-mutated tumor antigens recognized in anti-tumor immunity A range of can be profiled.

[0068] Protection from infectious diseases T cells are thought to mediate protection against a wide range of infectious diseases. There is a strong association between the HLA-B57 allele and selected controls for HIV. A promising determinant of virus control involves CD8 T cells and specific target antigens. The techniques disclosed herein can be used to control specific clinical outcomes, such as controlled exposure to malaria. or systematically assess CTL specificity in patients with immunity to selected controls of HIV. Profile to identify correlates of protection and inform vaccine design It is possible.

[0069] This platform will identify and characterize effective T cell epitopes to Some algorithms can also contribute to improving the design of MHC molecules. Although the peptides are present to predict their affinity for a given peptide, they are not likely to be productively recognized by T cells. There is no understanding of other characteristics that make certain peptides more likely to be resistant to steroids. The techniques disclosed in the present application allow for the production of antibodies that are productively presented by target cells and expressed by the patient's T cells. This allows the discovery of numerous T cell epitopes recognized by these antigens. This knowledge can then be used to characterize effective T cell epitopes. This knowledge can be used to generate optimized vaccines and to combat cancer and infectious diseases (e.g., HIV) , cytomegalovirus infection, and malaria) can.

[0070] Cytotoxic lymphocytes and NK cells In some embodiments, the cytotoxic lymphocytes are cytotoxic T cells. Cytotoxic T cells express their endogenous receptors, which may be either CD4 or CD8. The antigen receptors can be expressed in a variety of ways, including but not limited to, or can be modified to express an exogenous antigen receptor of interest. In some embodiments, the exogenous receptor is a T cell (e.g., The specificity of T cells is determined by the arrangement of their T cell receptors. By transferring the TCR from one T cell to another T cell, Transferring new TCR specificity while preserving the effector function of recipient cells This is the basis of TCR therapy in general. The TCR derived from CD8 T cells, when transferred into donor CD4 cells, It can drive the effector function of T cells (Ghorashian et al., J Immunol, 194(3): 1080-1089(2015)). As demonstrated herein, CD4 T cell-derived By transferring the TCR corresponding to the target antigen into donor CD8 cells, the target antigen is presented on MHC class II. , which confers GzB-mediated cytotoxic activity against antigens recognized by the CD4 TCR. (See Figure 10 herein.) In some embodiments, exogenous T cells The cell receptors are derived from helper T cells (Th1 or Th2) or regulatory T cells. Other types of cytotoxic cells, such as cytotoxic cells, may be used in the assay, and these cells The cytotoxic lymphoma used in this method can be identified. The spheres may be clonal or mixed populations. Alternatively, or in addition, the spheres may be clonal or mixed populations. using natural killer (NK) cells engineered to express T cell receptors. It is possible.

[0071] Cytotoxic lymphocytes or NK cells can be obtained from a variety of sources. Cytotoxic lymphocytes are obtained from a biological sample.

[0072] sample In some embodiments, a "reaction sample" is a lysate of target cells or target cells containing a candidate antigen. The reaction sample contains a library of candidate antigens on the surface of target cells and T cells in the sample of interest. Additional buffers, salts, osmotic agents, etc. may also be included to promote coalescence.

[0073] A "biological sample" is a sample of a subject that contains cells of interest, such as cytotoxic lymphocytes or antigen-presenting cells. In an exemplary embodiment, the biological sample is a sample containing cytotoxic T cells (C TL) and / or natural killer cells. The organ or tissue may be obtained from any organ or tissue of an individual, so long as it contains In some embodiments, the biological sample is an autologous sample. It may originate from the location of autoimmunity, the site of an autoimmune reaction, tumor infiltration, viral infection, or lesion.

[0074] In some embodiments, the biological sample is treated to remove biological particles or unwanted cells. Methods for removing cells from blood or other biological samples are well known in the art. These methods are well known and may include, for example, centrifugation, ultracentrifugation, immunoselection, or precipitation. Some non-limiting examples of body samples include blood samples, urine samples, semen samples, lymph samples, cerebrospinal fluid samples, and the like. Sample, plasma sample, serum sample, pus sample, amniotic fluid sample, body fluid sample, stool sample, biopsy sample, needle aspirate biopsy Test samples, swab samples, mouthwash samples, oral mucosal samples, cancer samples, tumor samples, tumor infiltration, tissue The sample may be a tissue sample (e.g., skin), a cell sample, a synovial fluid sample, or a combination of such samples. In the methods described herein, the biological sample may be blood or tissue biopsy (e.g., tumor, autopsy, etc.). Preferably, the pathology is an autoimmune or other pathology.

[0075] APC Modifications APCs can be engineered, such as by transfection or genetic modification, to APCs express exogenous nucleic acids encoding co-antigens. APCs can be expressed by genes, proteins, chemical labels, downregulating and / or inhibiting the expression of a composition of interest, such as an exogenous nucleic acid encoding a reporter molecule; The gene may be further modified to upregulate or inhibit the expression of the gene.

[0076] As mentioned above, APCs are involved in antigen recognition by cytotoxic lymphocytes and / or NK cells. The productive antigen recognition may be further modified to contain a molecular reporter that indicates For example, rather than directly measuring responding T cells, detection of activity resulting from antigen recognition In some embodiments, one or more of the amino acids further described herein are identified by A reporter of GzB activity, such as a GazB-based reporter, is used.

[0077] In the methods and compositions described herein, the APC further comprises an inhibitor of DNA degradation. In some embodiments, the inhibitor blocks DNA degradation directly by CAD. GzB induces genomic DNA degradation by caspase-activated deoxyribonuclease (CAD). This initiates caspase activation in target cells, leading to the internucleosomal degradation of A. The degradation of genomic DNA in some cases can be prevented by providing an inhibitor of CAD-mediated DNA degradation. The degradation of DNA during apoptosis can be delayed or inhibited in several ways. Protein inhibitors of caspase-activated deoxyribonucleases (IC) block the degradation of For example, in some embodiments, cells can be transformed by activated GzB. Caspase-activated deoxyribonuclease (CD3) is a novel nucleotide sequence that inhibits degradation of genomic DNA mediated by CD3. Some of the cells may be modified to express a protein inhibitor of catalysis (ICAD). In embodiments, the APC target is engineered to overexpress or increase the activity of ICAD. The mutant ICAD was expressed.

[0078] In some embodiments, ICAD is a mutant form that confers resistance to cleavage by caspases. mutations (e.g., D117E and / or D224E) and otherwise In the literature, these are referred to as caspase-resistant mutants (Sakahira et al., Arch Biochem Biology ofys. 2001 Apr 1;388(1):91-9;Enari et al., Nature. 1998 Jan 1;391(6662):43-50 (See Sakahira et al., Nature. 1998 Jan 1;391(6662):96-9). ICAD precursor An exemplary embodiment of the ATPase inhibitor (also known as DNA fragmentation factor subunit alpha or DFFA) The sequence is from the GenBank accession encoding NP_004392.1 (isoform 1). Nos. NM_004401.2 (transcript variant 1); and NP_998731.1 (A GenBank accession number NM_213566.1 (transcription bank) encoding the nucleotide isoform 2 An exemplary mature ICAD sequence is as follows: Residues D7 and D224 are as follows in the above case:

[0079] [ka]

[0080] Alternatively or additionally, the cells may be engineered to have a CAD knockout (e.g., using CRISPR). Disruption of the CAD gene; an exemplary reference gene sequence is RefSeqGene NG_029 098.1, in the range 5001–17026) or knockdown (e.g., shRN A, using inhibitory nucleic acids such as siRNA, LNA, or antisense Chemical or small molecule DNAse inhibitors, e.g., inhibit proteins that interact with nucleic acids mirin, a cell-permeable inhibitor of MRE11 nuclease, or ethidium bromide Intercalation dyes such as

[0081] Using caspase inhibition, we investigated the cleavage of ICAD and the cleavage of CAD that occurs during apoptosis. Caspase 3 can also block the activation of DFF45 (DNA fragmentation factor-45). / Cleaves ICAD (inhibitor of caspase-activated DNAse) to release the active enzyme CAD This initiates DNA degradation (Wolf et al., J Biol Chem. 1999 Oct 22; 274(43):30651-6). Therefore, cells were treated with caspase-3 knockout (e.g., CRISP Disruption of the caspase 3 gene using R; an exemplary reference gene sequence is available at RefSeqGe ne number NC_000004.12, complement of range 184627696~184649475 in vivo) or knockdown (e.g., shRNA, siRNA, LNA, or Exemplary sequences for human caspase 3 include: The column indicates NP_004337.2 (preproprotein of caspase-3 isoform). The coding sequence is NM_004346.3 (transcript variant 1) in GenBank; Isoforms can also be used. Chemical or small molecule caspase inhibitors can be used. It is also possible to use Z-VAD-FMK (benzyloxycarbonyl-Val-A la-Asp(OMe)-fluoromethyl ketone; Z-DEVD-FMK; Ac-DE VD-CHO;Q-VD-Oph(quinolyl-Val-Asp-OPh);M826(Ha n et al., The Journal of Biological Chemistry (277):30128-30136 (2002));Chu et al. al., J. Med. Chem., 2005, 48 (24), pp 7637-7647. Sulfonamide analogues; as described in Chen et al., J. Med. Chem., 2006, 49 (5), pp 1613-1623 isoquinoline-1,3,4-trione derivatives); and caspase proteins Protein or peptide inhibitors (e.g., mammalian XIAP (GenBank Refseq : NP_001158.2) or Cowpox CrmA (GenBank Ref seq: NP_001158.2). Caspase 3 is an important caspase for this purpose. However, in the absence of caspase 3, CAD is not activated during apoptosis. A report has been published showing that this is the case (Tang et al., J Biol Chem. 1998 Oct 30;273(4 4):28549-52), inhibitors of caspase 3 are exemplified, and other reports have shown inhibitors of other caspases. Therefore, other caspase inhibitors, e.g. For example, pan-caspase inhibitors, or inhibitors of executioner caspases (caspase 6 or 7) or inhibitors of initiator caspases (caspase 2, 8, 9, or 10) In some embodiments, the caspase inhibitors include caspase 3 and Similarly, other caspases, e.g., caspase 6, 7, 2, 8, and / or 9, may be inhibited. This will be the case.

[0082] A variety of methods are available for producing the desired modifications. Typically, vectors are used. The nucleic acid is introduced into the cell using the

[0083] Useful for transferring exogenous genes into target mammalian cells (e.g., by transformation) Many such vectors are available for generating the APCs and libraries described herein. The vectors can be episomal vectors, e.g., plasmids, cytomegaloviruses, and the like. It may be a vector derived from a virus such as a gallovirus or an adenovirus, or For example, vectors derived from retroviruses such as MMLV, HIV-1, and ALV. It may be integrated into the genome of the target cell by recombination or random integration. or lentiviral vectors, such as those based on FIV gag sequences, in human stem cells. It is also possible to transfect non-dividing cells, such as quiescent cells (Uchida et al. (1998) P (See, e.g., NAS95(20): 11939-44). In some embodiments, a retrovirus and an appropriate Suitable packaging cell line combinations may also find use, where capsid proteins are The protein is functional for infecting target cells. Normally, the cells and the virus are incubated in a culture medium. The cells are then incubated at RT for at least about 24 hours. In application, for example, at short intervals of 24 to 73 hours, or at least 2 weeks, the cells are grown in the culture medium. The cells are grown for 5 weeks or longer before analysis. The retroviruses produced are "defective," i.e., lack the viral proteins necessary for productive infection. Replication of the vector requires growth in a packaging cell line. is required.

[0084] Many viral vectors or virus-associated vectors are known in the art. Such vectors can be used to carry nucleic acid constructs into cells. Constructs, including viral and lentiviral vectors, are used to infect or transduce cells. Adenovirus, adeno-associated virus (AAV), or herpes simplex virus It is integrated into the genome of a non-replicating defective virus, such as hepatitis C virus (HSV), and packaged The vector may or may not be integrated into the cell genome. Viral vectors that can be used include, but are not limited to, SIN lentiviral vectors. vectors, retroviral vectors, foamy virus vectors, adeno-associated virus (AA V) vectors, hybrid vectors and / or plasmid transposons (e.g. , Sleeping Beauty transposon system) or integrase-based vectors Other vectors that may be used in connection with alternative embodiments will be apparent to those skilled in the art. It will be clear that

[0085] The construct may also include viral sequences for transfection, if desired. Alternatively, the construct may be expressed in a vector capable of episomal replication, such as EPV and EBV vectors. It may also be incorporated into a vector.

[0086] The inserted material of the vectors described herein is such that expression control sequences are not included in the polynucleotide It may be operably linked to an expression control sequence if it controls and regulates the transcription and translation of the sequence. The term "operably linked" refers to an appropriate initiating site in front of the polynucleotide sequence to be expressed. The polynucleotide has a signal (e.g., ATG) and is under the control of an expression control sequence. Expression of the polynucleotide sequence and production of the desired polypeptide encoded by the polynucleotide sequence. This includes maintaining the correct reading frame to allow for the production of the desired peptide. Transcription of the inserted material controls expression of the recombinant gene in the cell type in which expression is intended. The inserted gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the The material is identical to or contains sequences that control transcription of the naturally occurring form of the protein. It will be understood that the promoters may be under the control of different transcriptional regulatory sequences. The motor sequence is recognized by or introduced into the cell's synthetic machinery, specifically It is required to initiate transcription of certain genes.

[0087] The promoter sequence may be a "tissue-specific promoter," which means that the promoter A selected nucleic acid sequence acts as a promoter, i.e., is operably linked to a promoter. refers to a nucleic acid sequence that regulates the expression of a selected nucleic acid sequence in a particular cell. This term refers to a gene that modulates the expression of a selected nucleic acid primarily in one tissue but not in others. It also encompasses so-called "leaky" promoters that do not cause expression in the same tissue.

