Techniques for generating cell-based therapeutics using recombinant t cell receptor genes

Abstract

The present application relates to technologies for generating cell-based therapeutics using recombinant T cell receptor genes. More specifically, the technologies of the present application generally relate to compositions and methods for producing libraries of recombinant T cell receptors (TCRs), and methods of their therapeutic applications. The compositions and methods of the present technology can be used to rapidly isolate antigen-specific TCR repertoires as personalized targeted therapies against cancer and viral infections.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980029596.9, filed on February 27, 2019, entitled "Technology for generating cell-based therapeutic agents using recombinant T-cell receptor genes". Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 637,240, filed March 1, 2018, the entire contents of which are incorporated herein by reference. US government support

[0003] This invention was made with government support under grant GM103418 from the National Institutes of Health. The government owns certain rights to this invention. sequence list

[0004] This application contains a sequence list, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on February 25, 2019, is named 104434-0179_SL.txt and is 206,931 bytes in size. Technical Field

[0005] This technology generally relates to methods for high-throughput isolation and manipulation of genes encoding T-cell receptors from single T cells as cell therapies, compositions of T-cell libraries, and methods for their therapeutic application. Background Technology

[0006] The following description is provided to aid the reader's understanding. The information provided and the references cited are not considered prior art to this method.

[0007] Humans possess thousands of T-cell receptors that provide key components of the adaptive immune system, and T-cell receptor responses have been proven to make a significant contribution to protection against diseases including viral infections, cancer, and autoimmune diseases. However, molecularly defined T-cell receptor therapies have a narrow impact on clinical care because the methods for translating T-cell receptor responses into treatments have not been adequately elucidated on a scale actually used for translational therapy. Current approaches rely on low-throughput T-cell receptor identification technologies, the cumbersome practice of cloning T-cell receptors, and the limited ability to guide the cellular state of T-cell libraries.

[0008] There is a need to develop methods for rapidly converting T-cell receptor responses into drugs for cell therapy, particularly for treating cancer. This technology addresses this need in part by leveraging the contribution of TCR libraries to adaptive immunity for the clinical treatment or prevention of cancer. Summary of the Invention

[0009] This technology generally relates to novel compositions and methods for generating recombinant TCR libraries, and their therapeutic applications. The compositions and methods of this technology can be used for the rapid isolation of antigen-specific TCR libraries for the development of personalized targeted therapies against cancer and viral infections.

[0010] Therefore, in one aspect, this technology provides a recombinant T-cell receptor (TCR) library vector comprising: (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from mRNA isolated from a single lysed T cell present in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent or reverse transcription-PCR (RT-PCR). Alternatively or optionally, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet.

[0011] In some embodiments of the vector, the first and second polynucleotides are operatively linked, optionally operatively linked via a linker polynucleotide. In some embodiments of the vector, the first and second polynucleotides have been operatively linked by reverse transcription and PCR amplification of the captured T-cell mRNA. In some embodiments of the vector, the first and second polynucleotides have been cloned into the vector backbone by cleavage at a target restriction endonuclease site naturally found in the TCR gene. In some embodiments, the target restriction endonuclease site appears at a low frequency in the TCR gene. In some embodiments, the first and second polynucleotides have been altered to incorporate at least one target restriction endonuclease site disclosed in Table 7 or 8. In some embodiments, the target restriction endonuclease site contains a silencing mutation.

[0012] In some embodiments of the vector, the mRNA capture agent is selected from the group consisting of poly(dT)-coated beads, oligonucleotide-coated beads, hydrogel beads, and printed oligomers on the surface of microarray pores. In some embodiments, the compartments are emulsion droplets or pores. In some embodiments, the pores are located in printed polymer slides, plastic plates, microtiter plates, or gels. In some embodiments, the volume of the compartment is 5 nL or less.

[0013] In some embodiments of the vector, the vector further comprises at least one polynucleotide encoding an expression control element, said polynucleotide being operatively linked to a first polynucleotide and / or a second polynucleotide. In some embodiments, the expression control element is selected from the group consisting of: promoters, p2a sequences, and IRES sequences. In a particular embodiment, the promoter is an EF1α promoter or a CMV promoter. In some embodiments, the polynucleotide encoding the expression control element is located between the first polynucleotide and the second polynucleotide.

[0014] In some embodiments of the carrier, the carrier is circularized. In some embodiments, the carrier is circularized before the expression control element is incorporated into the carrier. In other embodiments, the carrier is circularized after the expression control element is incorporated into the carrier. In some embodiments, the carrier is linear (e.g., not circularized).

[0015] In some embodiments of the vector, the expression control element has been incorporated near a prototype spacer adjacent motif (PAM). In other embodiments, the expression control element has been incorporated into the vector using a DNA-modifying enzyme selected from restriction enzymes or TALENs. In other embodiments, the vector further comprises one or more polynucleotides encoding transposons, said polynucleotides being operatively linked to at least one of a first polynucleotide and a second polynucleotide.

[0016] In some embodiments of the vector, the vector further comprises one or more of the following: a polynucleotide encoding a detectable marker, a polynucleotide encoding a selection marker, a polynucleotide encoding a switching mechanism for controlling the expression and / or activation of a first polynucleotide and a second polynucleotide, and a polynucleotide encoding a Kozak common sequence or enhancer.

[0017] In some embodiments of the vector, the vector backbone is selected from the group consisting of: retroviral vector backbones, lentiviral vector backbones, adenoviral vector backbones, and adeno-associated virus vector backbones. In some embodiments, the vector may contain linear DNA for CRISPR / Cas9 integration. In some embodiments, the vector may contain DNA that can be incorporated into the host using recombinases. In some embodiments, the vector may contain intermediate DNA that can be incorporated into the host using transposases.

[0018] In some embodiments of the vector, the encoded T-cell receptor (e.g., TCRαβ or TCRγδ) is reactive to disease antigens or target cells. In some embodiments, the disease antigen is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71 (EV71), Ebola virus, hepatitis A (HAV), hepatitis B (HBV), cytomegalovirus (CMV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human T-lymphotropic virus (HTLV), influenza A virus, influenza B virus, Japanese encephalitis virus, leukemia virus, measles virus, molluscum contagiosum virus, sheep pox virus (orf) Virus, parvovirus, rabies virus, respiratory syncytial virus, Rift Valley fever virus, rubella virus, rotavirus, tick-borne encephalitis virus (TBEV), simian immunodeficiency virus, tobacco etch virus (TEV), varicella-zoster virus, smallpox virus, West Nile virus, Zika virus, and Chikungunya virus. In other embodiments, the disease antigen is a tumor antigen selected from the group consisting of: CD45, glypican-3, IGF2B3, kallikrein 4, KIF20A, lensin, meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tumor necrosis factor receptor superfamily member 10a (TRAILR1), receptor activator of nuclear factor κ-B ligand (RANKL), insulin-like growth factor 1 receptor (IGF1R), epithelial cell adhesion molecule (EpCAM), and carcinoembryonic antigen (CEA).

[0019] On the other hand, this technology provides recombinant cells comprising a vector, wherein the vector comprises (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of a single lysed T cell is isolated in the compartment using an mRNA capture reagent, optionally wherein the recombinant cell is a bacterial cell, a mammalian cell, or a yeast cell. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or optionally, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a droplet.

[0020] In one aspect, this technology provides a recombinant TCR vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are an associated pair, and wherein the first polynucleotide and the second polynucleotide are derived from mRNA of a single lysed T cell captured in a compartment by an mRNA capture reagent. In some embodiments, the plurality of vectors comprise a TCR library. In some embodiments, a single vector in the TCR vector library is selected for inclusion in the TCR library based on one or more of the following characteristics: TCR clonal universality, TCR enrichment characteristics determined in vitro, TCR binding specificity, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motif, and / or CDR3 gene motif. In some embodiments, the single vectors in the library are mixed at defined ratios to generate a synthetically derived TCR library.

[0021] On the other hand, this technology provides isolated immune cells comprising (a) a carrier backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell captured in a compartment by an mRNA capture reagent. In some embodiments, the immune cells are hematopoietic stem cells, hematopoietic progenitor cells, T cells, or natural killer (NK) cells.

[0022] In one aspect, this technology provides a cell population comprising a recombinant TCR vector library, the recombinant TCR vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet. In some embodiments, the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells.

[0023] On the other hand, this document also provides a method for preparing a recombinant TCR library, the method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet. In some embodiments, the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells. In some embodiments, the plurality of vectors are circularized at certain steps of the method and linearized at other steps.

[0024] In some embodiments of the method, a library is screened for specific binding to target cells. In some embodiments, the cells are cancer cells or virus-infected cells. In some embodiments, target cells are isolated from a subject.

[0025] In other embodiments of the method, a library is screened for specific binding to the antigen:MHC complex. In some embodiments, the antigen of the antigen:MHC complex is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), and enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus. In other embodiments, the antigen of the MHC complex is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, Lengsin, Meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, EGFR, HER2, TRAILR1, RANKL, IGF1R, EpCAM, and CEA.

[0026] In some embodiments of the method, libraries are screened for T-cell phenotypic markers. In other embodiments of the method, libraries are screened for hematopoietic stem cell phenotypic markers. In other embodiments of the method, libraries are screened for natural killer cell phenotypic markers.

[0027] In some embodiments of the method, for an activity screening library in a co-culture system, wherein the co-culture system comprises at least one of: (a) a cancer cell line; (b) a plurality of cells infected with a known virus; (c) a plurality of tumor cells isolated from a cancer patient; (d) an immortalized cell line; or (e) a plurality of cells derived from a patient tissue biopsy sample.

[0028] In some embodiments of the method, the transformed cells are activated in vitro. In specific embodiments, activation is performed using one or more of the following stimulants: anti-CD3 antibody, anti-CD8 antibody, anti-CD27 antibody, IL-2, IL-4, IL-21, anti-PD1 antibody, anti-CTLA4 antibody, tumor cell lysate, cell co-culture with virus-infected cells, and tumor cell lines.

[0029] In some embodiments of the method, a cell population is transformed with transcription factors. Transcription factors can influence the behavior or phenotype of the transformed cells. In some embodiments, the transcription factors are selected from the group consisting of: FOXP3, BLIMP-1, Helios, Ikaros, and TGF-β.

[0030] On the other hand, this document provides a recombinant TCR library prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet. In some embodiments, the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells.

[0031] In one aspect, the present technology provides a composition comprising a recombinant TCR library and a vector, the recombinant TCR library being prepared by a method comprising transforming a cell population with the vector library, the vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the vector is a pharmaceutically acceptable vector. In some embodiments, the mRNA of a single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a droplet.

[0032] In one aspect, this document provides a method for treating a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a microdroplet.

[0033] On the other hand, this document provides a method for treating cancer in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a microdroplet.

[0034] In some implementation schemes, the cancers include: acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; AIDS-related cancers; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rod-shaped tumors; brain cancer; basal cell carcinoma of the skin; bile duct cancer; bladder cancer; bone cancer; breast cancer; bronchoma; and Burkitt lymphoma. Lymphoma; Carcinoid tumors (gastrointestinal); Germ cell tumors; Primary CNS lymphoma; Cervical cancer; Bile duct cancer; Chordoma; Chronic lymphocytic leukemia (CLL); Chronic myeloid leukemia (CML); Chronic myeloproliferative neoplasm; Colorectal cancer; Craniopharyngioma; Cutaneous T-cell lymphoma; Ductal carcinoma in situ (DCIS); Endometrial cancer; Ependymoma; Esophageal cancer; Sensory neuroblastoma; Extracranial germ cell tumor; Gonadal extragerminoma; Ocular cancer; Intraocular melanoma; Retinoblastoma; Fallopian tube cancer; Malignant fibrous histiocytoma and osteosarcoma of bone; Gallbladder cancer; Gastric cancer; Gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors (GIST); Germ cell tumors; Gestational trophoblastic disease; Hairy cell leukemia; Head and neck cancer; Cardiac tumors; Hepatocellular carcinoma; Langerhans cell histiocytosis. Cell); Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumor, pancreatic neuroendocrine tumor; kidney cancer; laryngeal cancer; leukemia; lip and oral cancer; liver cancer; lung cancer; lymphoma; male breast cancer; malignant fibrous histiocytoma and osteosarcoma of bone; melanoma; Merkel cell carcinoma; mesothelioma; metastatic cancer; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides; myelodysplastic syndrome, chronic myeloproliferative neoplasm; nasopharyngeal carcinoma; neuroblastoma; non-Hodgkin lymphoma. Lymphoma; Non-small cell lung cancer; Oral and oropharyngeal cancer; Osteosarcoma; Ovarian cancer; Pancreatic cancer; Pancreatic neuroendocrine tumor; Papilloma; Paraganglioma; Sinus cancer; Parathyroid cancer; Pharyngeal cancer; Pheochromocytoma; Pituitary adenoma; Pleuroblastoma; Prostate cancer; Rectal cancer; Recurrent cancer; Renal cell carcinoma; Retinoblastoma; Rhabdomyosarcoma; Salivary gland cancer; Sarcoma; Ewing sarcoma; Kaposi sarcoma; Osteosarcoma; Uterine sarcoma; Sézary syndrome; Skin cancer; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma of the skin; Squamous neck carcinoma; Gastric cancer; T-cell lymphoma; Testicular cancer; Laryngeal cancer; Nasopharyngeal carcinoma; Hypopharyngeal carcinoma; Thymic carcinoma; Thyroid cancer; Urethral cancer; Uterine cancer; Vaginal cancer; Hemangioma; Vulvar cancer; or Wilms tumor.

[0035] In one aspect, this document provides a method for inhibiting tumor growth in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a droplet. In some embodiments, the tumor is a solid tumor.

[0036] On the other hand, this document provides a method for treating a viral infection in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a microdroplet. In some implementations, the viral infection is caused by viruses selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), and enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus.

[0037] In some embodiments, the treatment methods provided herein further include administering one or more additional doses of the recombinant TCR library or composition to the subject.

[0038] In some implementations, the recombinant TCR library contains cells that are autologous or allogeneic to the subject being treated.

[0039] In some implementations, the subjects are humans, animals, non-human primates, dogs, cats, sheep, mice, horses, or cows. In a particular implementation, the subjects are humans. Attached Figure Description

[0040] Figure 1 Gel images of the full-length TCR α:β variable region were obtained by overlap extension-PCR (OE-PCR). Following OE-PCR, the PCR products were purified and analyzed by DNA gel electrophoresis. The TCR α:β amplicon (approximately 1000 bp) was visible and further enriched using nested PCR. After DNA gel electrophoresis, gel extraction was performed to obtain the TCR α:β amplicon. 5 ng of purified PCR product was used as a template for semi-nested PCR.

[0041] Figure 2 Gel electrophoresis of nested PCR products is shown. Following DNA electrophoresis, the TCRα:β amplicons were excised and purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research). The purified amplicons were then subjected to zero-blunt cloning (Thermo Fisher Scientific) to analyze the TCRα:β amplicon sequence. Sequence identification was performed using the NCBI IGBLAST T cell receptor gene database.

[0042] Figure 3 This image shows a vector map of the exemplary pLVX-CMV-TCR1-pTert-iCas9 vector. The vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV6-2 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV38-1, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, an i-cysteine ​​9 suicide switch controlled by a tet-inducible promoter and flanked by a cHS4 isolator sequence, and a WPRE enhancer.

[0043] Figure 4 The vector map of pLVX-CMV-TCR2-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV5-8 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV40, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, an i-cysteine ​​9 suicide switch controlled by a tet-inducible promoter and flanked by a cHS4 isolator sequence, and a WPRE enhancer.

[0044] Figure 5The vector map for pLVX-CMV-TCR3-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV10-3 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV13-2, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, an i-cysteine ​​9 suicide switch controlled by a tet-inducible promoter and flanked by a cHS4 isolator sequence, and a WPRE enhancer.

[0045] Figure 6 The vector map for pLVX-CMV-TCR4-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV28-1 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV38-1, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, an i-cysteine ​​9 suicide switch controlled by a tet-inducible promoter and flanked by a cHS4 isolator sequence, and a WPRE enhancer.

[0046] Figure 7 The vector map for pLVX-CMV-TCR5-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV24-1 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV38, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, an i-cysteine ​​9 suicide switch controlled by a tet-inducible promoter and flanked by a cHS4 isolator sequence, and a WPRE enhancer.

[0047] Figure 8 The vector map for pLVX-TCR1-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV6-2 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV38-1, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, and a WPRE enhancer.

[0048] Figure 9 The vector map for pLVX-TCR2-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV5-8 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV40, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, and a WPRE enhancer.

[0049] Figure 10 The vector map for pLVX-TCR3-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV10-3 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV13-2, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, and a WPRE enhancer.

[0050] Figure 11 The vector map for pLVX-TCR4-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV28-1 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV38-1, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, and a WPRE enhancer.

[0051] Figure 12 The vector map for pLVX-TCR5-pTert-iCas9 is shown. This vector contains an ampicillin resistance gene, 5' and 3' LTRs, a CMV promoter, TCRβ sequences (TRBV24-1 leader peptide, TRBV25, TRBC) and TCRα sequences (TRAV38, TRAV35, and TRAC), a p2A sequence inserted between TCRβ and TCRα, an IRES sequence following the TCRα sequence, an mCherry fluorescent marker, and a WPRE enhancer.

[0052] Figure 13 The vector map used for the original pLVX-EF1α-IRES-mCherry vector is shown.

[0053] Figure 14The vector map of the pLVX-EF1α-IRES-mCherry vector used for modification is shown, in which excess AgeI, SphI, NheI and MluI restriction sites were eliminated by site-directed mutagenesis.

[0054] Figure 15 This image shows the DNA electrophoretic analysis of T cell receptor (TCR) amplicons (α:β strands) from single-cell emulsion overlap extension RT-PCR. The left panel indicates the PCR amplification protocol for paired α:β strand TCR amplicons. The right panel shows the DNA electrophoresis results of the first half-nested PCR, the second half-nested PCR, and Mi-seq PCR. T cell receptor amplicons are highlighted with red squares. Mi-seq primers annealed in the FR3 region of the TRBV gene, producing approximately 550 bp paired α:β PCR amplicons for naturally paired DNA sequence analysis.

[0055] Figure 16 This provides an overview of the distribution of the linked α:β T cell receptor genes. Following high-throughput sequencing, the raw DNA sequences were quality filtered and annotated using the NCBIIgBLAST and CDR3 motif algorithms, paired via α and β strands, and compiled into a TCR library.

[0056] Figure 17 The expression of mutant anti-HIV-Nef-Rm9 TCRs and their affinity for pMHC are shown, wherein these mutant TCRs include leader peptides that differ from the original wild-type anti-HIV-Nef-Rm9 leader peptide. These non-natural leader peptides all have silenced mutant DNA sequences to include restriction enzyme cleavage sites (the original and non-silent mutant leader peptide DNA sequences and the introduced restriction cleavage sites are summarized in Tables 7-8).

[0057] Figure 18 The expression of mutant anti-HIV-Nef-Rm9 TCRs and their binding affinity to pMHC are shown. These mutant TCRs contain either a non-silent mutation or a silent mutation in the TCR constant region for incorporation into the restriction cleavage site used for TCR cloning (see all silent mutations in Table 7 and non-silent mutations in Table 8).

[0058] Figure 19a This demonstrates the isolation of human effector T cells used for tumor-specific T cell analysis in humanized mouse models and cancer patients. Figure 19b The Vα:Vβ gene was shown in 31,718 isolated, RT-PCR amplified, and NGS-analyzed human CD8+ TCR clusters.

[0059] Figure 20The lentiviral TCR cell display platform demonstrates this technology. The TCRα:β gene, separated by p2a, is inserted into the multiple cloning site (MCS) of the IRESTCR expression vector. Lentiviral particles (expressed on the cell surface) of the J.RT3 / CD8 cell line transfected with packaging and envelope vectors in HEK293T cells are generated using the TCR gene. Flow plot: Cells with anti-B antibodies stained with HIV Nef B-0702 RM9 (RPQVPLRPM) (SEQ ID NO:1) 0702 NEF TM9 TCR J.RT3 / CD8.

[0060] Figure 21A This shows the size of RKO tumors in humanized mice treated with immune checkpoint inhibitors. TCR screening will be performed to identify antigen-specific TCRs targeting RKO cells. Figure 21B The image shows the treatment group in a mouse colon cancer model with an identified TCR, which will be used to evaluate antigen-specific immunotherapy.

[0061] Figure 22 This illustrates the workflow for testing and optimizing antigen-specific cancer immunotherapy in a PDX mouse model. (Left) Patient tumors are isolated and cultured as mouse xenografts, cryopreserved in LN2, and propagated in the culture. (Middle) TCR genes are collected and screened for PDX cells. (Right) Antitumor TCR genes are used as gene therapy in a PDX model to test and optimize therapeutic efficacy. Detailed Implementation

[0062] Immune checkpoint inhibitors have facilitated tremendous progress in cancer therapy, highlighting the crucial role of immune cells, particularly CD8+ T cells, in controlling tumor growth. However, while recent advances in immune receptor sequencing have provided insights into the clonal rank-based characterization of adaptive immunity, the inability to sequence and functionally screen paired TCR α and β chains hinders the collection of broader functional data on tumor immunosuppression and complicates the discovery and rapid therapeutic application of anti-cancer T cell receptor genes for gene therapy (Bonter, K. et al.). Regen. Med. 12, 623-636 (2017); Malherbe, L., Ann. Allergy. Asthma. Immunol. 103, 76-79 (2009); Ahmad, TA et al., Vaccine Rep. 6, 13-22 (2016); Maus, MV, etc. Annu. Rev. Immunol. 32, 189-225 (2014); Yee, C. J. Transl. Med.3, 17 (2005)). To date, due to the need for sorted live primary T cells, the clonal and functional analysis of tumor-specific T cells has been virtually limited to a few common peptide-MHC combinations for each sample (Malherbe, L., Ann. Allergy. Asthma. Immunol. 103, 76-79(2009); Ahmad, TA et al., Vaccine Rep. 6, 13-22 (2016); Sanchez-Trinchado, JL et al., Journal of Immunology Research (2017)). The fact that repeated screening is necessary for neoantigen discovery because each tumor sample has its own unique somatic mutation profile further complicates the situation where the number of effectively screenable peptide-MHCs is very small (Martincorena, I. and Campbell, PJ). Science 349, 1483-1489 (2015); Tran, E. et al. Science aad1253 (2015); Choudhury, NJ et al., Eur. Urol. Focus 2, 445-452 (2016). Alternative approaches (such as T-cell proliferation and ELISPOT) rely on live T cells, which are extremely limited in many patient samples and have limitations in cell growth rate, time required for application as a cell-based therapy, specificity, and maintenance of an appropriate T-cell phenotype (Bonter, K. et al.). Regen. Med. 12, 623-636 (2017); Maus, MV, etc. Annu. Rev. Immunol. 32, 189-225 (2014); Yee, C. J. Transl. Med. 3, 17 (2005); Redeker, A. & Arens, R., Front. Immunol. 7, (2016)).