[0088] The cell type of APC is not particularly limited. The basic requirement is that APC has a specific target antigen on MHC I. Endogenous processing and presentation of antigens (e.g., expression of proteasomes, TAP transporters) The key to this is the proper functioning of the immune system (expression of MHC molecules, and proper folding and transport of MHC molecules). This is believed to be true for almost all human and other mammalian cells. The system of the present invention utilizes single-chain MHC-peptides that circumvent the need for endogenous antigen processing. It can also be used with fusion proteins (Yu et al., Immunol 168(7): 3145-3149(2002)). However, this requires an increase in library size and the peptides are endogenous. APC may lose information about whether the data is actually reproduced. Methods for transducing vectors using lentiviral / retroviral transduction or other methods are well known in the art. also has the ability to efficiently introduce exogenous DNA into cells by transfection. should be.

[0089] For the IFP-based GzB reporter system described herein, the APC is an IFP protein. The proteins are mature and can fluoresce. strain expresses sufficient levels of biliverdin, an important cofactor for IFP maturation Biliverdin can also be supplemented exogenously (e.g., by increasing the amount of endogenous biliverdin). by increasing expression or by adding biliverdin to the cells), Additional cells can be used. Cells suitable for IFP reporter-based expression are: , well known in the art, including but not limited to, HEK 293T, MelJuso, MD A-MB231, MCF7, and NTERA2. It has been reported in the literature that it also functions in neurons and hepatocytes (Yu et al., Nature Communications 5:3626(2014)).

[0090] For autologous screening, primary dendritic cells and primary B cells can be used If an IFP-based reporter is used, supplemented biliverdin may be necessary. Biliverdin can be extracted as needed using methods well known in the art. The cells may be supplied accordingly.

[0091] In some embodiments, the APCs of the compositions and methods described herein are MHC-deficient. i.e., they do not express endogenous MHC. This prevents the expression of MHC expressed by APCs. The process of T cell responses restricted to specifically selected MHC alleles is engineered to For example, by introducing a single MHC allele, Any response produced will be represented by this one allele, and therefore any further deconvolution Such results can be interpreted without the need for endogenous MHC alleles. If a set of genes exists, it is not easily obtained. This is because T cells from different patients Alternatively, T cells with different MHC alleles can be generated by introducing a new MHC of interest. This also allows for the reuse of the same target cells for profiling cells. This also reduces background killing activity by T cells that recognize other antigens. It was determined herein that the level of MHC expression on target cells is a predictor of T cell activation. By starting with MHC-deficient target cells, signaling It is possible to fine-tune the amount of MHC on the surface to optimize the null noise. In embodiments, K562 cells, HEK293 cells, HEK293 T cells, U2O S cells, MelJuso cells, MDA-MB231 cells, MCF7 cells, NTERA2a Cells, dendritic cells, and primary autologous B cells are used.

[0092] Thus, the compositions and methods described herein are directed to T cells, NK cells, and cellular recognition. The present invention can be applied to any other cell for which proteases are to be delivered. Experiments detailed in the Examples section demonstrate that CD8+ T cell antigens and natural killer cell recognition This demonstrates the feasibility of a method to identify factors that confer CD4+ T cell immunity. Genma can be identified by direct screening of cytotoxic CD4 T cells or by non-toxic TCR derived from CD4 T cells is transferred to cytotoxic CTL (e.g., CD8 T cells) (as much as possible) These can be characterized by their introduction into the host (with co-expression of CD4).

[0093] APC target library For example, in generating a library of candidate antigens to be introduced into MHC target cells, Construction of large, genome-scale libraries of sequences related to the expression of encoded polypeptides. General methods for construction are known to those skilled in the art. Some examples of such methods are Xu GJ, Kula T, Xu Q, Li MZ, the contents of which are incorporated herein by reference in their entirety. Vernon SD, Ndung'u T, et al. Comprehensive serological profiling of human populace ations using a synthetic human virome. Science. 2015;348(6239);Larman HB, Zhao Z, Laserson U, Li MZ, Ciccia A, Gakidis MA, et al. Autoantigen discovery with a synthetic human peptidome Nat Biotechnol. 2011;29(6):535-41. Epub 2011 / 05 / 24. do i: 10.1038 / nbt.1856. pmid:21602805;Zhu J, Larman HB, Gao G, Somwar R, Zhang Z, Laserson U, Ciccia A, Pavlova N, Church G, Zhang W, Kesari S, Elledge SJ. Protei n interaction discovery using parallel analysis of translated ORFs(PLATO).Nat Bi otechnol. 2013 Apr;31(4):331-4. doi: 10.1038 / nbt.2539.

[0094] Also provided herein is a library of APC target cells comprising a plurality of candidate antigens. In some embodiments, the target cells are useful for identifying activated APCs, such as those described herein. In some embodiments, the reporter constructs further comprise one or more reporter constructs for use in In some embodiments, the APC target cells are CA Further examples include inhibitors of D-mediated DNA degradation. Numerous representative examples are described herein. For example, In some embodiments, the target cells are transfected with GzB to inhibit degradation of genomic DNA. Exogenous inhibitors of caspases activated by the caspases or CADs such as those described herein may also be used. For example, in some embodiments, the knockout of GzB is Caspase-activated DNAse (CAD) is activated by caspase-activated deoxyribonuclease (CD). It is inhibited by the inhibitor of ribonuclease (ICAD) or its mutants. In one embodiment, the ICAD mutation is D117E, where the aspartic acid at position 117 In some embodiments, the ICAD has the mutation D224E. Further included are those in which the aspartic acid at position 224 is replaced with glutamic acid. In an embodiment, the isoform of ICAD is identified by GenBank accession number O00273- 2. Other isoforms of ICAD may also be used in the methods described herein. In some embodiments, the compositions and methods may be modified to produce acceptable results. The active inhibitor is wild type.

[0095] In some embodiments, the candidate antigen is encoded by genomic DNA. may be isolated from a subject (e.g., a human) or from an infectious organism, or a combination thereof. In some embodiments, the subject is healthy. In some embodiments, the subject is diseased. In some embodiments, the infectious organism may be, but is not limited to, a bacterium, a virus, a fungus, Pathogens include fungi, protozoa, and multicellular parasites. The plurality of candidate antigens from which the library is generated may be selected from healthy subjects or from diseased subjects (e.g., Diseases including, but not limited to, cancer, autoimmune diseases, cardiovascular diseases, infectious diseases, etc. ) represents a substantially complete set of antigens derived from the genome of a subject having In some embodiments, the plurality of candidate antigens may be from a pathogen or group of pathogens, a virus, a bacterium, or a fungus (e.g., Substantially intact peptides derived from any pathogenic virus, bacterium, or fungus represents a set of

[0096] In some embodiments, the nucleic acid sequence includes, but is not limited to, an open reading frame (ORF) collection. sections, genome-wide peptide libraries, and application-specific custom libraries In some embodiments, a library of antigens such as a gel electrophoresis library of candidate antigens can be used. In some embodiments, genome-wide detection is used. Alignment of the genome (e.g., 90 amino acid fragments with 45 amino acid overlaps) (including 259,345 peptides across the entire human proteome in In some embodiments, the library is a human genome-wide peptide library of The 56 arrays with 28 amino acid overlaps are used to display the viral flora side by side. Proteomes of all viruses annotated in amino acid fragments that infect humans A virus flora-like pattern (containing 93,904 peptides displayed side-by-side) In some embodiments, the library is a CMV proteome-based library. Alignment (e.g., 56 amino acid fragments with 28 amino acid overlap) and aligned across all confirmed and predicted human cytomegalovirus proteins. CMV genome-wide peptide library (containing 5764 peptides) It's Braly.

[0097] Antigens will most likely be encoded by a single copy of DNA. Typically, there are 10 to 1,000 molecules per cell that are produced, processed, and However, even a single peptide on the cell surface may cause cell damage. This system allows for the production of very low replication numbers of virulent proteins that can be productively recognized by host lymphocytes. It is also functional against surface-expressed antigens.

[0098] In various embodiments, the library of target cells containing candidate antigens comprises about 10 2 from about 10 14 The target cells are included.

[0099] In some embodiments, each target cell encodes a unique candidate antigen. In embodiments, target cells contain one or more unique candidate antigens per cell, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 9, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , 85, 90, 95, 100, or more, or any number therebetween, inclusive Any range (e.g., 5-10) of candidate antigens can be encoded. When using antigens with higher background levels, screening may result in higher background levels. In this case, the method involves only one antigen per cell (preferably the one recloned from the first pass). The method may include a step of performing a second screening with the target antigen. .

[0100] In an exemplary embodiment, the library comprises about 1 x 10 2 from about 10 14 target cells, Approximately 1×10 3 from about 10 14 target cells, approximately 1 x 10 4 from about 10 14 target cells, Approximately 1×10 5 from about 10 14 target cells, approximately 1 x 10 6from about 10 14 target cells, Approximately 1×10 7 from about 10 14 target cells, approximately 1 x 10 8 from about 10 14 target cells, Approximately 1×10 9 from about 10 14 target cells, approximately 1 x 10 10 from about 10 14 target cells , about 1×10 11 from about 10 14 target cells, approximately 1 x 10 12 from about 10 14 Individual targets cells, approximately 1 x 10 13 from about 10 14 target cells, or approximately 1 x 10 14 target cells The libraries of target cells described herein include any one or more of: At least about 10 2 from about 10 14 a candidate antigen, wherein a sufficient amount of the target cells are Contains unique candidate antigens for efficient library screening. Between 10 and 10,000 representatives were used, and each candidate antigen was This means that the protein is presented by cells of the

[0101] In various embodiments, each target cell is about 10 2 from about 10 14 Contains candidate antigens of molecules. In an exemplary embodiment, each target cell contains approximately 1 x 10 of the candidate antigen. 2 from about 10 14 Individual copies , approximately 1 x 10 of candidate antigen 3 from about 10 14 Approximately 1 x 10 replicates of candidate antigen 4 from about 10 14 Approximately 1 x 10 replicates of candidate antigen 5 from about 1014 Approximately 1 x 10 replicates of candidate antigen 6 from about 10 14 Approximately 1 x 10 replicates of candidate antigen 7 from about 10 14 Individual replication, candidate antigen Approximately 1 x 10 8 from about 10 14 Approximately 1 x 10 replicates of candidate antigen 9 from about 10 14 Complex of individuals Approximately 1 x 10 of candidate antigens were prepared. 10 from about 10 14 Approximately 1 x 10 replicates of candidate antigen 11 from about 10 14 Approximately 1 x 10 replicates of candidate antigen 12 from about 10 14 100 copies of the candidate antigen 1×10 13 from about 10 14 10 replicates, or approximately 1 x 10 of the candidate antigen 14 Contains duplicates .

[0102] In various embodiments, the candidate antigens are nucleic acid sequences that are about 21 to about 150 nucleotides in length. In a further embodiment, the candidate antigen is encoded by a sequence of about 24 to about 150 nucleotides. nucleotide length, about 30 to about 150 nucleotides long, about 40 to about 150 nucleotides long, about 50 from about 150 nucleotides in length, from about 60 to about 150 nucleotides in length, from about 70 to about 150 nucleotide length, about 80 to about 150 nucleotides, about 90 to about 150 nucleotides long, about 100 to about 150 nucleotides long, about 110 to about 150 nucleotides long, about 1 20 to about 150 nucleotides in length, about 130 to about 150 nucleotides in length, about 140 to about 150 nucleotides in length or encoded by a nucleic acid that is about 150 nucleotides in length In some embodiments, the ORF or nucleic acid encoding the candidate antigen is longer than 150 nt. stomach.

[0103] In some embodiments, the candidate antigens displayed on the surface of the target cell are at least 8, 9, 10 In other embodiments, the candidate antigens are at least 20, or at least 11 amino acids in length. at least 30, at least 40, at least 50, at least 60, at least 70, at least At least 80, at least 90, at least 100, at least 150, at least 200 , at least 250, at least 300, at least 350, at least 400, less Both are 450 amino acids in length or longer. Upon expression, longer antigens (e.g. The target cells are then exposed to short peptides of 8 to 11 amino acids (several hundred amino acids). The audio is then processed.

[0104] In some embodiments, the candidate antigen is a complete ORF (eg, several hundred amino acids in length). Full-length candidate antigens are not necessarily presented in their entirety on the surface of APCs. is expressed by cells and processed endogenously to shorter peptides that are displayed on the cell surface. Then, after identifying APCs that have nucleic acids encoding such long candidate antigens, the identified Various fragments of the selected candidates may then be further screened.

[0105] In various embodiments, the candidate antigen is present in a concentration range of about 1 fM to about 100 μM, about 1 pM to about 100 μM, μM, about 100 nM to about 100 μM, about 1 μM to about 100 μM, about 1 μM to about 10 K of about 1 μM, about 1 pM to about 100 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM d In some embodiments, the candidate antigen binds to lymphocytes at a K d And lymph Bind to the sphere.

[0106] Screening libraries of target cells to identify antigens Candidate antigens are those that bind to MHC class I or class II molecules on cytotoxic lymphocytes. The resulting vectors are expressed in a library of APCs for presentation to the target vectors. , conditions suitable for recognition of any target cells presenting these cognate antigens, such as in the reaction mixture. The target cytotoxic lymphocytes (e.g., CTLs) are mixed under a microscope with the target cytotoxic lymphocytes (e.g., CTLs) under a microscope. The lymphocytes initiate the process of killing the target cell (e.g., CTLs inhibit the activity of serine proteases The target cells release cytotoxic granules containing granzyme B (GzB), which is a cytotoxic agent. Using reporters of the death process initiated by the nuclei (e.g., intracellular GzB activity), Genomic DNA is isolated from the recognized target cells. The antigens that were presented and recognized are then cloned. The nucleic acid to be read is identified (e.g., by PCR amplification and next-generation sequencing).

[0107] Furthermore, candidate antigens for identifying antigens specific to T cells (e.g., CTLs) can be identified. Described herein are methods for screening libraries of target cells comprising: The method includes the steps of: (i) providing a library of target cells as described herein; (ii) contacting the library of target cells with a biological sample containing cytotoxic T cells (CTLs); (iii) isolating target cells bound to the CTLs, (iv) C, wherein binding of the candidate antigen in the target cell results in the desired property; DNA is extracted from isolated target cells containing an antigen that specifically binds to the TL and produces the desired effect. In some embodiments, the method comprises isolating specific CTLs in the sample. Enriching (e.g., via PCR amplification) and identifying suitable candidate antigens In some embodiments, the present invention further comprises a step of detecting the presence of a target gene (e.g., via sequencing). The methods for screening described herein are iterative. Candidate antigens specific for the

[0108] In various embodiments, desirable properties include, but are not limited to, physically detectable a chemically detectable change, a chemically detectable change, an optically detectable change, or a combination thereof In some embodiments, the desired property is target binding. Activity or target binding-induced activity, e.g., catalytic activity or altered catalytic activity Inhibitory activity, activating activity, or alteration of inhibitory or activating activity; Switching activity The structure may be modified to switch the structure or activity; or the activity may be cooperative.

[0109] Identifying recognized APCs In some embodiments, GzB protease activity is used as a marker of recognized APCs. GzB is a cytotoxic agent secreted into APCs recognized by cytotoxic lymphocytes. GzB is a cytotoxic protease that inhibits caspase activity and apoptosis in APCs. Previous studies have shown that GzB released into target cells during cytolytic killing induces Thus, proteolysis of the GzB target is completed, forming a solid matrix that acts as the basis for the reporter. To detect GzB activity, the enzyme activity was demonstrated to be as follows: Molecular reporters of GzB activity, such as Cytotoxic T lymphocytes are typically not activated by the general apoptotic pathway. Other proteases secreted by lymphocytes (granzymes A, K, M) or in target cells Other enzymes or proteins, such as TEV protease engineered into T cells, are secreted into the T cells. Those skilled in the art will appreciate that other markers of recognized APCs, such as ATPases, can be used. will recognize.

[0110] The reporter molecules described herein can be used to indicate increased granzyme B activity. In some embodiments, the method comprises detecting a signal from a detectable label of a reporter molecule. In some embodiments, the method comprises quantifying the signal based on the quantified signal. In some embodiments, the method comprises enriching the target cell population. introducing one or more mutations into one or more candidate antigens having the property; Includes.

[0111] In some embodiments, the method comprises the steps of contacting, isolating and The method may include iteratively repeating one or more of the steps of determining For example, it may include 1, 2, 3, or 4 or more screening steps.

[0112] Reporter of granzyme B activity Additionally, molecular reporters of granzyme B activity, examples of which are described herein, as well as , nucleic acids encoding molecular reporters, and cells containing nucleic acids and / or molecular reporters. Cells (eg, APCs) are provided herein.

[0113] Granzyme B (GzB) is a protease secreted into target cells by CTLs. GzB mediates the cleavage of substrates, including effector caspases and downstream caspase substrates. It cleaves the target protein and induces apoptosis in the target cell.

[0114] In some embodiments, the reporter comprises a GzB cleavage site (e.g., V) linked to a detection molecule. GPD, SEQ ID NO: 1). This term also includes fusion polypeptides containing such a polypeptide of Gzb. As used herein with reference to a reporter, a "detector molecule" refers to a molecule that detects cleavage at the Gzb cleavage site. These are molecules that are released by cleavage and have activities such as enzyme activity, binding activity, or light emission. Once activated by cleavage, the activity can be measured by assays such as those described herein. can be detected (e.g., detection of a detectable label, detection of an enzymatic activity such as CRE). or affinity tag detection). GzB proteolytically cleaves aspartic acid P4 to P1 amino acids with N-terminus, Ile / Val, Glu / Met / Gln, P Preference is given to substrates containing ro / Xaa. Uncharged amino acids are preferred at P1', and P2' Preferably, the GzB cleavage sequence used is Ser, Ala, or Gly. are cleaved by GzB but not by caspases, e.g., V GPD (SEQ ID NO: 1; Choi and Mitchison, PNAS, 110(16): 6488-6493(2013)). In some embodiments, other GzB cleavage sequences, such as those described in Casciola-Rosen et al., Journal of f IETD (SEQ ID NO: 6) described in Biological Chemistry, 282(7):4545-4552(2007) is used.

[0115] Generally, reporters emit a fluorescent signal, e.g., only after GzB-mediated cleavage of the reporter. This results in a detectable signal that is recognized by CTLs and accepts GzB. This makes it possible to isolate the cells.

[0116] In some embodiments, the detection molecule is an infrared fluorescent protein (IFP). In its morphology, IFP is functionally separated into N fragments (N-IFPs) by a GzB cleavage site. and C-fragment (C-IFP), and further contains a green fluorescent protein located on the N-terminal side of C-IFP. The N-fragment of the protein (N-GFP) and the green fluorescent tag located at the C-terminal end of N-IFP were The C-GFP fragment of the protein (C-GFP) is flanked on both sides, resulting in the formation of N-GFP and C-GFP. P forms a constitutively active (fluorescent) molecule, one embodiment of which is shown in Figures 11-13. In particular, Figures 11 and 12 are diagrams illustrating the reporter and the mechanism by which it functions. is the nucleic acid and encoded amino acid sequence of the reporter molecule. The inactive IFP in is itself fused to split GFP (Figure 11). , which is constitutively fluorescent and provides a marker for the presence of the reporter. It acts to stabilize IFP both before and after activation of cleavage. GFP itself is not cleaved and does not respond to the presence of GzB. This inactive IFP is The wild-type IFP was split and N was isolated as described in [PubMed] et al. PNAS 112(11): 3338-3343(2015). The N-terminal half and C-terminal half are inverted, and the N-terminal half and C-terminal half are separated by a linker. The linker was created to allow the two halves of the protein to fold properly. However, upon cleavage of the linker region, I The halves of the FP can come together and mature to produce a protein that is fluorescent.

[0117] In this GzB reporter, the linker between the IFP halves is specifically regulated by GzB. It is cleaved by the cleavage enzyme but replaced with an amino acid sequence that is not cleaved by other proteases. As a result, GzB activity is detected by quantifying the mature IFP signal in each cell. GzB is an external protease delivered by CTLs and is an inactive I The reporter is activated by cleaving the linker that separates the FP protein portions. The GFP in the reporter is not cleaved, resulting in a constitutive fluorescent signal. vinegar.

[0118] The term "functionally separated" is used herein to refer to a GzB cleavage site, This refers to separating portions of a molecule at the cleavage site, thereby inactivating the molecule. In this case, cleavage enhances or restores function (fluorescence). A pair of chromophores that form a RET pair or in which one of the fluorophores is quenched Does not include.

[0119] Allows detection of GzB activity in target cells that are productively recognized by CTLs Several alternative GzB reporters are contemplated that would serve this purpose. Alternatively or in combination with the fluorogenic protease reporters described above, Target cells recognized by the GFP or mCHerr gene can be isolated. A small 16 amino acid peptide (GFP11) derived from y was used to express GFP or mCH The peptide lacking erry can be activated when fused to a protein. where it is inactive, but becomes active when released by cleavage of granzymes. See, e.g., Kamiyama et al., Nat Commun. 2016;7:11046.

[0120] In some embodiments, the molecular reporter comprises an affinity tag (e.g., a flag epitope). Detection is by the reporter GzB, which acts as a substrate for GzB. The affinity tag is based on staining against the antibody target, which is only revealed after cleavage. The tag may be located C-terminal to the GzB cleavage site, such that the tag is not cleaved at the GzB site. It is functional only in the presence of the Flag epitope recognized by the M1 flag antibody (e.g., One embodiment is illustrated in Figure 7A. Prior to cleavage, an internal tag (e.g., Flag epitope) is not recognized by the M1 Flag antibody and is an N-terminal Flag epitope However, after GzB cleavage, the tag is exposed at the N-terminus of the C-terminal cleavage fragment. The cells can be exposed and stained using an appropriate binding partner (eg, M1 antibody). In some embodiments, the molecular reporter further comprises a GFP located proximal to the internal tag. In some embodiments, the GFP is located at the C-terminus of the tag.

[0121] In some embodiments, the molecular reporter is a vesicle having a linker comprising a GzB cleavage site. It is a plasma membrane protein linked to an endothelial cell (ER) retention sequence. The target is retained in the ER. Cleavage of the linker releases the ER retention sequence and the target is released into the plasma. This results in the transport of the protein to the membrane, where it reacts with the extracellularly applied antibody. (or an antigen-binding fragment thereof). APCs expressing the epitope can be isolated or purified. The protein degradation signal is determined by the accessibility of the epitope, e.g., the level of The antigen is converted to the presence of a transporter protein by granzyme cleavage, which then transports the antigen to an antibody or other target. The binding moiety of the antigen becomes accessible to the antigen when the antigen produces or binds to a unique binding site. This is because it changes the location of the molecule (inside the cell or in the protein) so that it can bind to the molecule. Any binding protein that is Pairs of interacting proteins fused to a protein that interferes with the synthesis of the protein can be used. Granzyme cleavage of the blocking segment allows the release of the protein by its binding partner. Instead of antibodies, nucleic acid aptamers can also be used.

[0122] This is achieved by using an affinity reagent (FA) such as a fluorescent antibody against a reporter protein. bound to SC) or by affinity columns (e.g., MACS cell separation Direct capture of GzB-positive cells in the column allows for isolation of GzB-positive cells. As a result, CD4, CD19, CD20, CD40, or other However, other proteins such as Myc tag, Flag tag, HA tag, and histidine tag Any reporter tag not endogenously present on the cell surface, including tagged versions of Proteins can be used. See, e.g., Kimple, ME, Brill, AL, Pasker, RL ( 2013) Overview of Affinity Tags for Protein Purification. Curr Protoc Protein Sci . 73: See Unit-9.9.

[0123] In some embodiments, the detection molecule is an enzyme. Chid (e.g., MGVKVLFALICIAVAEASSGSSGDYKDDDDKPV QPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQ (SEQ ID NO: 7 )) operably linked to an enzyme (e.g., CRE recombinase) such that expression The protein, which is the fusion protein, is then found only at the plasma membrane of the expressing cell. The enzyme and membrane-attached protein are separated by the GzB cleavage site, resulting in The enzyme is then released from the plasma membrane. An example of this is shown in the C Cre recombinase is a protease reporter. It is inactive when tethered to the membrane by the peptide but is activated by GzB cleavage and The Cre gene is released to enter the nucleus, where it activates a reporter of Cre activity. In this state, a reporter in the APC nucleus is expressed using Cre-mediated recombination of a LoxP reporter. In some embodiments, Cre activity is detected via activation of a cellular reporter. In this method, Cre activity in recognized target cells is detected by a reporter gene (G Turn on the cells, e.g., by FACS, or with antibiotics (e.g., FP, puromycin). The selection of the cells allows the isolation of the cells. The genomes derived from these cells are then The DNA can be isolated. The GFP / RFP inversion cassette is expressed in response to Cre activity. 1 is an example of a cellular reporter that emits a fluorescent signal. In some embodiments, a LoxP reporter Recombination of the promoter allows PCR amplification of the antigen cassette in recognized cells. A primer structure is generated. Productively recognized antigens are targeted after treatment with cytotoxic cells. Identification by Illumina sequencing of PCR products derived from target cells This approach is illustrated in Figure 6A. Other enzymes useful for use as exogenous molecules are TEV protease and transcription factors. In some embodiments, the membrane attachment signal peptide is MALPVTALLLPLALL LHAARPSQ (SEQ ID NO: 8).

[0124] Additionally, a system for the detection of granzyme B activity in APCs is described herein. Such a system involves two distinct proteins that interact to exert granzyme B activity. This system preferably utilizes the method of the present invention. A fusion polypeptide containing a CRE recombinase linked to a plasma membrane attachment peptide as described in the specification. The CRE recombinase and membrane-attachment peptide are linked by a GzB cleavage site. The system further comprises a reporter of CRE activity as described herein. The reporter of CRE activity can be expressed in a head-to-head orientation as GFP and and RFP, and may be a nucleic acid sequence flanked on both sides by LoxP sites. The system uses a CRE-activated primer containing an inactive primer flanked on both sides by LoxP sites. a nucleic acid sequence encoding an expressible form of a candidate antigen located proximal to a target antigen recognition sequence; Thus, CRE-induced transposition of the LoxP site generates a functional primer recognition sequence. This CRE-mediated inversion event may occur by PCR and It can be detected directly in genomic DNA by sequencing. In this method, a Cre-mediated inversion event allows PCR amplification of the antigen-presenting cassette. The appropriate primer orientation can be generated. (without any selection) and PCR from this bulk gDNA This allows GzB to be accepted, Cre to be activated, and reverse primers surrounding the antigen-presenting cassette to be inserted. This method is used in qPCR to amplify the antigen-presenting cassette only from target cells that express the antigen. We then quantified the frequency of inversion events in a proof-of-concept experiment in Figure 6. These two methods are They can be used separately or in combination.

[0125] sign Suitable detector molecules that can be incorporated into the reporter molecules described herein include, but are not limited to: However, radioisotopes, fluorophores, chemiluminescent materials, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzymes Examples of suitable inhibitors include enzyme inhibitors, dyes, metal ions, and metal sols.

[0126] Any fluorescent polypeptide (also referred to herein as a fluorescent label) may be used to detect Suitable fluorescent polypeptides may be suitable for use as labels. A detectable signal can be assessed (e.g., serotonin) and quantitatively (equivalent to fluorescence). It would be something that would be easily brought about.

[0127] Exemplary fluorescent polypeptides include, but are not limited to, yellow fluorescent protein (YF P), cyan fluorescent protein (CFP), GFP, mRFP, RFP (t dimer 2), H CRED, or any mutant thereof (e.g., enhanced fluorescence or a modified emission spectrum) Fluorescent proteins (fluorescent proteins modified to produce a shift in the fluorescence intensity), analogs, or derivatives are included. Further preferred fluorescent polypeptides, and specific examples of those listed herein, include: They are provided and well known in the art.