[0063] The methods and compositions described herein directly address these bottlenecks and provide the first comprehensive sequence- and function-based annotation of epitope-specific T-cell responses in patients, offering novel molecular-scale techniques to guide the development of targeted cancer therapeutics. The compositions and methods of this technique can generally be used to rapidly isolate antigen-specific TCR libraries, which can be screened, modified, and used as personalized targeted therapies against, for example, cancer and viral infections. The targeted therapies described herein can be developed and administered much faster than currently available cell-based therapies (in as little as five days to two weeks). Furthermore, these therapies encompass a variety of different TCRs with a greater potential for immune evasion, a mechanism in which target cells can evade immune detection by blocking targeting epitopes or antigens.

[0064] Furthermore, specific embodiments of the methods and compositions described herein offer the following unique advantages over previously described methods for generating TCR libraries: (1) In some embodiments, a method is described herein in which TCR libraries can be grown as colonies in bacteria, and numerous colonies can be selected, sequenced, and mixed together for re-delivery as defined products; (2) In some embodiments, isolation and characterization steps are described herein that minimize the possibility of PCR reaction error variants that may be included in the final pharmaceutical product, thereby reducing the risk that the final therapeutic cell composition may contain uncharacterized and potentially very dangerous variants that could induce side effects; (3) In some embodiments, a suicide switch is described herein to detect complications and off-target effects. (4) In some embodiments, the cells are pre-stimulated before administration to achieve or enhance the desired TCR function in vivo; (5) In some embodiments, the method includes co-expressing transcription factors in cells to influence the ability of T cells to develop into an effective anticancer phenotype; (6) In some embodiments, the in vitro activation technique may be modified as needed depending on the patient’s specific response to the therapy; and (7) In some embodiments, the disclosed method may be used to provide the patient with repeated doses of cell therapy if the disease condition persists or recurs.

[0065] It should be understood that, in order to provide a substantial understanding of this technology, certain aspects, patterns, implementation schemes, variations and features of this method are described below with varying degrees of detail.

[0066] In implementing this method, many routine techniques from molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA were used. See, for example, Sambrook and Rus. cell Edited (2001) Molecular Cloning: A Laboratory Manual Molecular Cloning: A Laboratory Manual), 3rd edition; edited by Ausubel et al. (2007) Contemporary Molecular Biology Program ( Current Protocols in Molecular Biology ) series; enzymatic methods ( Methods in Enzymology ) series (Academic Press, NY); MacPherson et al. (1991) PCR1: Practical Methods ( PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: Practical Methods; Harlow and Lane (eds.) (1999) Antibodies, Laboratory Manual Antibodies, A Laboratory Manual ); Freshney (2005) Animal Cell Culture: A Basic Technical Manual ( Culture of Animal Cells: A Manual of Basic Technique ), 5th edition; edited by Gait (1984) Oligonucleotide Synthesis ( Oligonucleotide Synthesis ); US Patent No. 4,683,195; Hames and Higgins edited (1984) Nucleic acid hybridization ( Nucleic Acid Hybridization Anderson (1999) nucleic acid hybridization; Hames and Higgins editing (1984) transcription and translation. Transcription and Translation ) ； Immobilized cells and enzymes ( Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) Practical Guide to Molecular Cloning A Practical Guide to Molecular Cloning Miller and Calos edited (1987) gene transfer vectors for mammalian cells ( Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); edited by Makrides (2003) Gene transfer and expression in mammalian cells ( Gene Transfer and Expression in Mammalian Cells ); Mayer and Walker (eds., 1987) Immunochemical methods in cell and molecular biology ( Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., eds. (1996) Weil's Handbook of Experimental Immunology ( Weir’s Handbook of Experimental Immunology Methods for detecting and measuring the level of peptide gene expression products (i.e., gene translation level) are well known in the art and include the use of peptide detection methods (such as antibody detection) and quantitative techniques. (See also Strachan and Read, Human Molecular Genetics) Human Molecular Genetics ), Second Edition (John Wiley & Sons, NY, 1999).

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this art pertains. Unless expressly specified otherwise, the singular forms “a (a, an)” and “described” as used in this specification and the appended claims include a plural of indicators. For example, reference to “a cell” includes a combination of two or more cells, etc. Generally, the nomenclature used herein and the laboratory procedures described below in cell culture, molecular genetics, organic chemistry, analytical chemistry, nucleic acid chemistry, and hybridization are those well-known and commonly used in the art. All references cited herein are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, or patent application were expressly and individually incorporated by reference in their entirety for all purposes.

[0068] Unless otherwise stated or clearly indicated from the context (unless the number will be less than 0% or more than 100% of the possible value), the term “about” as used herein, when referring to the number, generally includes the number falling within the range of 1%, 5%, or 10% in either direction (greater or less).

[0069] As used herein, “administering” a drug or medicine to a subject includes any route by which a compound is introduced or delivered to a subject to perform its intended function. Administration may be carried out via any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, or local administration. Administration includes self-administration and administration by another person.

[0070] An "adjuvant" is one or more substances that stimulate the immune system. In this case, an adjuvant is used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants may be administered to the subject before, in combination with, or after the administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulatory complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponins), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

[0071] As used herein, the term "antibody" generally refers to immunoglobulins or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, such as mammals like humans, goats, rabbits, and mice, and in non-mammalian species (such as shark immunoglobulins). The term "antibody" includes both complete immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to the molecule of interest (or a highly similar set of molecules of interest), while substantially excluding binding to other molecules (e.g., a binding constant for the molecule of interest that is at least 10 greater than the binding constant for other molecules in the biological sample). 3 M -1 At least 10 4 M -1 Or at least 10 5 M -1 (Antibodies and antibody fragments). The term "antibody" also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugated antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Company, Rockford, Ill.); Kuby, J. Immunology , 3rd edition, WH Freeman & Co., New York, 1997.

[0072] More specifically, an antibody is a polypeptide ligand that contains at least a variable region of a light chain immunoglobulin or a variable region of a heavy chain immunoglobulin, said variable region specifically recognizing and binding to an epitope of an antigen. Antibodies are composed of heavy and light chains, each with a variable region called a variable heavy chain (VLC). H ) region and variable light chain (V L ) area. V H District and V L Together, these regions are responsible for binding antigens recognized by antibodies. Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains (lambda (λ) and kappa (κ)). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains constant and variable regions (also referred to as “domains”). Together, the heavy chain variable regions and light chain variable regions specifically bind antigens. The light chain variable regions and heavy chain variable regions contain “framework” regions interrupted by three hypervariable regions (also known as “complementarity-determining regions” or “CDRs”). The extent of the frame regions and CDRs has been defined (see Kabat et al., Protein Sequences of Immunological Concern).Sequences of Proteins of Immunological Interest (See, U.S. Department of Health and Human Services, 1991, which is incorporated herein by reference). The Kabat database is now kept online. The framework region sequences of different light or heavy chains are relatively conserved within species. The framework region of an antibody, a combination of constitutive light and heavy chains, predominantly adopts a β-sheet conformation and the CDR forms a loop that connects to the β-sheet structure and, in some cases, forms part of the β-sheet structure. Thus, the framework region serves to form a scaffold that positions the CDR in the correct orientation through interchain non-covalent interactions.

[0073] As used herein, the "complementarity-determining region" or "CDR" refers to the region of an antibody or TCR primarily responsible for binding to an antigen or an epitope of the antigen:MHC complex. CDRs are also known as hypervariable regions. The CDRs of individual TCRs or antibody chains are typically referred to as CDR1, CDR2, and CDR3 (numbered sequentially from the N-terminus), and are often identified by the chain in which a particular CDR is located. Antibodies and TCRs with different specificities (i.e., different binding sites for different antigens) have different CDRs. Only a limited number of amino acid sites within a CDR directly participate in antigen binding. These sites within a CDR are called specificity-determining residues (SDRs).

[0074] As used herein, "antigen" refers to a molecule to which an antibody can selectively bind. Target antigens can be proteins (e.g., antigenic peptides), carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. Target antigens can be polypeptides or peptide mimics. Antigens may also be administered to animals to generate an immune response in the animals. In some embodiments, the antigen comprises one or more epitopes. In some embodiments, the antigen or an epitope derived from the antigen may be loaded into an MHC class I or MHC class II complex.

[0075] As used herein, the term "autologous" refers to cells that have been isolated and administered back to the same subject (e.g., recipient, donor, or host). "Allogeneic" refers to non-autologous cells.

[0076] As used herein, “binding affinity” refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K0). dAffinity can be measured using standard methods known in the art, including those described herein. Low-affinity complexes contain antibodies that generally tend to dissociate easily from the antigen, while high-affinity complexes contain antibodies that generally tend to remain bound to the antigen for a longer period.

[0077] As used herein, the term "B cell" refers to a type of lymphocyte in humoral immunity within the adaptive immune system. The primary functions of B cells are antibody production, serving as antigen-presenting cells, releasing cytokines, and developing into memory B cells upon activation through antigen-antigen interactions. B cells differ from other lymphocytes (such as T cells) in the presence of B cell receptors on their cell surface. B cells can be isolated or obtained from commercially available sources. Non-restrictive examples of commercially available B cell lines include the following: AHH-1 (ATCC® CRL-8146™), BC-1 (ATCC® CRL-2230™), BC-2 (ATCC® CRL-2231™), BC-3 (ATCC® CRL-2277™), CA46 (ATCC® CRL-1648™), DG-75 [DG-75] (ATCC® CRL-2625™), DS-1 (ATCC® CRL-11102™), EB-3 [EB3] (ATCC® CCL-85™), Z-138 (ATCC number CRL-3001), DB (ATCC CRL-2289), Toledo (ATCC CRL-2631), Pfiffer (ATCC CRL-2632), SR (ATCC CRL-2262), JM-1 (ATCC CRL-10421), NFS-5 C-1 (ATCC CRL-1693); NFS-70 C10 (ATCC CRL-1694), NFS-25 C-3 (ATCC CRL-1695) and SUP-B15 (ATCC CRL-1929). Other examples include, but are not limited to, cell lines derived from anaplastic and large cell lymphomas, such as DEL, DL-40, FE-PD, JB6, Karpas 299, Ki-JK, Mac-2A Ply1, SR-786, SU-DHL-1, SU-DHL-2, SU-DHL-4, SU-DHL-5, SU-DHL-6, SU-DHL-7, SU-DHL-8, SU-DHL-9, SU-DHL-10 and SU-DHL-16, DOHH-2, NU-DHL-1, U-937, Granda 519, USC-DHL-1, RL; and cell lines derived from Hodgkin's lymphomas, such as DEV, HD-70, HDLM-2, HD-MyZ, HKB-1, KM-H2, L 428, L 540, L1236, SBH-1, SUP-HD1, SU / RH-HD-l. Non-limiting exemplary sources of such commercially available cell lines include the U.S. Type Culture Collection or ATCC (www.atcc.org / ) and the German Center for Microbiology and Cell Culture Collection (https: / / www.dsmz.de / ).

[0078] As used herein, the term "association" refers to a correspondence between two molecules (e.g., between a receptor and its ligand). In the context of TCRs, an "association pair" refers to the relationship between two distinct TCR polypeptides or polynucleotides encoding polypeptides derived from a single T cell (e.g., TCRα and TCRβ chains derived from a single T cell). "Association" can also refer to the relationship between a TCR and its specific antigen-MHC complex with which it binds.

[0079] As used herein, the term "CRISPR" refers to a sequence-specific genetic manipulation technique using the cluster-dependent regular interval short palindromic repeat pathway (CRISPR). CRISPR can be used for gene editing and / or gene regulation, as well as for simply targeting proteins to specific genomic locations. Gene editing is the genetic engineering process of altering the nucleotide sequence of a target polynucleotide by introducing deletions, insertions, or base substitutions into a polynucleotide sequence. In some respects, CRISPR-mediated gene editing utilizes non-homologous end joining (NHEJ) or homologous recombination pathways. Gene regulation refers to increasing or decreasing the production of specific gene products, such as proteins or RNA.