[0128] Biotin-based labels also find use in the methods disclosed herein. Biotinylation of molecules is well known, e.g., biotinylation of proteins, nucleic acids, carbohydrates, and carboxylic acids. A number of biotinylation agents are known, including amine-reactive and thiol-reactive agents for biotinylation. see, for example, chapter 4, Molecules, which is hereby incorporated by reference. See the Probes Catalog, Haugland, 6th Ed. 1996. Biotinylated materials are a detectably labeled biotin binding partner, such as avidin or streptavidin; Similarly, numerous heptenylation reagents are known. do.

[0129] Exemplary affinity tags suitable for use include, but are not limited to, monocyte Adaptor protein (MONA) binding peptide, T7 binding peptide, streptavidin Protein A (Nilsson et al., EMBO J. 4: 1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione S-tran Spherase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952(1985)), substance P , FLAG peptide (Hopp et al., Biotechnology 6: 1204 (1988)), or other antigenic enzymes. Generally, the term "protein" refers to a protein or a binding domain. See, for example, J. Am. Chem. Soc. and Purification 2:95 (1991). DNA encoding an affinity tag Molecules were obtained from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, Available from the University of New York, NJ.

[0130] Identification of antigens on activated APCs Furthermore, the detection of recognized antigen presentation by antigen-presenting cells to cytotoxic lymphocytes A system for this purpose is provided herein. The system comprises an exogenous gene encoding a candidate antigen. The candidate antigen comprises an antigen-presenting cell or a plurality of antigen-presenting cells containing nucleic acid, As described in the document, MHC class I and / or M The system is expressed and presented by HC class II molecules. or a molecular reporter of granzyme B activity as described herein. In some embodiments, the system further comprises: In some embodiments, the cytotoxic lymphocytes described herein are further included. The antigen-presenting cells of the system express an inhibitor of CAD-mediated DNA degradation, e.g., an expressed form of ICA It further contains the D gene.

[0131] As described herein, presented on target APCs recognized by cytotoxic lymphocytes Improved productive antigen recognition leads to recognizable changes within the APC. The detection is used in the identification of the APC and the ultimate determination of the antigen it expresses. Identification of the target cell and its antigen is achieved by detecting the reporter, thereby identifying the target cell. This is achieved by using high-throughput systems to isolate and / or sort identified cells. It can be done.

[0132] The isolating and / or selecting steps described herein are within the skill of the art. Various methods and / or devices known in the art, such as flow cytometry (e.g. , fluorescence-activated cell sorting (FACS) or Raman flow cytometry), fluorescence microscopy, Optical tweezers, micropipettes, affinity purification and microfluidic magnetic separation devices In some embodiments, the method can be performed using detectably labeled When the target cells are fluorescently labeled target cells, FASC can be utilized to identify one or more Cells can be qualitatively sorted based on the fluorescent signal. When target cells containing complementary antigens specifically bind to their cognate T cells, the target cells are transformed into red An extracellular fluorescent signal (e.g., an activated IFP-GFP fusion protein encoded by the target cell) Using FACS, the cells are analyzed based on infrared fluorescent signals. One or more sorting gates or The present invention utilizes threshold levels in conjunction with one or more detection molecules to detect a wide range of target cells - T cells. Furthermore, the stringency of the screening may be varied to provide a quantitative selection for cell interactions. The facility can be used, for example, to modulate the concentration of the target and set the position of the sorting gate. Therefore, quantitative control is possible.

[0133] For example, the fluorescent signal may be used to identify the target antigen of a candidate to a cytotoxic lymphocyte (e.g., CTL). Selection gates and / or stringency conditions, if related to binding affinity to produce a compound with a desired affinity or a desired affinity range for the target. In some cases, the antigen with the highest affinity may be selected as the candidate antigen. In some cases it may be desirable to isolate a specific library from a series. In this case, candidate antigens with binding affinities within a particular range may be isolated.

[0134] treating cells identified as having the recognized antigen to isolate exogenous nucleic acid; In some embodiments, the exogenous nucleic acid can be a known primer sequence (e.g., a primer sequence for the nucleic acid). The vectors are isolated by PCR amplification using a recombinant vector (known from transfection into APCs). Alternatively, RT-PCR can be used to amplify the transcribed form of the antigen cassette. When the antigen is expressed episomally (as part of the viral genome or a plasmid) , episomal DNA can be captured as a method to isolate antigen-presenting cassettes. Determination of specifically recognized antigens can be achieved using high-throughput systems such as DNA sequencing. This can be achieved by using a system.

[0135] Several DNA sequencing techniques, including fluorescence-based sequencing methodologies are known (e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor Laboratory Press, 2004). (See, eg, Wood Harbor, NY). In some embodiments, automated systems, as understood in the art, are used. In some embodiments, high throughput sequencing techniques as described herein are utilized. The RT system allows for parallel sequencing of split amplicons (e.g., PCT International Publication No. In some embodiments, the method is used to provide the DNA sequencing is performed using parallel oligonucleotide extension (see, e.g., U.S. Patent No. 5, (See U.S. Patent Nos. 750,341 and 6,306,597) An additional example of a sequencing technique is the Church Polony technique ( Mitra et al.,2003,Analytical Biochemistry 320,55-65;Shendure et al.,2005 Scienc e 309,1728-1732; U.S. Patent No. 6,432,360; U.S. Patent No. 6,485,9 44 and U.S. Pat. No. 6,511,803), 454 picotitrpi sequencing technology (Margulies et al., 2005 Nature 437, 376-380; U.S. Patent Application Publication No. 2005 / 0130173), Solexa single base addition technology (Bennett et al. l., 2005, Pharmacogenomics, 6,373-382; U.S. Patent No. 6,787,308; U.S. Patent No. No. 6,833,246), Lynx Massively Parallel Signature Sequencing technology (Brenner et al., (2000). Nat. Biotechnol. 18:630-634; U.S. Patent No. 5,695,9 34; U.S. Patent No. 5,714,330), and Adessi PCR Co. Ronnie technique (Adessi et al. (2000). Nucleic Acids Res. 28, E87; WO 000189 (See pamphlet no. 57).

[0136] Next-generation sequencing (NGS) methods are less costly than older sequencing methods. They share a common feature of massively parallel, high-throughput strategies aimed at achieving high throughput (e.g., Voelkerding et al.,Clinical Chem.,55:641-658,2009;MacLean et al.,Nature Rev.Mic (See, e.g., J. Biol., 7:287-296). NGS methods typically use template amplification. Methods that require amplification can be broadly divided into those that do and those that do not. , pyrosequencing, which is commercially available as a 454 technology platform from Roche ings (e.g., GS 20 and GS FLX), commercially available from ILLUMINA™ The Solexa platform, which is supported by APPLIED BIOSYSTEM Supported Oligonucleotides commercially available from S Ligation and Detection™ (SOLiD) Platform Non-amplification techniques, also known as single molecule sequencing, include HE HELISCOPE™, marketed by LICOS BIOSYSTEMS™ Platform, and VISIGEN™, OXFORD NANOPORE™ ECHNOLOGIES LTD., and PACIFIC BIOSCIENCES ( These are exemplified by the latest platforms commercially available under the trademarks (Trademarks).

[0137] Pyrosequencing (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLe an et al., Nature Rev. Microbial., 7:287-296; U.S. Pat. No. 6,210,891 No. 6,258,568), template DNA is fragmented and The ends are repaired, ligated to an adapter, and an oligonucleotide complementary to the adapter is inserted. In situ capture of a single template molecule on a bead carrying a Each bead carrying a single template type is clonally amplified. The template is then compartmentalized into a vesicle and transfected using a technique called emulsion PCR. The emulsion is broken after amplification and the beads are then subjected to sequencing. The elution reaction is carried out in individual wells of a picotiter plate, which acts as a flow cell during the reaction. The ordered and repeated introduction of each of the four dNTP reagents results in the sequencing enzyme and The appropriate dNT is introduced into a flow cell in the presence of a light-emitting reporter such as luciferase. In the event that P is added to the 3' end of the sequencing primer, the resulting ATP production The emission causes a disruption of luminescence within the well, which is recorded using a CCD camera. Read lengths of greater than or equal to 400 bases can be achieved, with 10 6 sequence reads can be achieved, resulting in up to 500 million base pairs (Mb) of sequence.

[0138] SOLEXA / ILLUMINA platform (Voelkerding et al., Clinical Ch em., 55:641-658, 2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S. Patent No. Nos. 6,833,246; 7,115,400; and 6, In the case of 969,488, sequencing data is collected in the form of shorter read lengths. In this method, single-stranded fragmented DNA is end-repaired and 5'-phosphorylated. A single A base is then added to the 3' end of the fragment in a Klenow-mediated manner to generate blunt ends. A-addition is the process of adding template to the surface of the flow cell to be studded with oligonucleotide anchors. T-overhang adaptor used subsequently to capture the rate adaptor molecule The anchor facilitates the addition of oligonucleotides used as PCR primers. However, due to the length of the template and its proximity to other nearby anchor oligonucleotides, PCR extension leads to the "arching" of the molecule, with adjacent anchor oligonucleotides These DNA fragments hybridize with the DNA fragments and form a bridge structure on the surface of the flow cell. The loop is denatured and cleaved. The forward strand is then degraded using a reversible dye terminator. The sequence of the incorporated nucleotides is determined by the sequence of the next nucleotide after the addition of dNTP. The fluorescence after incorporation can be detected with the fluorophore and block removed before the crosslink. The sequence read length is determined by the overall length of the sequence reads, which exceeds 1 billion base pairs per analysis run. The output ranges from 36 nucleotides to over 50 nucleotides.

[0139] SOLID(TM) technology (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacL ean et al., Nature Rev. Microbial., 7:287-296; US Pat. No. 5,912,148 Sequencing of nucleic acid molecules using chromatographic techniques (e.g., U.S. Pat. No. 6,130,073) is also possible. In addition, the fragmentation of the template, ligation of oligonucleotide adapters, and binding to beads This is involved in the attachment of the genus to the host and the clonal amplification by emulsion PCR. The bead-bearing plate is immobilized on the derivatized surface of a glass flow cell and attached to an adapter A primer complementary to the oligonucleotide is annealed. Rather than utilizing the primer for 3' extension, the primer is used to insert two probe-specific bases followed by The interrogation probe contains six degenerate bases and one of four fluorescent labels. Instead, it is used to provide a 5' phosphate group for ligation to the nucleotide. In the LID™ system, the interrogation probes are located at the 3' end of each probe. It has 16 possible combinations of two bases at the 5′ end and one of four fluorophores at the 5′ end. The color of the fluorophore, and therefore the identity of each probe, corresponds to a particular color-space coding scheme. After multiple (usually seven) rounds of probe annealing, ligation, and fluorophore detection , denaturation, and then adding a primer that is offset by one base relative to the initial primer. In this method, the template sequence is used for a second round of sequencing. The template bases are interrogated twice to form a highly The sequence read length is an average of 35 nucleotides, and the entire output The put exceeds 4 billion bases per sequencing run.

[0140] In certain embodiments, nanopore sequencing (see, e.g., Astier et al., J. Am. C hem.Soc.2006 Feb 8;128(5)1705-10) is used. The theory behind this is that a nanopore is immersed in a conductive fluid and a potential (voltage) is applied to it. Under these conditions, the flow of ions through the nanopore A small current due to conduction can be observed, and the amount of current is very sensitive to the size of the nanopore. As each base of the nucleic acid passes through the nanopore, each of the four bases is connected to a separate nanopore. A change occurs in the magnitude of the current passing through the pore, thereby determining the position of the DNA molecule to be determined. This makes it possible to perform a query.

[0141] In certain embodiments, H by HELICOS BIOSCIENCES™ ELISCOPE(TM)(Voelkerding et al.,Clinical Chem.,55:641-658,2009;Mac Lean et al., Nature Rev. Microbial., 7:287-296; U.S. Patent No. 7,169,560 No. 7,282,337; U.S. Patent No. 7,482,120; U.S. Patent No. 7,501,245; U.S. Patent No. 6,818,395; U.S. Patent No. Patent Nos. 6,911,345; 7,501,245) are used. The template DNA is fragmented and the final adenosine carrying the fluorescent label is The denatured polyadenylated template fragment is polyadenylated at the 3' end. The captured nucleotides are ligated to poly(dT) oligonucleotides on the surface of the antibody cell. The initial physical location of the template molecule is recorded by a CCD camera, and then the label is The DNA is cleaved and washed away. Sequencing is performed using polymerase and fluorescently labeled DNA fragments. The incorporation event is achieved by sequential addition of dNTP reagents. The signal was captured by a CCD camera before each dNTP addition. Sequence read lengths were 2.5 times higher with an overall output of over 1 billion base pairs per analytical run. It ranges from 5 to 50 nucleotides.

[0142] Ion Torrent technology is based on the detection of hydrogen ions released during DNA polymerization. It is a DNA sequencing method based on the method (e.g., Science 327(5970):1190(2010); US Patent Application Publication No. 2009 / 0026082; Patent Application Publication No. 2009 / 0127589 Specifications; Specification No. 2010 / 0301398; Specification No. 2010 / 0197507 ; Specification No. 2010 / 0188073; and Specification No. 2010 / 0137143 (See the manual.) The microwells contain the template DNA strands to be sequenced. Beneath the layer of microwells is a hypersensitive ISFET ion sensor. All layers are made of CMOS semiconductor chips similar to those used in the electronics industry. When a dNTP is incorporated into the growing complementary strand, a hydrogen ion is released The homopolymer repeats in the template sequence, triggering a hypersensitive ion sensor. When present, multiple dNTP molecules are incorporated during a single cycle. The corresponding number of hydrogens added leads to a proportionally higher electron signal. It differs from other sequencing technologies in that it does not use nucleotides or optical components The Ion Torrent sequencer has a per-base precision of approximately 100 Mb per run. The yield of 50-base reads is approximately 99.6%. The read length is 100 base pairs. The accuracy is approximately 98% for homopolymer repeats of length 5.

[0143] Another exemplary nucleic acid sequence that can be employed for use in connection with the methods described herein is The sequencing method was developed by STRATOS GENOMIC, Inc. This sequencing process involves the use of XPANDOMERS™. Typically, this involves providing a daughter strand produced by template-directed synthesis. Generally, each subunit comprises a tether, at least one probe, or a nucleic acid base. residue, and at least one selectively cleavable bond, It contains multiple subunits linked in a sequence corresponding to a portion of a contiguous nucleotide sequence. The selectively scissile bond is cleaved to produce XPA chains longer than the subunits of the daughter strand. XPANDOMER™ is typically used to obtain the target nucleic acid. Analyzing the genetic information in a sequence corresponding to all or part of a contiguous nucleotide sequence The XPANDOMER (trademark) ) reporter element is detected. Further details are found in, for example, "High Speed ​​by Extension," filed June 19, 2008. High Throughput Nucleic Acid Sequencing Published U.S. patent application entitled "CID SEQUENCING BY EXPANSION" No. 2009 / 0035777, which is incorporated by reference in its entirety. and is incorporated herein by reference.

[0144] Another state-of-the-art single molecule sequencing method is the VISIGEN™ platform (Voelkerding et al., Clinical Chem., 55:641-58, 2009; U.S. Patent No. 7,329,492 No.; U.S. Patent Application No. 11 / 671,956; U.S. Patent Application Publication No. 11 / 78 1166), including real-time sequencing by synthesis using VIS In the IGEN platform, fixed, primed DNA templates are visualized by fluorescent The modified polymerase is subjected to chain extension using a fluorescent acceptor molecule to form a base pair. Upon addition, detectable fluorescence resonance energy transfer (FRET) occurs.

[0145] Unless otherwise defined herein, the chemical and Allen et al., Remington: The Science and Practice of Pharmacy 22 nd ed., Pharmaceutical Press (September 15, 2012);Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biolo gy 3 rded., revised ed., J.Wiley & Sons (New York, NY 2006);Smith, March's Adva nced Organic Chemistry Reactions, Mechanisms and Structure 7 th ed., J.Wiley & So ns (New York, NY 2013);Singleton, Dictionary of DNA and Genome Technology 3 rd ed ., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning ing: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spr. ng Harbor, NY 2012) provides those of skill in the art with a general guide to many of the terms used in this application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2 nd ed., Cold Spring Harbor Press (Cold Spring Harbor rbor NY, 2013);Kohler and Milstein, Derivation of specific antibody-producing t issue culture and tumor lines by cell fusion, Eur.J.Immunol.1976 Jul, 6(7):511-9 Queen and Selick, Humanized Immunoglobulins, U.S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy , Nature 1988 Mar 24, 332(6162):323-7. Furthermore, where the context requires otherwise, Unless required otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0146] Other than in the operating examples, or unless otherwise indicated, the ingredients or All numbers expressing quantities regarding or reaction conditions are in all instances modified by the term "about." It should be understood that the term "about" is used to describe the present invention. In the case of percentages, this means ±5%.

[0147] In one aspect, the compositions, methods, and components thereof described herein are essential. and is open to the inclusion of elements not specified, whether required or not ( In some embodiments, the composition, method, or each thereof Other elements included in the description of the constituent elements must be materially related to its basic and novel characteristics. Consisting essentially of (consisting of) (essentially of)). This refers to the steps in the described method and the This applies equally to compositions and components. In some embodiments, the compositions described herein The product, method, and each component thereof are essential to the component, composition, or method. It is intended to be exclusive of any element not considered to be ("consisting of"). ting of)").

[0148] All patents, patent applications, and publications identified herein are incorporated by reference, for example, as if fully set forth in their entirety, and are not to be construed as limiting the scope of the present invention. The present application is hereby incorporated by reference to describe and disclose the methodology described in such publications which may be used. These publications are hereby expressly incorporated herein by reference in their entirety for their disclosure prior to the filing date of this application. The present invention is provided for illustrative purposes only. Nothing in this respect is to be construed as a substitute for prior invention or for any As an admission that the inventors are not otherwise entitled to antedate such disclosure. Any statements as to the date or content of these documents should not be construed as implied. All statements in this application are based on the information available in this application and are as of the date of these documents. It does not constitute any admission as to the correctness of the content.

[0149] The present invention is further illustrated by the following examples, which should not be construed as further limiting. should not be interpreted. [Example]

[0150] material and method The following materials and methods were used in the examples herein. Preparation of T cells Clonal T cells that recognize the IV9 epitope (amino acids 309-31 of HIV Pol) 7, ILKEPVHGV) kindly provided by Bruce Walker. The cells were cultured in 10% FBS (Gibco), 1% penicillin-streptomycin (Gibco o) and cultured in RPMI containing 50 U / ml human recombinant IL-2 (Roche). In the presence of anti-CD3 (OKT3, 0.1 μg / ml), the cells were irradiated with 20E6 (50 Gy) T cells were expanded by culturing 1E6 T cells with allogeneic PMBCs. I did.

[0151] For TCR regeneration, the RosetteSep CD8 T cell purification kit (Stem Primary CD8 T cells were purified from donor blood using a ELISA kit (Diagnostic Cell). The cells were activated using anti-CD3 / anti-CD28 magnetic beads (Invitrogen) At the same time, a gene encoding the TCR of interest and a Zesty Green (Zsg) fluorescent marker was inserted. The transduced cells were transduced with an antiviral vector. Five days later, the Zsg signal was detected. FACS (BD FACSAria™ I) based on the FITC channel I) was selected.

[0152] NK cells were purified using the RosetteSep NK cell purification kit (StemCell). The antibody was purified from donor blood using IL-16 as a marker and added to RPMI and 10% FBS at 100 U / ml. Activated in 2 for 24 hours.

[0153] Cytotoxicity assay Hmy2.CIR-HLA-A2 target cells were cultured using CFS according to the manufacturer's protocol. E (Invitrogen) and 10 μg / ml of IV9 peptide (NeoBio Cells were pulsed with PB (NeoBioLab) or control peptide (NeoBioLab) for 1 hour. The cells were washed three times with S. 5E4 target cells were seeded per well of a 96-well plate. Mix with 10-fold excess IV9 T cells, spin down at 300g for 2 minutes, and incubate at 37°C for 4 minutes. The cells were resuspended by pipetting and diluted 1:20 The cells were incubated with diluted 7-AAD (BD Biosciences) for 10 minutes. Target cells were identified by CFSE staining (FITC), and dead cells were stained with PerCP-Cy5.5. LDH assay was performed on a BD FACSAria™ II channel. After 4 hours of incubation, the supernatant was collected and analyzed according to the manufacturer's protocol (Pierre et al., 2011). ce).

[0154] Fluorogenic GzB reporter The fluorogenic GzB reporter was expressed in the iTEV-HO1 vector (To et al. PNAS 112(11): The TEV cleavage site in the GzB cleavage sequence (VGPDFGR (sequence Choi, PJ and Mitchison, TJ (2013)PN AS 110(15): 6488-6493). The new reporter was analyzed using pHAGE TRex hygromycin. Cloned into a lentiviral expression vector and inoculated into HEK293T cells at an MOI of approximately 1. The cells were selected with 200 μg / ml hygromycin for 4 days. Target cells were distinguished from T cells based on GFP signal, and reporter activation was confirmed by AP Increased fluorescence in the C-Cy7 channel (BD FACSAri™ a II) It was detected.

[0155] Cre reporter of GzB activity Signal peptide (MALPVTALLLPLALLLHAARPSQ (SEQ ID NO: 8) ), flag tag (DYKDDDDK (SEQ ID NO: 10)), CD8 transmembrane domain, Gz B cleavage site (VGPDFGR (SEQ ID NO: 9)), and Cre recombinase A construct encoding a membrane-tethered Cre fusion was generated. This construct was synthesized (IDT) and expressed in p Cloned into the ENTR vector (ThermoFisher) and then pHAGE CMV hygro destination vector of virus The resulting vector was cloned and introduced into K562 target cells by lentiviral transduction. As a sex reporter, it contains GFP and RFP in a head-to-head orientation and is transfected with loxP. We used a vector flanked by GFP-activating sites, which allows for activation of GFP and loss of RFP. A reporter cassette for the presence of Cre, allowing fluorescent detection of Cre activity by Furthermore, qPCR primers (illustrated in Figure 14 and sequences shown in Figure 15) were designed, This reporter cassette enabled the detection of the same inversion event by qPCR rather than by fluorescence detection. The kit was cloned into the pHAGE CMV lentiviral vector and the lentivirus was Transduction of reporter cells (200ug / ml hygromycin selection for 4 days) Finally, the caspase-resistant D117E ICAD gene was introduced into the cells using lentivirus. Transduction of the target cells with 40 μg / ml of blasticidin selection for 5 days Target cells were mixed with a 2:1 excess of activated primary NK cells for 4 hours. GeneJE Genomic DNA was purified using a T™ purification kit and the Cre reporter inversion was Quantification of PCR amplification products by qPCR using inversion-specific primers The inversion cassette contains, among other things, RFP and GFP for fluorescent detection of Cre activity. Although the signal was contained in the chromatogram itself, no fluorescent signal was used in this experiment. Regardless, the reporter cassette was normalized to the set of quantified primers.

[0156] Antibody-based GzB reporter HA tag (YPYDVPDYA (SEQ ID NO: 11)), GzB cleavage site (VGPD (SEQ ID NO: 12)), No. 1), a fusion of a Flag tag (DYKDDDDK (SEQ ID NO: 10)), and GFP Constructs encoding the vectors were synthesized (IDT) and inserted into the pENTR vector (ThermoFish er) and then cloned into pHAGE T by Gateway cloning. The expression vector was cloned into the rex neo expression vector. or control empty vector transfected and then co-cultured with primary NK cells for 2 h Cell lysates were collected and run on a 4-12% Bis-Tris gel, and the gel was probed with M1 anti-Flag antibody. The resultant was blotted with PBS (Sigma Aldrich).

[0157] CD4 TCR testing The alpha and beta chains of the OB1a.12 TCR (MB) separated by the P2A sequence P peptide) was cloned into the pHAGE EF1a-PGK-Zsg vector. As a control, the alpha and beta chains of the TCR (Ko) targeting the IV9 peptide were lowo, W. et al. (1999) Journal of Immunology, 162:7525-7533) were cloned into the same vector. The cells were then stimulated again with anti-CD3 / anti-CD28 magnetic beads (Invitrogen). Primary CD8 T cells were infected with lentivirus expressing OB1a.12 or control TCR. After 5 days, Zsg-positive T cells were sorted by FACS. 12 or control T cells were cultured in HEK293T cells expressing the fluorescent GzB reporter alone. Single-chain MHC1 with MBP peptide or mutants of MBP peptide Activation of the GzB reporter was detected by FACS after 4 hours. Ta.

[0158] Mixing Experiment A 56 amino acid fragment containing the IV9 epitope (GAKALTDIVPLTREAE LELAENKEILKEPVHGVYYDSAKELIAEVQKQGLDQWTYQ ; SEQ ID NO: 12) was cloned into the pHAGE CMV Purolentiviral expression vector HEK293T cells expressing a fluorogenic GzB reporter were transfected with lentivirus The mice were transduced with either IV9 or a control lentivirus (MO The cells were transduced with approximately 1 μg of puromycin and selected for 3 days with 1 μg / ml of puromycin. Cells expressing the Ce gene were transfected according to the manufacturer's protocol (Invitrogen). Cells were labeled with llTrace™ violet cell dye and compared with unlabeled control cells at various ratios. The mixture was mixed and seeded into a 96-well plate. After 12 hours of growth, the mixture was diluted with IV9 at a 10:1 ratio. T cells were added and the cells were co-cultured for 4 hours. The cells were suspended and analyzed for GFP, APC-Cy7, and DAPI (violet dye). Analysis was performed by flow cytometry (BD FACSAria™ II).

[0159] IV9 T cell screening A 56 amino acid fragment aligned across the complete proteomes of 10 HIV strains. A library of 2494 oligos encoding fragments was synthesized (Agilent). The nucleotide sequence was amplified using the following primers: HIV_lib_F 5' ggggacaagtttgtacaaaaaagcagg ctcaAGAATTCTCCGTGGC (SEQ ID NO: 13) HIV_lib_R 5' ggggaccactttgtacaagaaagctgg gtcagctagttaCACTCGAGAGCTCAC (SEQ ID NO: 14) The sections in capital letters represent our antibodies. The region directly complementary to the original cassette is indicated (lowercase letters indicate regions added by PCR). The library was then purified using LR clonase to pHAG E CMV N-FlagHA IRES puro destination vector Two replicates of 30E6 target cells (GzB reporter and ICAD) were cloned into HEK293T cells expressing HIV-1 were transduced with an HIV peptide library at an MOI of approximately 0.2. The cells were transfected with 3E6 target cells from each replicate and selected with 1 μg / ml puromycin for 3 days. The cells were co-cultured with 10E6 activated IV9 T cells for 12 hours. Resuspend the GzB reporter-activating target cells by FACS. Genomic DNA was selected using the GeneJET™ kit (Thermo). The peptide cassettes were purified from the sorted cells and the 3E6 pre-sorted control. Amplify using primer: T_sell_PCR1_F 5' CCAGTCAGGTGTTGATGCTCGGGG ATCCAGGAATTCAGTTTGTACAAAAAAGCAGGCTCA (Sequence No. No. 15);T_cell_PCR1_R 5' CGAGCTTATCGTCGTCAT CCCCACTTTGTACAAGAAAGCTGGGTCA (SEQ ID NO: 16), 1 ul The PCR1 product was analyzed as previously described (Xu, et al., (2015) Science, 348(6239), a aa0698), which was used as a template for two library prep PCRs. The samples were pooled, gel extracted, and subjected to single-end 300bp sequencing on an Illumina MiSeq. The reads were aligned using BWA and the input The abundance of each peptide in the sorted population relative to its frequency was calculated.