[0080] As used herein, the term “guide RNA” or “gRNA” refers to a guide RNA sequence used to target CRISPR complexes to specific nucleotide sequences, such as specific regions of the cellular genome. Techniques for designing target-specific gRNAs and donor therapeutic polynucleotides are well known in the art. For example, Doench, J. et al. Nature biotechnology 2014; 32(12):1262-7; Mohr, S. et al. (2016) FEBS Journal 283: 3232-38; and Graham, D. et al. Genome Biol. 2015; 16: 260. gRNAs include fusion polynucleotides comprising CRISPR RNA (crRNA) and trans-activated CRIPSPR RNA (tracrRNA); or polynucleotides comprising CRISPR RNA (crRNA) and trans-activated CRIPSPR RNA (tracrRNA); or optionally consisting substantially of the aforementioned fusion polynucleotides or polynucleotides, or further comprising them. In some respects, gRNA is synthetic (Kelley, M. et al. (2016) J of Biotechnology 233 (2016) 74-83).

[0081] As used herein, the term "detectable biomarker" refers to at least one biomarker capable of generating a detectable signal, directly or indirectly. A non-exhaustive list of exemplary biomarkers includes, for example, enzymes that generate detectable signals by colorimetry, fluorescence, or luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent or luminescent dyes; groups having an electron density detectable by electron microscopy or by their electrical properties (such as conductivity, galvanometry, voltammetry, and impedance); detectable groups, such as those whose molecules are large enough to induce detectable changes in their physical and / or chemical properties, such as those detectable by optical methods (such as diffraction, surface plasmon resonance, surface variation, and contact angle variation) or physical methods (such as atomic force spectroscopy and tunneling effects) or radioactive molecules (such as...). 32 P, 35 S or 125 I) to achieve this.

[0082] As used herein, the term "purification marker" refers to at least one marker that can be used for purification or identification. A non-exhaustive list of such markers includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy5, Cy5.5, Cy7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluor, FITC, TRITC, or any other fluorescent dye or hapten.

[0083] As used herein, the term "effective amount" or "therapeutic effective amount" of a composition is an amount sufficient to achieve the desired therapeutic effect, such as an amount resulting in a reduction of symptoms associated with the treated disease (e.g., a disease or medical condition related to cancer or a viral infection). The amount of the composition of this technology administered to a subject will depend on the type and severity of the disease and individual characteristics such as general health status, age, sex, weight, and tolerance to the drug. It will also depend on the extent, severity, and type of the disease. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors. The composition of this technology may also be administered in combination with one or more additional therapeutic compounds. In some embodiments, an effective amount refers to the amount of cells of this technology that are partially or completely effective in neutralizing cancer or viral infections.

[0084] As used herein, the term "epitope" refers to a protein determinant that specifically binds to an antibody or TCR. Epitopes typically consist of chemically active surface groups on the side chains of molecules such as amino acids or sugars and usually possess specific three-dimensional structural characteristics and specific charge properties. The difference between conformational epitopes and non-conformational epitopes is that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0085] As used herein, “expression” includes one or more of the following: gene transcription into precursor mRNA; splicing and other processing of precursor mRNA to produce mature mRNA; mRNA stability; translation of mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if appropriate for expression and function.

[0086] As used herein, “elevated expression” means an increase in gene or protein expression compared to a control or reference sample (e.g., an increase of at least 2-fold, about 2-fold to about 150-fold, 5-fold to 150-fold, 5-fold to 100-fold, 10-fold to 150-fold, 10-fold to 100-fold, 50-fold to 150-fold, 50-fold to 100-fold, 75-fold to 150-fold, or 75-fold to 100-fold compared to a control or normal reference sample). “Decreased expression” means an overall decrease in gene or protein expression compared to a control or reference sample (e.g., 20% or greater, 50% or greater, or 75%, 80%, 85%, 90%, 95% or greater). Increases or decreases in gene or protein expression can be determined using any useful method known in the art or described herein (e.g., ELISA). For therapeutic applications, “decreased” can refer to a decrease in the level of a peptide or protein associated with a condition (e.g., T1 proteinosis, TBI, or stroke). For diagnostic or monitoring applications, "reduction" can refer to a decrease in the level of protein or nucleic acid detected by diagnostic or monitoring methods.

[0087] As used in this article, the term "gene" refers to a segment of DNA containing all the information about the regulated biosynthesis of RNA products, including promoters, exons, introns, and other untranslated regions that control expression.

[0088] As used herein, nonhuman proteins in the form of “humanized” (e.g., mouse TCRs) are chimeric proteins containing minimal sequences derived from nonhuman homologues of the protein. Most humanized proteins are human TCRs, in which the variable region residues of the recipient are replaced by variable region residues from a nonhuman species (such as a mouse, rat, rabbit, or nonhuman primate) (donor TCR) with desired specificity, affinity, and ability.

[0089] When used in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percentage "identity" as used herein refers to two or more identical sequences or subsequences, or sequences with a specified percentage of identical amino acid residues or nucleotides (i.e., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of identity in a specified region (e.g., the nucleotide sequence encoding the antibody described herein or the amino acid sequence of the antibody described herein) when compared and aligned for maximum correspondence in a comparison window and specified region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are referred to as "substantially identical." The term also refers to, or may be applied to, complementary sequences of the test sequence. The term also includes sequences with deletions and / or additions, as well as sequences with substitutions. In some implementations, identity exists in regions of at least about 25 amino acids or nucleotides in length, or of 50-100 amino acids or nucleotides in length.

[0090] A “Kozak concordant sequence” or “Kozak sequence” is an mRNA sequence recognized by ribosomes as a translation initiation site. A Kozak sequence contains a start codon (also called an initiation codon) and additional flanking nucleotides for initiating translation. The start codon specifies the N-terminal methionine amino acid of the polypeptide to be translated. Vertebrate Kozak concordant sequences are known in the art (e.g., Kozak, M. 1987 NucleicAcids Res. 15(20): 8125-48). In some embodiments, the Kozak sequence can be modified to be “strong,” meaning that the nucleotide sequence closely matches the concordant sequence, particularly at nucleotides +4 and -3 relative to the first nucleotide. A “sufficient” Kozak sequence has only one of these matching nucleotides, while a “weak” Kozak sequence has none. The strength of the Kozak sequence is directly related to the amount of polypeptide translated from the expressed mRNA. Typically, strong Kozak sequences lead to more efficient translation of expressed mRNAs, while fewer peptides are transcribed from mRNAs with weak Kozak sequences.

[0091] As used in this article, "major histocompatibility complex" or "MHC" refers to cell surface proteins that present antigens to T cells. Class I MHC molecules are recognized by CD8+ T cells. Class II MHC molecules are recognized by CD4+ T cells. MHC molecules loaded with antigens or their epitopes are called antigen:MHC complexes.

[0092] As used herein, the term "NK cell" (also known as natural killer cell) refers to a type of lymphocyte that originates in the bone marrow and plays a key role in the innate immune system. NK cells provide a rapid immune response against virally infected cells, tumor cells, or other stress cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface. NK cells can be isolated or obtained from commercially available sources. Non-limiting examples of commercial NK cell lines include the NK-92 line (ATCC® CRL-2407™) and NK-92MI (ATCC® CRL-2408™). Other examples include, but are not limited to, NK strains HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplary sources of such commercially available cell lines include the U.S. Center for Type Culture Collection or ATCC (http: / / www.atcc.org / ) and the German Center for Microbiology and Cell Culture Collection (https: / / www.dsmz.de / ).

[0093] As used herein, the term "operably linked" refers to two or more polynucleotides that are part of the same nucleic acid molecule. In some embodiments, the linked polynucleotides are appropriately positioned and oriented to initiate transcription from the same expression control element. In some embodiments, the transcription of the polynucleotides operably linked to an expression control element (e.g., a promoter) is controlled, regulated, or influenced by the expression control element.

[0094] As used herein, the term "pharmaceuticalally acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption-delayed compounds, etc., compatible with drug administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, edited by A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0095] As used herein, the terms "polynucleotide" or "nucleic acid" refer to any RNA or DNA, which may be unmodified or modified. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA, which may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, a polynucleotide refers to a triple-stranded region comprising RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone is modified for stability or other reasons.

[0096] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers containing two or more amino acids linked together by peptide bonds or modified peptide bonds (i.e., peptide isosteres). A polypeptide can refer to both a short chain, commonly called a peptide, glycopeptide, or oligomer, and a long chain, commonly called a protein. Polypeptides may contain amino acids in addition to the 20 amino acids encoded by genes. Polypeptides contain amino acid sequences modified by natural processes (such as post-translational processing) or by chemical modification techniques well known in the art. Such modifications are described in detail in basic textbooks and more detailed monographs, as well as in a large body of research literature.

[0097] When a polypeptide, peptide, polynucleotide, or cell has been removed from another cell or cell component using physical, mechanical, or chemical methods, it is considered "isolated" or "substantially pure." An isolated polypeptide, peptide, polynucleotide, or cell (e.g., isolated cells), a "substantially pure" or "substantially pure and isolated" polypeptide, peptide, polynucleotide, or cell is generally considered to have been removed from the cell component and is substantially pure when at least 60% by weight of it does not contain proteins and naturally occurring organic molecules bound to it. The polypeptide may be at least 75%, 80%, 85%, 90%, 95%, or 99% by weight pure. Substantially pure polypeptides, peptides, polynucleotides, or cells (e.g., substantially pure antibodies or fragments thereof) can be obtained using standard techniques, such as by expressing recombinant nucleic acids encoding the polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any suitable method, such as by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0098] When used in reference to, for example, cells or nucleic acids, proteins or vectors, the term “recombinant” as used herein indicates that the cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein or by altering the native nucleic acid or protein, or that the material is derived from cells that have been so modified. Thus, for example, recombinant cells express genes not found in native (non-recombinant) cell forms, or native genes that are otherwise abnormally, poorly, or not expressed at all.

[0099] As used in this article, “single” therapeutic application means the simultaneous or substantially simultaneous administration of at least two active ingredients via different routes.

[0100] As used herein, “repeated” therapeutic application refers to the administration of the active ingredient at different times, via the same or different routes of administration. More specifically, sequential use refers to the complete administration of the active ingredient before the initiation of a second application of the same or different active ingredient. Thus, one of the active ingredients may be administered minutes, hours, days, months, or years before the second application.

[0101] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably and refer to humans or non-human animals, such as livestock (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), wild animals (bats, raccoons, foxes, skunks, squirrels, chipmunks, mice, rabbits, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). In some embodiments, the subject is a mammal. In a particular embodiment, the subject is a human.

[0102] As used herein, the term "switch" refers to a mechanism by which the expression, activation, or stability of a recombinant TCR or a component of a recombinant TCR is controlled (i.e., a mechanism that "turns" the TCR "on" or "off"). Switching mechanisms include, but are not limited to, TCR expression systems that require co-expression of more than one construct to be activated, suicide switches, safety switches, and TCRs that require polymerization for activation. In some embodiments, the switch is inductive.