[0160] CMV sub-library screening This screen was performed as the IV9 T cell screen described above with the following modifications: Briefly, NLV2 TCR (Schub et al., J Immunol, 183:6819-6830 (2009) ) was synthesized as a gBlock fragment (IDT) and used in pHAGE EF1a Zsg DE The cloned vector was then packaged into a lentivirus and transfected with the IgG. The complete protein of the Merlin strain of CMV was then transduced into CD8 T cells. A library of 5,784 oligos encoding the ohmics was then prepared on a releasable microarray (T The CMV NFlagHA was synthesized by Wist Biosciences and pHAGE Cloned into puro DEST lentiviral vector and packaged into lentivirus The cells were then cultured at an MOI of approximately 0.5 and selected with 1 μg / ml puromycin for 3 days. ), HLA-A2 target cells (MHC Null HEK293T / dmICAD-bs d / iGzB-hyg / HLA-A2).

[0161] Three replicates of 10E6 CMV target cells were co-incubated with 50E6 NLV2 T cells for 12 hours. After culturing, IFP-positive target cells were sorted (FACSAria™ II). The sorted cells were spun down at 500g for 5 minutes and the gDNA was extracted using GeneJET (commercially available). The DNA was extracted using a genomic DNA purification kit (Thermo). Adapter and multiplex index were used for the above IV9 T cell screening. The samples were analyzed using an Illumina MiS PCR kit. The reads were then subjected to high-throughput sequencing using BWA. and calculate the abundance of each peptide in the selected population versus the input frequency. did.

[0162] Microbiota-wide screening This screen was performed as the IV9 T cell screen described above with the following modifications: Briefly, pp65-specific primary T cells were kindly provided by Kim Lyerly. The VirScan library (Xu et al. Science, 348(6239), aaa0698( 2015)) pHAGE CMV NFlagHA puro DEST lentivirus Cloned into vector and packaged into lentivirus at an MOI of approximately 0.5 (selected with 1 μg / ml puromycin for 3 days), and HLA-A2 target cells (MH C Null HEK293T / dmICAD-bsd / iGzB-hyg / HLA-A 2) was transduced.

[0163] Four replicates of 120E6 virus-biota target cells were co-cultured with 120E6 T cells for 12 hours. The cells were cultured and then IFP-positive target cells were selected (FacsAria™ II). The separated cells were spun down at 500g for 5 minutes, and the gDNA was extracted using GeneJET (trademark ) Genomic DNA was extracted using a genomic DNA purification kit (Thermo). The PIP and multiplex index were compared with the IV9 T cell screening described above. The samples were run in triplicate using an Illumina MiSe PCR kit as described previously. The reads were subjected to high-throughput sequencing using BWA. and calculate the abundance of each peptide in the selected population relative to the input frequency. Ta.

[0164] CMV library-pair library screening This screen is a modified version of the CMV sublibrary screen described above with the following modifications: Memory T cells were isolated from PBMCs of donor no. 224 (starting cells of 76E6). , Astarte Biologicals) and grown as described above.

[0165] Four replicates of 30E6 CMV target cells were co-cultured with 25E6 T cells for 8 hours. Afterwards, IFP-positive target cells were sorted (FacsAria™ II) and processed as described above. I understood.

[0166] Tiling mutagenesis screen This screen was performed as the IV9 T cell screen described above with the following modifications: Briefly, pp65-specific primary T cells were kindly provided by Kim Lyerly. The four T cell epitopes (pp65 epitope: NLVPMVAT) were provided. 3,376 oligos (C, V) encoding a complete set of single amino acid mutants The library (TL_mut library) was then transferred onto a releasable microarray (Twis The CMV NFlagHA purified protein was synthesized at NIH Biosciences and analyzed by pHAGE. Cloned into the DEST lentiviral vector and packaged into lentivirus and selected with 1 μg / ml puromycin for 3 days at an MOI of approximately 0.2. HLA-A2 target cells (MHC Null HEK293T / dmICAD-bsd / i GzB-hyg / HLA-A2).

[0167] Three replicates of 25E6 CTL_mut target cells were co-cultured with 25E6 T cells for 12 hours. The cells were cultured, and then IFP-positive target cells were sorted (FacsAria™ II) and It was processed as described above.

[0168] Two rounds of selection screening This screen was performed as the IV9 T cell screen described above with the following modifications: Briefly, IV9-specific "HA" TCR (Kolowos et al., J Immunol 162:7525 -7533 1999) was synthesized as a gBlock fragment (IDT) and used in pHAGE EF1a Z Cloned into sgDEST lentiviral vector and packaged into lentivirus The antibodies were then transduced into primary CD8 T cells.

[0169] Three replicates of 5E6 target cells were co-cultured with 40E6 “HA” T cells for 10 hours. IFP-positive target cells were then sorted (FacsAria™ II) and analyzed as described above. was processed.

[0170] To perform the second round of selection, PCs derived from each of the three screening replicates were The R1 product was then back-bagged into the pHAGE CMV NFlagHA puro DEST vector. The vector was cloned, packaged into lentivirus, and transfected with HL at an MOI of approximately 0.2. A-A2 target cells were transduced. Each of the three recloned libraries was generated. One copy of the 5E6 target cells expressing the 5E6 gene was co-cultured with 25E6 "HA" T cells for 10 hours. IFP-positive target cells were then sorted (FacsAria™ II) and analyzed as described above. The following sequencing and read alignments were performed once and twice: The abundance of peptides recovered after selection was compared to the input library before selection.

[0171] Screening Optimization Details To preserve genomic DNA after isolation of IFP-positive cells, keep sorted cells on ice at all times. The cells were maintained, spun down, and frozen within 4 hours of sorting.

[0172] An optimization run was performed immediately prior to each screening to provide optimal signal-to-noise for target detection. Briefly, a known T cell antigen (pulsed peptide, final 10 μg / ml) was used. MHC-matched target cells (expressing the iGzB reporter) were incubated with 100 μg of iGzB in the presence or absence of 100 μg of iGzB. , and co-cultured for 4 hours with serial dilutions of the T cells used for screening. Activation was determined under each condition by flow cytometry, and background activation (P<0.05) was The ratio of on-target activation (absence of pulsed antigen) to on-target activation (presence of pulsed antigen) was calculated. The optimal T cell:target cell ratio was selected for library screening.

[0173] To reduce the number of cells presenting multiple antigens, target cells were incubated at a multiplicity of infection of 0.2–0.5. The cells were transduced with the lentiviral library at a multiplicity of infection (MOI).

[0174] To allow for robust detection of antigen sequences, the sample should be cultured at least twice the number of cells sorted. Deep sequencing (i.e., 100,000 cells selected by FACS) (200,000 reads per sample if separated).

[0175] To allow clear separation of T cells and target cells by FACS, target cells were Before co-culture with cells, the cells were stained with CellTrace™ Violet dye (Invitrogen) en).

[0176] [Example 1] Identifying T cell antigens from complex libraries by high-throughput sequencing Compositions and methods for Compositions and methods for comprehensive, genome-wide identification of target antigens for T cells are provided herein. This technique involves the use of antibodies against MHC class I molecules on target cells. Lentiviral delivery of candidate antigens is then used to induce this live target cell. The cytotoxic T lymphocytes (CTLs) are then mixed with a sample of the desired cytotoxic T lymphocytes (CTLs) and the CTLs are then injected with the allogeneic antigen. This allows time for the CTL to recognize any target cells that exhibit the antigen. Upon recognition, the CTL initiates the killing process. It contains the serine protease granzyme B (GzB) to initiate the Release of cytotoxic granules. Using a reporter of intracellular GzB activity, we identify the recognized target. Isolate genomic DNA from the cells. Finally, PCR amplification and next-generation sequencing (NGS) are performed. S) allows comprehensive identification of the antigens presented by these cells. Quantitative sequencing readout of antigens recognized by the input population of CTLs This approach is illustrated in Figure 1.

[0177] A method for identifying candidate antigens specific for cytotoxic T cells involves identifying the cognate antigen of the CTL. To demonstrate that the present invention can robustly enrich target cells, a proof-of-concept experiment was performed. For development and testing purposes, HLA A * HI restricted to 0201 V CTL clones highly specific for pol peptide IV9 (Pol residues 476-484) CTL clones were obtained from MH cells pulsed with the allogeneic but non-control peptide. In C-matched target cells, as detected by 7-AAD staining for membrane permeability , was able to induce apoptosis (Fig. 2A).

[0178] The candidate antigens encoded by the genes are effectively transferred onto MHC I molecules by target cells. This allows for efficient presentation of the target cell library. Lentiviral expression of a single copy of a 56 amino acid fragment of HIV pol containing the nucleotide sequence Tests were conducted to determine whether this product allows for efficient processing and presentation of the IV9 peptide. Expression of this fragment was shown to be cytotoxic as determined by LDH release assay. It was observed that the IV9 antibody was sufficient to confer target cell recognition by IV9 CTL (Fig. 2 B), which demonstrated the feasibility of generating a targeted cell library.

[0179] To isolate DNA from target cells that were productively recognized by CTLs, We developed a reporter assay for productive antigen recognition in GzB protease activity. was used as a readout / marker for recognized target cells. GzB was Thus, caspases are cytotoxic proteases secreted into recognized target cells. GzB reporters induce activation and apoptosis in the general apoptotic pathway. Therefore, the target cells are not activated by the CTL, which means that only the killed target cells are isolated by the CTL. This means that the antigen-independent bacterium in the method described herein can be Previous studies have shown that the background noise of target cells is reduced during cytotoxic killing. GzB released into the vesicles leads to the complete proteolysis of GzB targets. This was demonstrated, suggesting a robust enzymatic activity that served as the basis for the reporter.

[0180] To detect GzB activity, the method described in To et al. PNAS 112(11): 3338-3343 (2015) was used. Based on this work, we developed a new fluorogenic GzB reporter protein and characterized it with two domains. Modified infrared fluorescent proteins that cannot be matured due to a constrained peptide linker between the nuclei. The protein was prepared by proteolytic cleavage of this linker. This allows for optimal folding, resulting in up to a 1000-fold increase in fluorescence. AS 112(11): 3338-3343(2015) used this reporter to measure caspase activity and TE activity. The activity of the reporter gene as described herein was successfully detected. We modified the reporter to detect GzB cleavage instead. Target cells stably expressing a live GzB reporter were generated. Co-culture with IV9 CTLs This resulted in infrared fluorescence detection in target cells pulsed with IV9 but not in the control. This led to an increase in protein signal (Fig. 3), consistent with efficient detection of GzB activity.

[0181] The platform described herein allows target cells presenting the cognate antigen of CTLs to be isolated. To demonstrate that the GzB repo can be enriched, a reconstitution experiment was carried out. Target cells expressing the IV9 peptide were labeled with violet cell stain. These cells were then cultured in a medium that also expresses the GzB reporter but does not present the control peptide. The complex was analyzed using various ratios of IV9 to control cells mixed with unstained target cells. The mixed cells were then used to simulate an antigen library with increased diversity. The target cells were co-cultured with β-glucan and GzB-activated cells were isolated. Using this method, all target cells that activated the GzB reporter expressed IV9 peptide. The enrichment of target cells was calculated. This experiment is illustrated in Figure 4.

[0182] The results of the experiment are shown in Figure 4B and demonstrate potent enrichment of target cells presenting the cognate IV9 antigen. When an initial dilution of 1:1000 was used (the lowest we tested), At this dilution, a 21-fold enrichment of target cells presenting the cognate antigen was observed. A library of 1,000 different antigens was simulated under these conditions, and the platform Forms can be used to identify targets from a highly complex library of candidates This demonstrates that:

[0183] The final step in applying this method is to extract the genomic DNA of the recognized target cells. However, the goal is to enable the recovery of intact antigen libraries from Gz. B initiates caspase activation in target cells and catalyzes the synthesis of caspase-activated deoxyribonucleic acid (CDN). This causes internucleosomal degradation of genomic DNA by CAD. is normally inactivated by the protein inhibitor of CAD (ICAD), a caspase substrate. Overexpression of activated but caspase-resistant (D117E) ICAD inhibits ICAD activity during apoptosis. It has been shown to block DNA degradation (Sakahira et al., Nature. 1:391(6662 ):96-9. 1998). Caspase-resistant D11 cells were generated by lentiviral transduction and selection. Target cells modified to express the 7E ICAD gene were used. This strategy allows for the recovery of intact genomic DNA from apoptotic cells. Demonstrate that it will become.

[0184] This platform allows enrichment of CTL targets from complex antigen libraries. To demonstrate that this approach can be successfully implemented, T cell clones were screened against targets. We performed sequencing across the complete proteomes of 10 HIV strains in 56 amino acid steps. A library of 2,494 peptide fragments was created, which were displayed side by side. The vector was cloned into a lentiviral vector and transfected with our GzB reporter and mutant D1 The mutant ICAD with 17E and D224E was introduced into target cells expressing the mutant ICAD. The library of target cells was then co-cultured overnight with the IV9 CTL clone to induce GzB expression. The target cells that activated the transporter were isolated by FACS. PCR was performed to amplify the antigen cassette from the genomic DNA of the cells, and the selected cells qPCR was performed to quantify the enrichment of selected peptides in the The experiment is illustrated in Figure 5A and the results are shown in Figure 5B. Significant and reproducible enrichment of the peptide was observed, while the control antigen was not significantly enriched. This indicates that this platform can be used to target CTLs. It is possible to screen complex antigen libraries using these Target cells can be comprehensively detected by using next-generation sequencing. Demonstrate the following.

[0185] [Example 2] Identifying T cell antigens from complex libraries by high-throughput sequencing Use this platform to discover CTL targets from complex antigen libraries To demonstrate that this is possible, we performed a reconstitution script for the target of the T cell clone of interest. The complete proteomes of 10 HIV strains were obtained in 56 amino acid steps. We generated a library encoding 2,494 peptide fragments that are displayed across the entire genome. This library was cloned into a lentiviral vector and used in our GzB repo The target cells expressing the ICAD mutant with the D117E and D224E mutations were The library of target cells was then co-transfected with the IV9 CTL clone overnight. Cultured target cells with activated GzB reporter were analyzed by fluorescence-activated cell sorting (FACS). The antigen cassette was amplified from the genomic DNA of the input and selected cells. PCR was used to enrich for cells recognized by the T cell clone. Illumina sequencing was performed to characterize the identified antigens.