[0103] As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play a crucial role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of T cell receptors on their cell surface. T cells can be isolated or obtained from commercially available sources. "T cells" encompass all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses. Non-limiting examples of commercially available T-cell lines include the cell lines BCL2 (AAA) Jurkat (ATCC® CRL-2902™), BCL2 (S70A) Jurkat (ATCC® CRL-2900™), BCL2 (S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL-2899™), Neo Jurkat (ATCC® CRL-2898™), and the TALL-104 cytotoxic human T-cell line (ATCC number CRL-11386).In addition, examples include, but are not limited to, mature T-cell lines such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T-cell lines such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, and MOLT 13. MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1, T ALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, TLBR-2, TLBR-3 and TLBR-4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCCTIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4;11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and cutaneous T-cell lymphoma lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), and HuT102 (ATCC TIB-162). Nulleukemia cell lines, including but not limited to REH, NALL-1, KM-3, and L92-221, are another commercially available source of immune cells, similarly derived from other leukemia and lymphoma cell lines (such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, and U266 myeloma). Non-limiting exemplary sources of such commercially available cell lines include the U.S. Type Culture Collection or ATCC (http: / / www.atcc.org / ) and the German Center for Microbiology and Cell Culture (https: / / www.dsmz.de / ).

[0104] As used herein, the term "T-cell receptor" or "TCR" refers to a heterodimeric cell surface protein of the immunoglobulin superfamily associated with an invariant protein of the CD3 complex involved in mediating signal transduction. The TCR consists of two associated protein chains: an alpha (α) chain and a beta (β) chain (encoded by TRA (Entrez gene: 6955) and TRB (Entrez gene: 6957), respectively), or a gamma (γ) chain and a delta (δ) chain (encoded by TRG (Entrez gene: 6965) and TRD (Entrez gene: 6964), respectively). Each chain comprises two extracellular domains: a variable (V) region and a constant (C) region. The constant region is adjacent to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the antigen:MHC complex. Both the α and β chains of the TCR have three hypervariable regions, or complementarity-determining regions (CDRs), within their variable domains. Another hypervariable region exists on the β chain (HV4), which typically does not contact the antigen and is therefore not considered a CDR. CDR3 is the major CDR responsible for recognizing processed antigens, although it has also been shown that CDR1 of the α chain interacts with the N-terminal portion of the antigenic peptide, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC. Although CDR4 of the β chain is not considered to be involved in antigen recognition, it has been shown to interact with superantigens. The constant domain of the TCR consists of short linker sequences in which cysteine ​​residues form disulfide bonds that connect the two chains.

[0105] The diverse TCR repertoire in subjects is achieved through V(D)J recombination, a somatic recombination mechanism that rearranges variable (V) gene segments, conjugation (J) gene segments, and diversity (D) gene segments. In humans, the TRA locus contains 54 TRAV (V) segments, 61 TRAJ (J) segments, and a unique constant TRAC (C) segment. The TRB locus contains 64–67 TRBV (V) segments, 2 TRBD (D) segments, 14 TRBJ (J) segments, and 2 TRBC (C) segments. The TRG locus contains 12–15 TRGV (V) segments, 5 TRGJ (J) segments, and 2 TRGC (C) segments. The TRD locus is embedded within the TRA locus and contains 8 TRDV or TRAV / DV (V) segments, 3 TRDD (D) segments, 4 TRDJ (J) segments, and one TRDC (C) segment. Non-limiting examples of TCR amino acid sequences are known in the art and are provided herein.

[0106] Human TCR (α) chain TRAC segment: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO: 2) Human TCR (β) chain TRBC1 region: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 3) Human TCR (β) chain TRBC2 region: DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 4) Human TCR (γ) chain TRGC1 region: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO: 5) Human TCR (γ) chain TRGC2 region: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKDSYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLGRTAFCCNGEKS (SEQ ID NO: 6) Human TCR (δ) chain TRDC segment: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO: 7) As used herein, the term "therapeutic agent" is intended to mean a nucleic acid, recombinant TCR, vector, cell, or cell population that, when present in an effective amount, produces the desired therapeutic effect on a subject in need.

[0107] This encompasses one or more amino acid sequence modifications of the TCR described herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the TCR. Amino acid sequence variants of the TCR are prepared by introducing appropriate nucleotide changes into the TCR nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the TCR. Any combination of deletions, insertions, and substitutions may be performed to obtain the TCR of interest, provided that the obtained TCR possesses the desired properties. Modifications also include alterations to the protein glycosylation pattern. Sites of most interest in substitution-induced mutagenesis include hypermutation regions, but FR alterations are also covered. “Conserved substitutions” are shown in the table below.

[0108]

[0109] As used herein, “specifically binding” means a molecule that recognizes and binds to another molecule (e.g., an antigen:MHC complex) but substantially does not recognize and bind to other molecules (e.g., a TCR). The terms “specifically binding,” “specifically binding to,” or “specifically targeting” a particular molecule (e.g., a particular cell, antigen, epitope, or antigen:MHC complex) as used herein can be, for example, by the molecule to which it binds having at least about 10 -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or larger K d The term "specific binding" can also refer to the binding of a molecule (e.g., a TCR) to a specific cell, antigen, epitope, or antigen:MHC complex while substantially not binding to any other cell, antigen, epitope, or antigen:MHC complex. For example, the affinity of a TCR for one antigen:MHC complex can be, for example, at least 10 to 100 times greater than its affinity for another antigen:MHC complex (e.g., 10...). 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 Or 10 10 times).

[0110] As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different hosts, including but not limited to plasmids, viruses, granules, bacteriophages, BAC, YAC, etc. In some embodiments, plasmid vectors may be prepared from commercially available vectors. In other embodiments, viral vectors may be generated from baculoviruses, retroviruses, adenoviruses, AAVs, etc., according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector.

[0111] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters can be, for example, constitutive, inducible, repressive, or tissue-specific. A "promoter" is a control sequence, a region in a polynucleotide sequence that controls the initiation and rate of transcription. It may contain genetic elements that regulate the binding of proteins and molecules, such as RNA polymerases and other transcription factors. Non-limiting examples of promoters include p2A, CMV, and E1α.

[0112] The term "transduction" (or "transduction"), when used in the context of recombinant cell generation, refers to the process of introducing a foreign nucleotide sequence into a cell. In some implementations, this transduction is accomplished via a vector.

[0113] As used herein, “treating” encompasses treating the disease or condition described herein in a subject (such as a person) and includes: (i) suppressing the disease or condition, i.e., preventing its development; (ii) alleviating the disease or condition, i.e., causing the remission of the condition; (iii) slowing the progression of the condition; and / or (iv) suppressing, alleviating, or slowing the progression of one or more symptoms of the disease or condition. “Treating cancer” means that cancer or cancer cells are, for example, reduced, decreased, cured, or placed in remission. “Treating a viral infection” means that a virus or viral load is, for example, reduced, decreased, cured, or placed in remission.

[0114] It should also be understood that the various treatment modalities for cancer and viral infections described in this article are intended to mean “substantial,” which includes complete treatment as well as less-than-complete treatment, and in which some biological or medically relevant outcome is achieved, such as extended lifespan of the subject. Treatment can be continuous, extended therapy to prevent recurrence, or, for emergency treatment, administration for a short period of time.

[0115] I. Composition provided by the present technology In one aspect, this technology provides a recombinant T-cell receptor (TCR) library vector comprising: (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are an associated pair, and wherein the first polynucleotide and the second polynucleotide are derived from mRNA isolated from a single lysed T cell present in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated using an mRNA capture reagent or reverse transcription-PCR (RT-PCR). Alternatively or optionally, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a droplet. In some embodiments, the mRNA of the single lysed T cell is isolated within the compartment without using an mRNA capture reagent.

[0116] The term "vector" refers to a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the genome of a target cell. In some embodiments, the vector is derived from or based on a wild-type virus. In other embodiments, the vector is derived from or based on a wild-type lentivirus, retrovirus, adenovirus, or adeno-associated virus. Examples include, but are not limited to, human immunodeficiency virus (HIV), human T-lymphotropic virus type 1 (HTLV-1), human T-lymphotropic virus type 2 (HTLV-2), human adenovirus (HadV-1 to 57), adeno-associated virus (AAV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and murine leukemia virus (MLV). It will be apparent that viral vectors according to this disclosure are not necessarily limited to components of a specific virus. Viral vectors may contain components derived from two or more different viruses and may also contain synthetic components.

[0117] In some embodiments of the vector, the vector backbone is selected from the group consisting of retroviral vector backbones, lentiviral vector backbones, adenoviral vector backbones, and adeno-associated virus vector backbones. The genome of the vector backbone contains components from the virus of its original source. For example, in some embodiments, the vector backbone contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. In some embodiments, these include gag and pol proteins derived from a specific retrovirus. In some embodiments, structural components of the vector backbone have been genetically or otherwise modified to provide desired useful properties. For example, the vector host range and target cell type can be altered by using different env genes in the vector production system to confer different target specificities on the vector.

[0118] In certain embodiments, the vector backbone is a lentiviral vector backbone, such as pLVX. Lentiviral vectors are based on or derived from tumor retroviruses (a subgroup of retroviruses containing MLV) and lentiviruses (a subgroup of retroviruses containing HIV). Non-limiting examples include ASLV, SNV, and RSV, all of which have been categorized into packaging and vector components for use in lentiviral vector particle production systems. Lentiviral vector particles according to this disclosure can be modified in form based on the genetic or other means (e.g., through specific selection of the packaging cell system) of a particular retrovirus.

[0119] Non-limiting exemplary vector backbones are known in the art, for example, see U.S. Patent Nos. 6,924,123; 7,056,699; 7,07,993; 7,419,829 and 7,442,551, which are incorporated herein by reference; and the Invitrogen spLenti series versions 4, 6 and 6.2 “ViraPower” system pHIV-7-GFP, generated and used in the laboratory by the City of Hope Research Institute; the “Lenti-X” lentiviral vector pLVX, manufactured by Clontech; pLKO.1-puro, manufactured by Sigma-Aldrich; pLemiR, manufactured by Open Biosystems; and pLV, generated and used in the laboratory by Charite Medical School, Institute of Virology (CBF), Berlin, Germany.

[0120] Certain retroviral sequences facilitate integration into the target cell genome (see, for example, U.S. Patent No. 6,924,123). Each retroviral genome contains genes called gag, pol, and env, which encode virion proteins and enzymes. These genes are flanked by regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. They also function as enhancer-promoter sequences capable of controlling viral gene expression. Capsidation of the retroviral genome occurs via a ψ sequence located at the 5′ end of the viral genome. The LTR itself is a single sequence that can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3′ end of the RNA. R is derived from a repeating sequence at both ends of the RNA, and U5 is derived from a sequence unique to the 5′ end of the RNA. The size of these three elements can vary considerably among different retroviruses. U3 contains most of the proviral expression control elements, including promoters and multiple enhancer sequences that respond to cellular transcriptional activation proteins and, in some cases, viral transcriptional activator proteins.

[0121] Regarding the structural genes gag, pol, and env themselves, gag encodes the virus's internal structural proteins. The Gag protein is processed by proteolysis into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT) (which contains DNA polymerase), associated RNase H, and integrase (IN), enzymes that mediate genome replication.

[0122] Viral particles are generated by expressing a vector RNA genome from a DNA construct encoding a vector RNA genome in a host cell. In some embodiments, components of the viral particle not encoded by the vector backbone are provided in a trans-form by additional nucleic acid sequences expressed in the host cell (“packaging system,” which typically includes one or both of the gag / pol and env genes). In some embodiments, the sequence set required to generate the viral vector particles is introduced into the host cell via transient transfection, integrated into the host cell genome, or provided using a packaging cell line. The techniques involved are known to those skilled in the art.

[0123] In some embodiments, the methods or processes for deriving the first and second polynucleotides result in structural features in the first and / or second polynucleotides that differ from those of other vectors and methods known in the art. For example, using an mRNA capture reagent in conjunction with a single compartment for lysing T cells allows the capture of the associated pair of TCR polynucleotides. Other non-limiting examples of structural features include restriction enzyme recognition sites, integration primer sites, and sequences derived from the mRNA capture reagent.