[0186] Figure 8 shows the relative abundance of each peptide in our library across two biological replicates of this experiment. Plot the fold enrichment after peptide sorting, with numerical annotations. The plotted peptides correspond to the known target epitopes of the IV9 CTL clone used. The most potent and most reproducible enrichment contains the IV9 epitope peptides, and almost all such peptides have at least moderate Furthermore, the most enriched Unbiased motif analysis of the selected peptides revealed that the most frequently occurring motif was the positive The precise IV9 epitope was identified (Fig. 9). Compositions and methods are used to accurately identify T cell targets from highly complex antigen pools Demonstrate that it is possible.

[0187] [Example 3] Application of the GzB reporter to CD4 T cells This approach can be used to identify CD4 T cells or other T cells that do not themselves have cytotoxic activity. We also obtained a step to demonstrate that cellular targets can be identified. This is achieved by introducing the T cell receptor (TCR) of Cytotoxic T cells then recognize and kill the target cells of the introduced TCR. The target cells recognized by the TCR of interest are then subjected to the same procedures as described herein. The GzB reporter gene can be used to identify the gene.

[0188] Using this technique, we were able to successfully generate CTLs that exhibit CD4 TCR specificity. Experiments were performed to demonstrate that lentiviral infection can be used to induce MHC class II deficiency. The primary CD4 TCR (Ob1A.12) recognizes the MBP peptide in the context of the IL-2 molecule. Cytotoxic CD8 T cells were engineered with the Ob1A.12 TCR but not with the control CD8 T cells not modified with TCRs recognize MBP peptides with appropriate MHC II molecules. The GzB reporter was specifically activated in target cells expressing the GzB receptor (Figure 10 These results suggest that the GzB reporter can identify targets for CD4 T cells and induce sensitization. Th1, Th2, and Treg CD4 in infectious diseases, cancer, and autoimmune settings We demonstrate that it is possible to identify T cell targets.

[0189] [Example 4] Alternative Granzyme B reporters As described herein, in target cells productively recognized by CTLs, Many GzB-based reporters are contemplated that allow for detection of GzB activity. For example, Several alternative reporters of GzB activity have been generated. or in combination with the fluorogenic protease reporters described above, Target cells recognized by the antibody can be isolated.

[0190] For example, an inactive, membrane-tethered Cre gene is activated by cleavage of its tether by GzB. One method for detecting GzB activity has been developed using recombinase. , releases Cre, enters the nucleus, and activates the reporter in response to T cell recognition. PCR of antigen cassette in cells recognized by Cre-mediated recombination of P reporter A primer structure is generated that allows amplification. Productively recognized antigens are then induced by cytotoxic cells. Illumina sequencing of PCR products derived from target cells after treatment with Sequencing of the PCR product from target cells after This approach is illustrated in Figure 6A.

[0191] To demonstrate the feasibility of this approach, tests were performed. Target cells were 1) induced on the tether; 2) membrane-tethered Cre recombinase with a GzB cleavage sequence in the membrane; 3) a reporter cassette containing two loxP sites that can be inverted by apoptosis; Expressing the D117E mutant form of ICAD to preserve genomic DNA during cis The GzB was engineered to be transduced into these target cells by natural killer cells. This resulted in Cre recombinase cleavage and reporter fragmentation, as detected by qPCR. This resulted in an approximately three-fold increase in inversion (Fig. 6B). They act similarly on NK cells, and possess the same perforin- and granzyme-mediated cytolysis mechanisms as NK cells. This uses the Cre recombinase to detect GzB activity. demonstrated the feasibility of recovering intact DNA from cells targeted for killing. This highlights our ability to achieve this.

[0192] Another alternative would be to use a caspase reporter rather than GzB. However, unlike the caspase reporter, the granzyme reporter does not express caspases. are not activated during T cell-mediated apoptosis and are more potent in the context of our T cell killing assays. Has low levels of background activation (not affected by positive signal, approximately 3 fold background reduction).

[0193] Another method developed to detect GzB activity is based on staining for antibody targets ( Whether the reporter is expressed in the cytoplasm or membrane, whether intracellular or extracellular Depending on which gene is targeted, this may result in the reporter acting as a substrate for GzB. The reporter is a GzB cleavage motif. Before cleavage, the internal Flag epitope The epitope is not recognized by the M1 Flag antibody, but rather by the N-terminal Flag epitope. However, after GzB cleavage, the Flag epitope is located in the C-terminal cleavage fragment. The N-terminus of the protein is exposed and can be stained using the M1 antibody. This approach is illustrated in Figure 7A. do.

[0194] This method allows NK cells (created as cytotoxic lymphocytes in this proof-of-concept experiment) to be isolated. Target cells expressing the GzB reporter with or without delivery of GzB by ELISA (using Western blot analysis of cell lysates using M1 Flag antibody revealed After exposure of the reporter to GzB delivered by NK cells, as detected by This resulted in a significant increase in the abundance of the M1 antibody target (Fig. 7B). by antibody staining and flow cytometry compatible with screening methods described in the literature. It can also be detected.

[0195] Yet another reporter of GzB activity in target cells is the ER-retention motif-containing cell membrane protein. Based on reporter protein sequestration within vacuoles. Cleavage of the ER retention motif by GzB. Upon this, the reporter protein is released and detected, for example by transporting it to the cell surface. This technique allows the isolation of proteins from the target cells. To demonstrate that the protein degradation can be successfully detected, we performed the analysis of the ER retention motif. CD4, which previously contained a TEV cleavage site, was used as a reporter protein. To this end, we used the C-terminal KKX, which has previously been reported to sequester proteins in the ER. X motif (where X is any amino acid), e.g., KKYL (SEQ ID NO: 17) (See Nilsson et al., Cell, 58(4): 707-718 (1989)). This reporter was fused to GFP to track expression. Co-culture with TEV constructs significantly increased cell surface CD4 expression. (Figure 25).

[0196] The KKXX reporter sequence used is as follows:

[0197] [ka]

[0198] [Example 5] Genome-wide screening identifies known and novel T cell targets Screening centers applying the described compositions and methods to various T cell populations. These screens were performed using the compositions and methods previously described. The precise target antigen of the engineered TCR was identified and, importantly, genome-wide scale We demonstrate the discovery of novel, biologically meaningful antigens of T cell populations.

[0199] To demonstrate accurate targeting of known TCRs, we used the N We synthesized a TCR that recognizes the LV epitope. This is because the TCR binds to the lentivirus phenotype. CMV is introduced into primary donor CD8 T cells by transduction, and these cells are used to Screening a library of 2882 candidate 56-mers aligned across the genome Two 56 amino acid peptides were identified in the library containing NLV epitopes. Only the top two scoring peptides in the library were enriched 7 to 20 times. (Figure 16 and Table 1). Notably, the other peptides were more than four-fold This experiment demonstrated that this platform The target of the "resurrected" TCR introduced into donor CD8 T cells was successfully identified. It was proven that this was the case.

[0200] [Table 1]

[0201] This platform allows for the screening of more complex sets of tens to hundreds of thousands of antigens. We performed a virome-wide screening to demonstrate that the virus can be screened. In this screen, HLA-A cells grown in the presence of NLV peptides were Primary T cells derived from a donor who was HIV-1 positive and CMV positive were used. A 55-amino acid stepwise alignment across the entire genomes of 206 virus species These cells were screened against a library of over 93,000 candidates. Only two 56-mers in the library containing NLV epitopes ranked in the top two positions. were enriched peptides, with enrichment ranging from 25 to 100 fold (Fig. 17 and and Table 2). Two further overlapping 56-mers were enriched 15- to 40-fold, The epitope of the 28 amino acid region shared by these two 56-mers is It was confirmed that approximately 2% of the CD8 T cells recognized the antibody (Figure 18). This platform identifies novel T cell targets on a genome-wide scale. Remarkably, of the 93,000 candidate lines screened, Braly has the entire human ORFeome collection (approximately 20,000 full-length ORFs) ) is more complex than the previous experiment. Furthermore, this experiment demonstrated that the antigen is recognized by a 2% subset of T cells. Novel targets, some of which have only recently been identified and validated, allow for characterization of multiple T cell populations at once. It was shown that it is possible.

[0202] [Table 2]

[0203] Next, we identified novel immunodominant targets of polyclonal T cells on a genome-wide scale. To demonstrate this, we performed a genome-wide library-to-library screen. Bulk memory T cells were purified from HLA-A2-positive, CMV-positive donors. These T cells were screened against a CMV-wide library of 2,882 epitopes. Six sets of overlapping 56-mers were identified that were reproducibly enriched (Table 3). All six of these candidate antigens show high activity in a 28 amino acid overlapping region. The affinity of the 2000-kDa antibody contains predicted HLA-A2 binders (Figure 20). Only 10% of the 8-amino acid stretch is a high-affinity HLA-A2 binder One of the enriched epitopes is predicted to contain It was confirmed that this was the dominant PP65 epitope, recognized by up to 0.3% of T cells. The five identified epitopes have not been previously reported. Novel T cell targets of clonal T cells are identified in a library-versus-library setting using genome-wide It was demonstrated that it can be identified on a scale.

[0204] [Table 3]

[0205] Table 3 shows the duplicates that were reproducibly enriched in the screen described in Figure 19. Six sets of 56-mers are shown. In the 6 / 6 case, a 28 amino acid overlap region (bold) is shown. There are predicted high affinity HLA-A2 binding epitopes found in In contrast, only 10% of the 28-mers contain high-affinity HLA-A2 binding epitopes. Epitopes 1336-1337 are predicted to have a well-established immunodominant sequence. The remaining five epitopes have not been reported previously. Ta.

[0206] This platform allows mapping of the TCR-epitope interaction landscape. To demonstrate that the nucleotide sequence is regulated by the nucleotide sequence, a comprehensive mutagenesis screen was performed. Primary T cells expanded with the above NLV were used. In addition to the two upstream and downstream residues, To screen all libraries of single amino acid mutants of a known target epitope, Mutations of upstream or downstream amino acids did not abolish T cell recognition. However, most mutations in the epitope itself reduce epitope recognition. This method allows accurate recognition of T cells in the context of larger antigens (Figure 6). The identified epitopes can be precisely mapped. Furthermore, this approach can be used to identify relevant can be used to search for off-target peptide sequences, significant, tolerated By mapping the TCR-interacting residues, we can identify potential off-target molecules for T cells. The target can be identified.

[0207] This platform allows for the identification of target epitopes through multiple rounds of screening. Continuous enrichment is possible. Recognizes the IV9 epitope of HIV polymerase. The gene encoding the TCR that binds the primary donor was synthesized and transduced by lentiviral transduction. The resulting cells were used to transduce the genomes of 10 strains of HIV. Screening a 56-mer library of 1,247 candidates tiled across To perform the second round of selection, the isolated antigens were amplified and expressed in lentiviral expression vectors. These were then transduced into the target vector by viral transduction. The screening was repeated using the same IV9 T cells. Increased enrichment of known targets of the TCR was observed in the second round of selection (Figure 21 Therefore, this platform can be used in multiple rounds of selection to improve antigen specificity. It can be improved.

[0208] [Example 6] Application of the platform to tumor-derived TCRs This platform will enable the identification of tumor-derived TCR targets. To demonstrate this, we performed a signal-to-noise analysis. and the efficacy of T cells to activate the reporter in the presence versus absence of their known antigen. For all viral screening, the actual screening Signal-to-noise measurements are highly predictive of the target enrichment observed in The T cells that we are studying for recognizing tumor antigens are gave a signal-to-noise ratio comparable to previously used antiviral T cells, and this screening This will further provide sufficient confidence in the identification of tumor-derived TCRs and other Various genome-wide human screens to identify known and novel targets of TCRs This becomes possible.

[0209] [Example 7] Inhibiting CAD-mediated DNA degradation A key challenge in granzyme-based detection of T cell activity is that granzymes Upon entry into target cells, the nuclease CAD degrades the characteristic nucleosomes of genomic DNA. The first step is to initiate apoptosis, which involves the breakdown of the nuclei between the nuclei. CAD is kept inactive by the inhibitor protein ICAD. It is a direct caspase substrate that is degraded during cis-cleavage and prevents CAD from degrading DNA. This platform prevents intact granzymes from receiving cells. This allows the recovery of DNA containing the antigen cassette, which occurs during early apoptosis. DNA degradation limits the ability to identify antigens that drive T cell recognition. This challenge is addressed by CA It has been determined herein that this can be overcome by inhibiting D-mediated DNA degradation. For example, in target cells, the overexpression of mutant ICAD protein A caspase-resistant version of granzyme inhibits CAD nuclease activity after granzyme delivery. We have determined that overexpression of this mutant ICAD prevents apoptosis. It was confirmed that laddering of genomic DNA after induction of cis was prevented (Fig. 23).

[0210] Inhibiting CAD-mediated DNA degradation to recover antigen information is a key screening method. It was determined that ICAD overexpression was important in the setting of The efficiency of antigen recovery in the different screening situations performed was calculated. Mapping the observed distribution of reads per antigen against cell number for a Poisson distribution , fits were used to estimate the total number of cells characterized in each screening replicate. The number of cells recovered by sequencing was then compared with the number of publicly available cells isolated by FACS. The net efficiency of antigen retrieval was determined by comparing the number of cells obtained with the known number of cells. Over six replications of screening, antigen information was recovered from only 1-2% of the sorted cells. In contrast, the screening performed for overexpression of mutant ICAD The cloning resulted in approximately 10-fold higher efficiency of antigen recovery (Figure 24). During screening, genomic DNA preparation, PCR, and sequencing steps These results suggest that the DNases, e.g., caused by overexpression of mutant ICAD, did not differ significantly. Inhibition of A degradation improved assay performance by approximately 10-fold, allowing for significantly more complex antigen ligation. This shows that screening of the brilliance is possible.