[0124] In some embodiments of the vector, the mRNA capture agent is selected from the group consisting of poly(dT)-coated beads, oligonucleotide-coated beads, hydrogel beads, and printed oligomers on the surface of microarray pores. For example, the mRNA capture agent can be a solid support, such as beads, containing an immobilized network of oligonucleotides or polymers, such as dextran and agarose. In some embodiments, the beads are silica beads or magnetic beads. In some embodiments, the mRNA capture agent contains oligonucleotides that hybridize with mRNA. For example, the oligonucleotides may contain at least one poly(T) and / or a primer specific to the transcript of interest. In some embodiments, the beads of the mRNA capture agent are smaller than the isolated single cells (e.g., T cells). In some embodiments, single T cells are chelated with the mRNA capture agent prior to T cell lysis. In other embodiments, chelation of single T cells with the mRNA capture agent is performed simultaneously with T cell lysis. Thus, in some embodiments, single T cells and the mRNA capture agent are separated into single microvesicles in an emulsion in the presence of a cell lysis solution.

[0125] In some embodiments, a single compartment has a volume of 100 nL or less, 50 nL or less, 40 nL or less, 30 nL or less, 20 nL or less, 10 nL or less, or 5 nL or less. In a particular embodiment, a single compartment has a volume of 5 nL or less. In some embodiments, a single compartment is a microdroplet or microbubble, optionally in an emulsion. In some embodiments, the compartment is a pore. In some embodiments, the pore is located in a printed polymer slide, plastic plate, microtiter plate, or gel. In some embodiments, the pore is sealed with a permeable membrane before T cell lysis or before RT-PCR. Compartmentalized preparation as described herein enables library characterization and minimizes the possibility of PCR erroneous variants that may be included in the final pharmaceutical product. This reduces the risk that the final therapeutic cell composition contains uncharacterizable and potentially very dangerous variants that can induce side effects and / or off-target TCR binding specificity.

[0126] In some embodiments, mRNA from a single lysed T cell is reverse transcribed into cDNA using any method known in the art. For example, in some embodiments, reverse transcription is performed using overlap extension (OE) reverse transcription PCR (RT-PCR). The OE-RT-PCR reaction mixture includes primers designed to produce a single PCR product containing cDNAs of two or more transcripts of interest covalently linked together. The primer design for OE-RT-PCR determines which transcripts of interest (e.g., TCR gene transcripts) expressed by a given cell are linked together. For example, in some embodiments, primers are designed to covalently link individual cDNAs from TCR strand transcripts associated with each other. Non-limiting examples of OE-RT-PCR reaction conditions are provided in Table 3 herein. Non-limiting examples of PCR primers suitable for performing the reaction to obtain ligated TCR cDNA are provided in Table 4 herein. The ligated cDNA product from the OE RT-PCR is recovered and used as a template for nested PCR, which amplifies the ligated transcripts of interest. Exemplary reaction conditions for nested PCR are provided in Table 5 herein. In some embodiments, the purified products of nested PCR are then sequenced and paired information is analyzed. In some embodiments, restriction and ligation can be used to ligate cDNAs of multiple transcripts of interest. In other embodiments, recombination can be used to ligate cDNAs of multiple transcripts of interest.

[0127] In some embodiments, the TCRα polypeptide comprises at least one TRAV segment and a TRAC segment, or equivalents thereof. In some embodiments, the TCRα polypeptide comprises at least one TRAV segment, a TRAC segment, and at least one TRAD segment. In some embodiments, the TCRα polypeptide comprises at least one TRAV segment, a TRAC segment, at least one TRAD segment, and at least one TRAJ segment. In some embodiments, the TCRα polypeptide comprises at least one TRAV segment, a TRAC segment, and at least one TRAJ segment.

[0128] In some embodiments, the TCRβ polypeptide comprises at least one TRBV segment and a TRBC segment, or equivalents thereof. In some embodiments, the TCRβ polypeptide comprises at least one TRBV segment, at least one TRBC segment, and at least one TRBD segment. In some embodiments, the TCRβ polypeptide comprises at least one TRBV segment, at least one TRBC segment, at least one TRBD segment, and at least one TRBJ segment. In some embodiments, the TCRβ polypeptide comprises at least one TRBV segment, at least one TRBC segment, and at least one TRBJ segment.

[0129] In some embodiments, the TCRγ polypeptide comprises at least one TRGV segment and a TRGC segment, or equivalents thereof. In some embodiments, the TCRγ polypeptide comprises at least one TRGV segment, at least one TRGC segment, and at least one TRGD segment. In some embodiments, the TCRγ polypeptide comprises at least one TRGV segment, at least one TRGC segment, at least one TRGD segment, and at least one TRGJ segment. In some embodiments, the TCRγ polypeptide comprises at least one TRGV segment, at least one TRGC segment, and at least one TRGJ segment.

[0130] In some embodiments, the TCRδ polypeptide comprises at least one TRDV segment and a TRDC segment, or equivalents thereof. In some embodiments, the TCRδ polypeptide comprises at least one TRDV segment, at least one TRDC segment, and at least one TRDD segment. In some embodiments, the TCRδ polypeptide comprises at least one TRDV segment, at least one TRDC segment, at least one TRDD segment, and at least one TRDJ segment. In some embodiments, the TCRδ polypeptide comprises at least one TRDV segment, at least one TRDC segment, and at least one TRDJ segment.

[0131] In some embodiments of the vector, the first and second polynucleotides are optionally operably linked via a linker polynucleotide. In some embodiments, the linker polynucleotide encodes a linker polypeptide. As used herein, the term "linker polypeptide" refers to any amino acid sequence comprising 1 to 10, or optionally 8, or optionally 6, or optionally 5 amino acids, repeated 1 to 10 times, or optionally up to about 8 times, or optionally up to about 6 times, or optionally about 5 times, or 4 times, or optionally 3 times, or optionally 2 times. For example, the linker may comprise up to 15 amino acid residues consisting of a pentapeptide repeated three times. In one aspect, the linker sequence is a (glycine-4serine)3 (SEQ ID NO:8) flexible polypeptide linker (which comprises three copies of gly-gly-gly-ser (SEQ ID NO:9)) or its equivalent. Non-limiting examples of adapter sequences known in the art include, for example, GGGGSGGGGGSGGGG (SEQ ID NO: 10) (and its equivalents); the tripeptide EFM; or Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO: 11), and their respective equivalents. In some embodiments of the vector, the first and second polynucleotides have been operatively ligated by reverse transcription and PCR amplification of the captured T-cell mRNA.

[0132] In some embodiments of the vector, a restriction enzyme that cleaves at a target restriction endonuclease site naturally found in the TCR gene is used to link and clone the first and second polynucleotides into the vector backbone. In some embodiments, the target restriction endonuclease site appears at a low frequency in the TCR gene. “Low frequency” means a site that appears less than 20 times, less than 15 times, less than 10 times, less than 5 times, or less than 2 times in the TCR gene. In some embodiments, the target restriction endonuclease site contains a silent mutation that does not alter the sequence of the expressed TCR polypeptide. Non-limiting examples of restriction endonuclease recognition sites are provided in Tables 6 and 7 herein. In some embodiments, the first and / or second polynucleotides have been modified to incorporate at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten target restriction endonuclease sites disclosed in Tables 6 or 7.

[0133] In some embodiments of the vector, the vector further comprises at least one, at least two, at least three, or at least four polynucleotides encoding expression control elements. As used herein, "expression control element" means a polynucleotide that directly or indirectly promotes, regulates, or affects the expression of a polynucleotide. In some embodiments, the expression control element activates the expression of a polynucleotide. In some embodiments, the expression control element maintains the expression of a polynucleotide. In some embodiments, the expression control element enhances the expression of a polynucleotide. In some embodiments, the expression control element stabilizes the transcript of a polynucleotide. In some embodiments, the expression control element represses the expression of a polynucleotide. In some embodiments, the activity of the expression control element is inducible. In some embodiments, the expression control element is operatively linked to a first polynucleotide and / or a second polynucleotide. In some embodiments, the expression control element is upstream (5') of the first polynucleotide and / or the second polynucleotide. In some embodiments, the expression control element is downstream (3') of the first polynucleotide and / or the second polynucleotide. In some embodiments, the polynucleotide encoding the expression control element is located between the first polynucleotide and the second polynucleotide. In some implementations, the expression control element is selected from the group consisting of: promoter, p2A sequence, enhancer, and internal ribosome entry site (IRES) sequence.

[0134] The p2A sequence is a short peptide (approximately 20 amino acids) that generates equimolar levels of multiple genes from the same mRNA. These peptides are thought to function by inducing ribosomes to skip the synthesis of peptide bonds at the C-terminus of the 2A element, resulting in a separation between the end of the 2A sequence and the downstream of the next peptide. The resulting "cleavage" occurs between glycine and proline residues found at the C-terminus, meaning the upstream cistron will have some additional residues added to the end, while the downstream cistron will begin with proline. In a particular embodiment, the p2A sequence is selected from the group consisting of: T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 12) P2A: (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 13) E2A: (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 14) F2A: (GSG)VKQTLNFDLLKLAGDVESNPG (SEQ ID NO: 15) A promoter is a regulatory polynucleotide that provides a control site for the regulated transcription of polynucleotides. In some embodiments, the promoter is selected from the group consisting of: CMV, EF1a, SV40, PGK1, UBC, MNDU3, human β-actin, CAG, TRE, UAS, Ac5, polyhedroprotein, CaMKIIa, GAL1, GAL10, GDS, ADH1, CaMV35S, Ubi, H1, and U6. In a particular embodiment, the promoter is either the EF1α promoter or the CMV promoter.

[0135] The internal ribosome entry site (IRES) is an RNA element that allows translation to be initiated in a cap-independent manner as part of the larger process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of the mRNA molecule because 5' cap recognition is required for the assembly of the initiation complex. The IRES element is often located in the 5' UTR, but can also appear elsewhere on the mRNA. In some embodiments, the IRES is an FMDV or EMCV IRES sequence. This document provides non-limiting examples of polynucleotides encoding IRES sequences (EMCV): TATGCTAGTACGTCTCTCAAGGATAAGTAAGTAATATTAAGGTACGGGAGGTATTGGACAGGCCGCAATAAAATATCTTATTTTCATTACATCTGTGTGTTGGTTTTGTGTGAATCGATAGTACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAA TAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTGGCCTAACTGGCCGGTACCTGAGCTCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCCTCGAGGATATCAAGATCTGGCCTCGGCGGCCAG (SEQ ID NO: 16) Enhancers are regions of DNA that can be bound by proteins (e.g., transcription factors) to increase the likelihood of transcription of a specific target polynucleotide. Enhancers are cis-acting. In some embodiments, they are 50-1500 base pairs in length. In some embodiments, they are located upstream or downstream of the target polynucleotide. In some embodiments, enhancers are selected from the group consisting of CENTG2 enhancers, GADD45G enhancers, and WPRE enhancers. In a particular embodiment, the enhancer is a warble mouse hepatitis virus (WHP) posttranscriptional regulatory element (WPRE), which is a DNA sequence that produces enhanced expression at the tertiary level upon transcription. WPRE is a triple regulatory element with γ, α, and β components. In some embodiments, the enhancer contains only the α component of the WPRE. In other embodiments, the enhancer contains the complete triple WPRE sequence. When used alone without the γ and β WPRE components, the activity of the α component is only 9% of that of the complete triple WPRE.

[0136] WPREα sequence: GCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGT (SEQ ID NO: 17) Complete WPRE sequence: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATA AATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTG CGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ IDNO: 18) In some embodiments of the carrier, the carrier is circularized. In some embodiments, the carrier is circularized before the expression control element is incorporated into the carrier. In other embodiments, the carrier is circularized after the expression control element is incorporated into the carrier.

[0137] In some embodiments of the vector, the expression control element has been incorporated near the prototype spacer adjacent motif (PAM). In these embodiments, the expression control element is inserted into the vector via a CRISPR / Cas-mediated mechanism. In other embodiments, a DNA-modifying enzyme selected from restriction enzymes or TALENs has been used to incorporate the expression control element into the vector.

[0138] In some embodiments, the vector further comprises one or more polynucleotides encoding transposons to facilitate integration of at least one of the first and second polynucleotides into target or host cells. The Sleeping Beauty transposase inserts the transposon into a TA dinucleotide base pair in the recipient's DNA sequence. For example, in some embodiments, transposons flanking the first and second polynucleotides facilitate integration at the TA dinucleotide into the recombinant cell genome or immune cell genome. In some embodiments, the vector backbone is derived from or comprises a nonviral vector. Advantages of nonviral vectors include ease and relatively low cost of producing sufficient quantities to meet the needs of an entire patient population, stability during storage, and lack of immunogenicity. A non-limiting example of a transposon system suitable for use in vectors of this technology is the Sleeping Beauty transposon system (see, for example, Kebraei, P. et al. (2017)). Trends in Genetics 33:852-70, which is incorporated herein by reference. The Sleeping Beauty transposon system consists of two components: (i) a transposon containing a gene expression cassette and (ii) a source of transposase. Sustained transcription of transgenes can be achieved by transposing the expression cassette from a plasmid into the genome. Exemplary Sleeping Beauty transposase vectors include, but are not limited to: pSBbi (Kowarz, E. et al., Biotechnol J. 10(4):647-53, obtained from Addgene), pCMV(CAT)T7-SB100 (Mátés, L. et al.), Nat Genet June 2009; 41(6):753-61, obtained from Addgene) and pT2 / LTR7 (Wang, J. et al., Nature . 18 December 2014; 516(7531):405-9, obtained from Addgene).

[0139] In some embodiments of the vector, the vector further comprises one or more polynucleotides encoding a detectable or purified marker. In a particular embodiment, the detectable marker is a fluorescent protein selected from the group consisting of: GFP, YFP, RFP, dTomato, cherry, Cy3, Cy5, Cy5.5, Cy7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexaflur, FITC, and TRITC.

[0140] In some embodiments of the vector, the vector further comprises one or more polynucleotides encoding a selection marker. In a particular embodiment, the selection marker confers a positive selective trait in eukaryotic cells, for example, blastomycin (…). bsd Gene), G418 / genetic mycin (neo Gene), Hygromycin B ( hygB Gene), puromycin ( pac Gene) or zeocin ( Sh bla (Genes). In some implementations, selection markers confer positive selective traits in bacterial cells, for example, β-lactamase Gene.

[0141] In some embodiments of the vector, the vector further comprises one or more polynucleotides encoding a switching mechanism for controlling the expression and / or activation of the first and second polynucleotides. In other embodiments of the vector, the recombinant TCR encoded by the first and second polynucleotides includes the switching mechanism. In a particular embodiment, the switching mechanism is a suicide switch, such as i-cysteine ​​9, which is a safe mechanism that can be activated to induce apoptosis or death in cells containing the TCR library.

[0142] For example, in some embodiments, the TCR may include an extracellular domain having a target-specific binding element comprising a marker, binding domain, or tag specific to a molecule other than a target antigen expressed on or by a target cell. In such embodiments, the specificity of the TCR is provided by a second construct comprising, or substantially comprising, a target antigen-binding domain and a domain recognized by, or bound to, a marker, binding domain, or tag on the TCR. See, for example, WO 2013 / 044225, WO 2016 / 000304, WO 2015 / 057834, WO 2015 / 057852, WO 2016 / 070061, US 9,233,125, and US 2016 / 0129109. In this way, T cells expressing TCR can be administered to the subject, but the T cells cannot bind to their target antigens until a second composition containing an antigen-specific binding domain is administered.

[0143] In other embodiments, the TCR is modified to require polymerization to be activated (see, for example, US 2015 / 0368342, US 2016 / 0175359, US 2015 / 0368360) and / or exogenous signals, such as small molecule drugs (US 2016 / 0166613, Yung et al., Science, 2015).

[0144] In some embodiments, the vector contains a polynucleotide encoding a “suicide switch” or “safety switch” to induce cell death in cells expressing the vector upon treatment (Buddee et al., PLoS One, 2013) or to downregulate TCR expression upon binding to a target antigen (WO 2016 / 011210). For example, the vector may contain a suicide gene conferring sensitivity to an antibody or prodrug that can be administered to stop TCR activity. In some embodiments, the antibody or prodrug is administered to a subject who has received a TCR library therapy in the event of an adverse event. Exemplary suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (HSV-TK) (Bonini et al., Science 276: 1719-1724 (1997)) which sensitizes cells to ganciclovir, inducible caspase 9 (i-caspase 9) which allows dimerization and activation of apoptosis when activated by a dimeric drug (Gargett et al., Front Pharmacol, 2014 5:235), and truncated EGFR (Wang et al., Blood 118: 1255-63 (2011)) which sensitizes cells to cetuximab.

[0145] In some embodiments of the vector, the vector further comprises one or more polynucleotides encoding a Kozak concordant sequence. In some embodiments, the Kozak concordant sequence is strong, sufficient, or weak.

[0146] In some embodiments of the vector, T cells are screened for reactivity with target cells or disease antigens prior to T cell lysis. In some embodiments of the vector, the TCR encoded by the vector has binding specificity to or is activated by the target cells or disease antigens. In some embodiments, the target cells are cancer cells, virus-infected cells, cells derived from a virus-infected subject, tumor cells, or cells from a tissue biopsy sample isolated from a subject suspected of having a viral infection or cancer. In some embodiments, cells are isolated from the subject. In some embodiments of the vector, TCRs are screened for specific binding to the disease antigen:MHC complex.

[0147] In some embodiments, the disease antigen is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), and enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus.

[0148] In other embodiments, the disease antigen is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, lensin, meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tumor necrosis factor receptor superfamily member 10a (TRAILR1), receptor activator of nuclear factor κ-B ligand (RANKL), insulin-like growth factor 1 receptor (IGF1R), epithelial cell adhesion molecule (EpCAM), and carcinoembryonic antigen (CEA).

[0149] For example only, in some implementations, the original target cloning vector (e.g., pLVX-EF1α-IRES-mCherry) may contain multiple restriction enzyme recognition sites, such as AgeI (2415), SphI (2331), NheI (8192), and MluI (6669) cleavage sites (the numbers indicate the locations of these cleavage sites). See also Figure 13 In some implementations, excess restriction enzyme sites for AgeI, SphI, NheI, and MluI can be eliminated by site-directed mutagenesis (see [link to implementation details]). Figure 14In some embodiments, the excess restriction enzyme sites to be removed from the target cloning vector may be any restriction enzyme sites listed in Table 7 or Table 8. In other embodiments, the excess restriction enzyme sites may be replaced by other known restriction enzyme sites that allow T-cell receptor-paired α:β clones to be cloned into linear or circular vector forms.

[0150] Original pLVX-EF1α-IRES mcherry vector sequence Modified pLVX-EF1α-IRES mcherry vector sequence Non-limiting examples of nucleic acid sequences of vectors disclosed herein are provided in Table 2 and in Figure 3-12 The explanation is as follows.

[0151] Table 2: Exemplary vector sequences

[0152] On the other hand, this technology provides recombinant cells comprising a vector, wherein the vector comprises (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of a single lysed T cell is isolated in the compartment using an mRNA capture reagent, optionally wherein the recombinant cell is a bacterial cell, a mammalian cell, or a yeast cell. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or optionally, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a droplet.

[0153] In one aspect, this technology provides a recombinant TCR vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding the paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet.

[0154] In some embodiments, the TCR vector library comprises a TCR library. Therefore, in some embodiments, the TCR vector library is a collection of all or part of different TCRs generated in a single donor or subject due to V(D)J rearrangement and / or T cell selection. In other embodiments, the TCR vector library of this disclosure comprises a defined set of TCRs specifically selected for their binding specificity or other desired characteristics. For example, the TCR vector library may comprise a subset of TCRs that specifically bind to a particular target cell, antigen, antigen:MHC complex, or combination thereof. In these embodiments, the TCRs may be derived from a single donor or subject, or from more than one donor or subject.

[0155] In some embodiments, each TCR in the TCR vector library is genetically distinct and contains different binding specificities. In some embodiments, the TCR vector contains about 2 to about 5 different TCRs, about 2 to about 10 different TCRs, about 5 to about 10 different TCRs, about 5 to about 15 different TCRs, about 5 to about 20 different TCRs, about 10 to about 30 different TCRs, about 10 to about 40 different TCRs, about 10 to about 50 different TCRs, about 20 to about 60 different TCRs, about 25 to about 75 different TCRs, about 50 to about 100 different TCRs, or more than 100 different TCRs.

[0156] Precise molecular composition can benefit the stability, efficacy, and / or safety of biotherapeutic agents. Therefore, in some embodiments, each vector and / or TCR in the TCR vector library can be characterized to determine the nucleic acid and / or amino acid sequence and / or its binding specificity. In some embodiments, molecular characterization is performed by nucleic acid sequencing of the vector or a portion of the vector (e.g., a first polynucleotide and a second polynucleotide). In some embodiments, molecular characterization is performed by mass spectrometry analysis of the TCR protein. In some embodiments, the TCR vector library is molecularly characterized by nucleic acid sequencing of 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 15 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more isolated colonies, each colony containing recombinant cells (e.g., bacterial cells) transformed with the TCR vector library. In some embodiments, a TCR vector library is perfected or selected by amplifying and sequencing single-colony nucleic acids from transformed recombinant cells, followed by mixing different vector colonies at defined ratios to generate a defined molecular library composition. In some embodiments, the selection of individual vectors to be included in the library is informed by aspects of the characterization and / or binding specificity of the starting library. Non-limiting examples of such aspects include TCR clonal universality, TCR enrichment characteristics determined in vitro, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motif, and / or CDR3 gene motif.

[0157] In some implementations, the vector ratios in the TCR vector library are adjusted or selected to optimize the therapeutic activity of the library. For example, a TCR vector library containing two TCRs may have a vector ratio of approximately 1:1, approximately 1:2, approximately 1:5, approximately 1:10, etc., for each vector. In another non-limiting example, a library containing three TCRs may have a vector ratio of approximately 1:1:1, approximately 2:1:1, approximately 2:2:1, etc., for each vector.

[0158] On the other hand, this technology provides isolated immune cells comprising (a) a carrier backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of a single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a droplet. In some embodiments, the immune cells are hematopoietic stem cells, hematopoietic progenitor cells, T cells, or natural killer (NK) cells.

[0159] In one aspect, this technology provides a cell population comprising a recombinant TCR vector library, the recombinant TCR vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet. In some embodiments, the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells. In some embodiments, the cell population comprises all or part of a subject's TCR library.

[0160] Another aspect of this technology relates to compositions comprising a vector and one or more vectors of the present invention. Optionally, the composition comprises a vector and one or more recombinant TCR vector libraries of the present invention. In another embodiment, the composition comprises a vector and one or more immune cells comprising one or more vectors of the present invention. In yet another embodiment, the composition comprises a vector and a cell population comprising a recombinant TCR vector library of the present invention. Well-known examples of vectors include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. For the purposes of this disclosure, the nature of the vector may be soluble or insoluble. Those skilled in the art will recognize other suitable vectors for binding vectors, cells, cell populations, or vector libraries, or will be able to identify such vectors using conventional experiments.