[0211] (References) Cameron, BJ et al. Identification of a Titin-Derived HLA-A1-Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3-Directed T Cells. Sci Trans Med 197ra103 (2013). Sakahira, H., Enari, M. & Nagata, S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96-99 (1998). Sekaly, R. The failed HIV Merck vaccine study: a step back or a launching point for future vaccine development? JEM 205 (1): 7, (2008). To, TL et al. Rationally designed fluorogenic protease reporter visualizes spati otemporal dynamics of apoptosis in vivo. PNAS 112(11): 3338-3343 (2015). Kolowos, W., Schmitt, M., Herrman, M., Harrer, E., Low, P., Kalden, J.R., Harrer , T. (1999) Biased TCR Repertoire in HIV-1-Infected Patients Due to Clonal Expan sion of HIV-1-Reverse Transcriptase-Specific CTL Clones. J Immunol 162:7525-7533 . Schub, A., Schuster, I.G., Hammerschmidt, W., Moosmann, A. (2009) CMV-Specific T CR-Transgenic T Cells for Immunotherapy. J Immunol, 183:6819-6830. Xu, GJ,* Kula, T.,* Xu, Q., Li, MZ, Vernon, SD, Ndung'u, T., Ruxrungtham, K., Sanchez, J., Brander, C., Chung, RT, O'Connor, KC, Walker, B., Larman, HB, Elledge, SJ (2015) Comprehensive serological profiling of human populations ions using a synthetic human virome. Science, 348 (6239), aaa0698

[0212] Other embodiments Although the present application has been disclosed in the context of certain specific embodiments and examples, those skilled in the art will appreciate that The embodiments of this application may include other alternative embodiments and aspects beyond those specifically disclosed. It is understood that this also extends to the use and / or application of the Deaf.

[0213] All patents, patent applications, patent application publications, and other materials referenced herein are hereby incorporated by reference. Materials, such as articles, books, specifications, publications, documents, articles, etc., are associated with them. any prosecution file history, any of which is inconsistent with or conflicts with this document; or limited with respect to the broadest scope of any claim now or hereafter associated with this document. for any purpose whatsoever except any of which may have any effect. is incorporated herein by reference in its entirety. For example, any of the incorporated materials Explanations, definitions, and / or uses of terms associated with and related to this document In the event of any inconsistency or conflict between the explanation, definition, and / or use of a word, If so, the explanation, definition, and / or use of the term in this document takes precedence.

[0214] It is understood that the embodiments of the present application disclosed herein are illustrative of the principles of the embodiments of the present application. It should be understood that other modifications that may be used may be within the scope of this application. By way of example, and not limitation, alternative configurations of the embodiments of the present application may be implemented in accordance with the teachings herein. Therefore, the embodiments of the present application may be utilized exactly as shown and described. It is not limited to what is listed.

Claims

1. a) one or more of the antigens expressed and presented by MHC class I or MHC class II molecules exogenous nucleic acids encoding a plurality of candidate antigens, b) a molecular reporter of granzyme B (GzB) activity, and c) caspase-activated deoxyribonuclease (CAD)-mediated DNA degradation, CAD knockout, or exogenous inhibitor of caspase knockout Antigen-presenting cells (APCs) comprising:

2. The exogenous nucleic acid is optionally a lentiviral vector, a retroviral vector, or stably introduced into the genome of the APC via a transposon. Item 1. An APC according to item 1.

3. 10. The method of claim 1, wherein the exogenous nucleic acid is flanked on both sides by predetermined primer recognition sequences.

2. APC according to claim 2.

4. The molecular reporter of GzB activity comprises a GzB cleavage site (VGPD, A fusion polypeptide comprising the sequence of any one of claims 1 to 3 (SEQ ID NO: 1). PC.

5. The molecular reporter may be a modified infrared fluorescent protein, a membrane-tethered CRE recombinase, , antibody-based reporter of GzB activity, ER retention-based reporter of GzB activity, cell a cell surface-detectable-based reporter of GzB activity, or a combination thereof.

4. APC according to claim 4.

6. The molecular reporter comprises a membrane-tethered CRE recombinase and the APC comprises LoxP and optionally, said exogenous 6. The method of claim 4, wherein the nucleic acid is located proximal to a CRE-activating primer recognition sequence. APC.

7. The exogenous inhibitor of CAD-mediated DNA degradation is expressed in an expressible form as a caspase-activating deoxyribonucleotide. Nucleic acid encoding the xyloribonuclease inhibitor (ICAD) gene, CAD or CAS Inhibitory nucleic acids targeting caspase-3, small molecule inhibitors of caspase-3, chemical DNAse inhibition or a peptide or protein inhibitor of caspase 3, or 7. The method according to claim 1, wherein the caspase knockout is a caspase 3 knockout. Item 1. APC according to item 1.

8. i) does not express endogenous MHC molecules and is engineered to express exogenous MHC molecules; and / or ii) HEK 562 cells, HEK 293 cells, HEK 293 T cells cells, U2OS cells, MelJuso cells, MDA-MB231 cells, MCF7 cells, NT ERA2 cells, LN229 cells, dendritic cells, and primary autologous B cells. The APC of claim 1 .

9. The candidate antigens are 8, 9, 10, 11, 20, 30, 50, 100, 200, or 3 9. The method of claim 1, wherein the amino acid sequence of the present invention is less than or equal to 0.00 amino acids in length. APC.

10. 9. The method of claim 1, wherein the candidate antigen is more than 300 amino acids in length. APC.

11. the exogenous nucleic acid encoding the candidate antigen is derived from the DNA of an infectious organism or a human; 11. An APC according to any one of claims 1 to 10.

12. The APC of claim 11 , wherein the human DNA is obtained from a cancer cell.

13. The infectious organisms include viruses, bacteria, fungi, protozoa, and multicellular parasites. The APC of claim 11, selected from the group consisting of:

14. Each APC encodes a candidate antigen, thereby targeting MHC class I and / or MHC class II antigens. A variety of exogenous antigens representing a library of candidate antigens expressed and presented by C class II molecules are A library of APCs according to any one of claims 1 to 13, comprising a gene encoding a targeting nucleic acid.

15. 15. The live vector of claim 14, wherein the exogenous nucleic acid is derived from an infectious agent or human DNA. Rally.

16. about 10 ２ from about 10 １４ 16. The method of claim 14 or 15, comprising: Ibraly.

17. A fusion polypeptide comprising a GzB cleavage site (VGPD, SEQ ID NO: 1) linked to a detection molecule. A molecular reporter of granzyme B activity, comprising:

18. The detection molecule may be an enzyme, a detectable label, an antibody binding antigen, or an affinity tag.

18. The molecular reporter of claim 17, wherein:

19. The detectable label may be an infrared fluorescent protein (IFP), a nucleic acid amplification target, or an antibody. The composition comprises a composition that is recognized, a composition that is released from the ER, and a composition that is present on the cell surface.

19. The molecular receptor of claim 18, which is detectable after GzB cleavage, selected from the group consisting of: Porter.

20. The IFP is functionally separated into N fragments (N-IFPs) and and C fragment (C-IFP), and further comprising a green fluorescent tag located on the N-terminal side of C-IFP. The N-fragment of the protein (N-GFP) and the green fluorescent protein located at the C-terminal side of N-IFP The N-GFP and C-GFP are flanked on both sides by a C fragment of the same structure (C-GFP). The molecular reporter of claim 13, which is structurally active.

21. The enzyme is a CRE recombinase, and the fusion polypeptide is a GzB cleavage polypeptide. The CRE recombiner is operably linked to plasma membrane attachment peptides separated by sites. The molecular reporter of claim 18, comprising an enzyme.

22. The affinity tag is a Flag epitope located C-terminal to the GzB cleavage site. so that the epitope binds to the M1 Flag antigen upon cleavage of the GzB site. and optionally a G located C-terminal to the flag epitope.

18. The molecular reporter of claim 17, further comprising a FP.

23. the GzB portion comprises an endoplasmic reticulum (ER) retention signal and an antibody-binding plasma membrane protein; Cleavage of this site removes the ER retention signal and optionally the antigen is transferred to CD4 0, CD4, CD19, CD20, or a tagged protein, optionally wherein the tag is a Myc tag, a Flag tag, an HA tag, or a histidine tag.

19. The molecular reporter of claim 18.

24. 24. A nucleic acid encoding a molecular reporter according to any one of claims 17 to 23.

25. 1. A system for detecting granzyme B activity in antigen-presenting cells, comprising: a) a fusion polypeptide comprising a CRE recombinase operably linked to a plasma membrane attachment peptide; The CRE recombinase and the membrane-attached peptide are separated by a GzB cleavage site. a fusion polypeptide, b) Encoding GFP and RFP in a head-to-head orientation and flanked by LoxP sites a reporter of CRE activity comprising a flanking nucleic acid sequence, and / or c) CRE active primers, including inactive primers flanked by LoxP sites. a nucleic acid sequence encoding an expressible form of a candidate antigen located proximal to a recognition sequence, a nucleic acid in which CRE-induced rearrangement of the LoxP site generates a functional primer recognition sequence array Including, the system.

26. Antigen presentation recognized by antigen-presenting cells to cytotoxic lymphocytes or NK cells 1. A system for the detection of a) i. MHC class I and / or cytotoxic lymphocytes and / or NK cells or exogenous nucleic acids encoding candidate antigens that are expressed and presented by MHC class II molecules. acid, ii. The activity of granzyme B (GzB) according to any one of claims 17 to 24. The molecular reporter of claim 25 or the system for detecting the activity of granzyme B. system, and iii. Inhibitors of CAD-mediated degradation an antigen-presenting cell (APC) comprising: b) A system comprising cytotoxic lymphocytes and / or NK cells.

27. The inhibitor of CAD-mediated degradation is an exogenous inhibitor of CAD-mediated DNA degradation, CAD knockout out, or caspase knockout, and optionally, said caspase knockout The subject is a caspase 3 knockout or an exogenous inhibitor of CAD-mediated DNA degradation. The inhibitor is an inhibitor of caspase-activating deoxyribonuclease (ICAD) in an expressible form. ) gene encoding nucleic acid, CAD or caspase 3 targeting inhibitory nucleic acid, caspase small molecule inhibitors of caspase-3, or peptide or protein inhibitors of caspase-3 27. The system of claim 26.

28. The antigen-presenting cells are K 562 cells, HEK 293 cells, HEK 293 T cells. , U2OS cells, MelJuso cells, MDA-MB231 cells, MCF7 cells, NTE 28. The method of claim 27, wherein the antibody is selected from the group consisting of RA2a cells, dendritic cells, and primary autologous B cells. The system described in

29. The cytotoxic lymphocytes include cytotoxic CD4 T cells and cytotoxic CD8 T cells.

27. The system of claim 26, wherein the cell is selected from the group consisting of:

30. The cytotoxic lymphocytes and / or NK cells are adapted to express an antigen receptor of interest.

30. The system of any one of claims 26 to 29, wherein the system is modified to:

31. The cytotoxic lymphocytes and / or NK cells are derived from non-cytotoxic CD4 T cells. Cytotoxic T cells and / or NK cells modified to express T cell receptors.

31. The system of claim 30.

32. Method for identifying antigens recognized by cytotoxic T cells and / or NK cells It is a law, a) an antigen-presenting cell (APC) or an APC according to any one of claims 1 to 16; The library is then subjected to the induction of one or more cytotoxic T cells (CTs) under conditions suitable for antigen recognition. L) and / or NK cells; b) by assaying granzyme B activity in the APCs. Identifying APCs that express the antigen, and measuring the granzyme activity compared to a suitable control. The increase in B activity indicates that the APC is recognized by the cytotoxic T cells and / or NK cells. demonstrating that the cells express the recognized antigen; c) from the APCs identified in step b), a nucleic acid encoding the recognized antigen. and isolating A method comprising:

33. Method for identifying antigens recognized by cytotoxic T cells and / or NK cells It is a law, a) treating an antigen-presenting cell (APC) or a library of APCs according to claim 23 with an antigen contacting the G with one or more CTLs under conditions suitable for recognition, Cleavage of the zB site removes the ER retention signal and inhibits transport to the plasma membrane. releasing the plasma membrane protein from the ER for b) by contacting said APC with an antibody that binds to said plasma membrane protein. isolating APCs expressing the recognized antigen and purifying the antibody-bound APCs. and, c) from the APCs isolated in step b), a nucleic acid encoding the recognized antigen. and isolating A method comprising:

34. The method of claim 1, further comprising the step of sequencing the nucleic acid isolated in step c).

34. The method according to claim 32 or 33.

35. The cytotoxic T cells and / or NK cells are obtained from a biological sample of a subject.

35. The method according to any one of paragraphs 32 to 34.

36. The biological sample may be blood, tumor, healthy tissue, ascites fluid, autoimmune location, tumor infiltration, virus 36. The method of claim 35, wherein the target site is selected from the group consisting of a site of a viral infection, a lesion, oral mucosa, and skin. How to post.

37. the biological sample is obtained from a site of infection or autoimmune reaction in the subject; 37. The method of claim 35 or 36.

38. 38. The method of claim 30, wherein the cytotoxic T cells are CD4 or CD8 cells. The method according to any one of claims 1 to 5.

39. The cytotoxic T cells and / or NK cells are adapted to express an antigen receptor of interest.

39. The method of claim 38, wherein the method is modified.

40. The cytotoxic T cells and / or NK cells are derived from non-cytotoxic CD4 T cells.

40. The method of claim 39, wherein the cell has been modified to express a cell receptor.

41. The step b) of identifying the phenotype of the present invention is characterized by a difference of at least 2-fold, at least 5-fold, or times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, detection of a fluorescent signal in said APC that increases by at least 1000-fold or more; 41. The method of claims 32 to 40, wherein the method is by extraction.

42. The identifying step b) can be performed by flow cytometry or affinity purification.

42. The method of claims 32 to 41,

43. The identifying step b) can be performed using fluorescence activated cell sorting (FACS) or affinity 43. The method of claims 32 to 42, wherein the method is by purification.

44. 44. The method of claim 32 to 43, wherein the isolating step c) is by PCR amplification. How to post.

45. The sequencing is performed by pyrosequencing or next generation sequencing.

45. The method of claims 33 to 44.

46. The library of APCs comprises at least 5,000 different candidate antigens.

46. ​​The method according to any one of items 32 to 45.

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