[0161] In some embodiments, the carrier is a pharmaceutically acceptable carrier. The pharmaceutical compositions of this disclosure, including but not limited to any of the claimed compositions, may comprise a combination of a carrier, library, cell or cell population as described herein with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure may be formulated for oral, intravenous, topical, enteral, and / or parenteral administration. In some embodiments, the compositions of this disclosure are formulated for intravenous administration.

[0162] II. Method for preparing the composition of this technology This document provides a method for preparing a recombinant TCR library, the method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet. In some embodiments, the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells.

[0163] In some embodiments of the method, a library is screened for specific binding to target cells. In some embodiments, the target cells are cancer cells, virus-infected cells, cells derived from a virus-infected subject, tumor cells, or cells from a tissue biopsy sample isolated from a subject suspected of having a viral infection or cancer.

[0164] In some embodiments of the method, libraries are screened for specific binding to the antigen:MHC complex. In some embodiments, the antigen of the antigen:MHC complex is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus. In other embodiments, the antigen of the MHC complex is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, Lengsin, Meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, EGFR, HER2, TRAILR1, RANKL, IGF1R, EpCAM, and CEA.

[0165] In some embodiments of the method, a library is screened for T cell phenotypic markers. In some embodiments, the T cell phenotypic markers identify the expression of one or more TCR complex components (e.g., TCRα, TCRβ, TCRγ, TCRδ, CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η). In some embodiments, the T cell phenotypic markers help identify specific T cell subsets, such as naive CD8+ T cells, naive CD4+ T cells, CD4+ T cells, CD8+ cytotoxic T cells, γ / δ T cells, NKT cells, Th1 cells, Th2 cells, Th9 cells, Th22 cells, T follicular helper cells, Th17 cells, and regulatory T cells. Antibodies specific to cell surface markers suitable for identifying these T cell subsets are known in the art and are available from, for example, R&D Systems. Screening can be performed using any method known in the art, including but not limited to ELISA, Western blot, Northern blot, PCR, qPCR, and flow cytometry.

[0166] In some embodiments of the method, an activity screening library is selected for use in a co-culture system comprising at least one of the following: (a) a cancer cell line; (b) multiple cells infected with a known virus; (c) multiple tumor cells isolated from a cancer patient; (d) an immortalized cell line; or (e) multiple cells derived from a patient tissue biopsy sample. In some embodiments, activity is measured by determining the co-conjugation of the recombinant TCR / CD3 complex with a co-stimulatory receptor (e.g., CD28). Co-conjugation of these receptors on the cell surface leads to intracellular signaling events and activation of nuclear transcription factors such as activated T cell nuclear factor (NFAT), NF-κB, and AP-1. Specifically, conjugation of the TCR / CD3 complex leads to phosphorylation and activation of PLC-g, intracellular calcium flux, and transcriptional activation of the NFAT pathway. In some embodiments, co-conjugation of TCR / CD3 with the co-stimulatory receptor CD28 leads to activation of ERK / JNK and IKB kinase (IKK), which in turn regulate transcriptional activation of the AP-1 and NF-κB pathways, respectively. The IL-2 promoter contains DNA binding sites for NFAT, NF-κB, and AP-1. Therefore, co-binding of TCR / CD3 and CD28 leads to the production of IL-2, which is commonly used as a functional readout for T cell activation. In some embodiments, other endpoints for measuring T cell activation include, but are not limited to, cell proliferation, cytotoxicity (target cell death), and the production of additional cytokines such as IFNγ. Kits suitable for measuring or detecting T cell activity are available, for example, from Promega (T Cell Activation Bioassay (IL-2) (a, b) (catalog numbers J1651 and J1655)).

[0167] In some embodiments of the method, the transformed cells are activated in vitro. In specific embodiments, activation is performed using one or more of the following stimulants: anti-CD3 antibody, anti-CD8 antibody, anti-CD27 antibody, IL-2, IL-4, IL-21, anti-PD1 antibody, anti-CTLA4 antibody, anti-CD3 / CD28 tetramer antibody, tumor cell lysate, cell co-culture with virus-infected cells, and tumor cell lines. T cell activation kits are available, for example, from Miltenyi Biotec (T cell activation / expansion kit, human, catalog number 130-091-441).

[0168] In some embodiments of the method, the cell population is transformed with transcription factors. In some embodiments, the transcription factors are selected from the group consisting of: forkhead box P3 (FOXP3, Entrez gene: 50943, RefSeq mRNA: NM_001114377; NM_014009), PR domain zinc finger protein 1 (BLIMP-1, Entrez gene: 639, RefSeq mRNA: NM_001198; NM_182907), Helios (IKZF2, Entrez gene: 22807, RefSeq mRNA: NM_001079526.1; NM_016260.2, Uniprot: Q9UKS7), Ikaros (IKZF1, Entrez gene: 10320, RefSeq mRNA ... mRNA: NM_001220765.2; NM_006060.6, etc.; Uniprot: Q13422) and transforming growth factor β1 (TGF-β1, Entrez gene: 7040, RefSeq mRNA: NM_000660). FOXP3 is a protein involved in the immune system response and is considered to act as a master regulator of the development and function of regulatory T cells. Regulatory T cells typically suppress immune responses. BLIMP-1 acts as a repressor of β-interferon (β-IFN) gene expression. Regulatory T cells release TGF-β1 to suppress the action of other T cells. The interleukin-1 and interleukin-2 dependent proliferation of activated T cells, as well as the activation of resting helper T cells and cytotoxic T cells, are prevented by the activity of TGF-β1. Similarly, TGF-β1 can inhibit the secretion and activity of many other cytokines, including interferon-γ, tumor necrosis factor-α (TNF-α), and various interleukins. It can also reduce the expression levels of cytokine receptors such as IL-2 receptors, thereby downregulating the activity of immune cells. This article provides non-limiting examples of transcription factor amino acid sequences: FOXP3 (UniProt Q9BZS1): MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP (SEQ ID NO: 31) BLIMP-1 (UniProt O75626): MLDICLEKRVGTTLAAPKCNSSTVRFQGLAEGTKGTMKMDMEDADMTLWTEAEFEEKCTYIVNDHPWDSGADGGTSVQAEASLPRNLLFKYATNSEEVIGVMSKEYIPKGTRFGPLIGEIYTNDTVPKNANRKYFWRIYSRGELHHFIDGFNEEKSNWMRYVNPAHSPREQNLAACQNGMNIYFYTIKPIPANQELLVWYCRDFAERLHYPYPGELTMMNLTQTQSSLKQPSTEKNELCPKNVPKREYSVKEILKLDSNPSKGKDLYRSNISPLTSEKDLDDFRRRGSPEMPFYPRVVYPIRAPLPEDFLKASLAYGIERPTYITRSPIPSSTTPSPSARSSPDQSLKSSSPHSSPGNTVSPVGPGSQEHRDSYAYLNASYGTEGLGSYPGYAPLPHLPPAFIPSYNAHYPKFLLPPYGMNCNGLSAVSSMNGINNFGLFPRLCPVYSNLLGGGSLPHPMLNPTSLPSSLPSDGARRLLQPEHPREVLVPAPHSAFSFTGAAASMKDKACSPTSGSPTAGTAATAEHVVQPKATSAAMAAPSSDEAMNLIKNKRNMTGYKTLPYPLKKQNGKIKYECNVCAKTFGQLSNLKVHLRVHSGERPFKCQTCNKGFTQLAHLQKHYLVHTGEKPHECQVCHKRFSSTSNLKTHLRLHSGEKPYQCKVCPAKFTQFVHLKLHKRLHTRERPHKCSQCHKNYIHLCSLKVHLKGNCAAAPAPGLPLEDLTRINEEIEKFDISDNADRLEDVEDDISVISVVEKEILAVVRKEKEETGLKVSLQRNMGNGLLSSGCSLYESSDLPLMKLPPSNPLPLVPVKVKQETVEPMDP(SEQ ID NO: 32) TGF-β1 (UniProt P01137): MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEW LSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 33) In some embodiments, the method further includes administering an effective amount of the recombinant TCR library prepared according to the method described herein to a subject in need. In some embodiments, the subject in need is suffering from cancer or a viral infection.

[0169] On the other hand, this document provides a recombinant TCR library prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a droplet. In some embodiments, the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells.

[0170] In one aspect, this technology provides a composition comprising a carrier and a recombinant TCR library prepared by methods as described herein. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0171] III. How to use The treatment methods described in this article provide a form for isolating and using specific TCRs that can be rapidly identified, amplified, and returned to the subject on a timescale associated with bedside therapies (e.g., weeks, rather than months).

[0172] Therefore, in one aspect, this document provides a method for treating a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore in a 96-well plate) or a microdroplet.

[0173] On the other hand, this document provides a method for treating cancer in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of the single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a microdroplet.

[0174] In some implementation schemes, cancer includes: acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; AIDS-related cancer; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rod-shaped tumors; brain cancer; basal cell carcinoma of the skin; bile duct cancer; bladder cancer; bone cancer; breast cancer; bronchoma; Burkitt lymphoma; carcinoid tumor (gastrointestinal); germ cell tumor; primary CNS lymphoma; cervical cancer; bile duct cancer; chordoma; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); chronic myeloproliferative neoplasm; colorectal cancer. Colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; ductal carcinoma in situ (DCIS); endometrial cancer; ependymoma; esophageal cancer; sensory neuroblastoma; extracranial germ cell tumor; gonadal germ cell tumor; ocular cancer; intraocular melanoma; retinoblastoma; fallopian tube cancer; malignant fibrous histiocytoma and osteosarcoma of bone; gallbladder cancer; gastric cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); germ cell tumor; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; cardiac tumors; hepatocellular carcinoma; Langerhans cell histiocytosis; Hodgkin's lymphoma; hypopharyngeal cancer; intraocular melanoma Pancreatic islet cell tumors, pancreatic neuroendocrine tumors; kidney cancer; laryngeal cancer; leukemia; lip and oral cavity cancer; liver cancer; lung cancer; lymphoma; male breast cancer; malignant fibrous histiocytoma and osteosarcoma of bone; melanoma; Merkel cell carcinoma; mesothelioma; metastatic cancer; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides; myelodysplastic syndrome, chronic myeloproliferative neoplasm; nasopharyngeal carcinoma; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer; oral cavity cancer, oropharyngeal cancer; osteosarcoma; ovarian cancer; pancreatic cancer; pancreatic neuroendocrine tumors; papilloma. Diseases; paraganglioma; sinus carcinoma; parathyroid carcinoma; pharyngeal carcinoma; pheochromocytoma; pituitary adenoma; pleural pulmonary blastoma; prostate cancer; rectal cancer; recurrent cancer; renal cell carcinoma; retinoblastoma; rhabdomyosarcoma; salivary gland carcinoma; sarcoma; Ewing sarcoma; Kaposi's sarcoma; osteosarcoma; uterine sarcoma; Cezary syndrome; skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; squamous neck carcinoma; gastric cancer; T-cell lymphoma; testicular cancer; laryngeal cancer; nasopharyngeal carcinoma; hypopharyngeal carcinoma; thymic carcinoma; thyroid cancer; urethral cancer; uterine cancer; vaginal cancer; hemangioma; vulvar cancer; or Wilms' tumor.

[0175] In one aspect, this document provides a method for inhibiting tumor growth in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from the mRNA of a single lysed T cell in a compartment. In some embodiments, the mRNA of the single lysed T cell is isolated in the compartment using an mRNA capture reagent. In other embodiments, the polynucleotide encoding a paired T cell receptor polypeptide is derived from a single cell without using an mRNA capture reagent. Additionally or alternatively, in some embodiments, the compartment containing the contents of a single lysed T cell is a micropore (e.g., a micropore within a 96-well plate) or a droplet. In some embodiments, the tumor is a solid tumor.

[0176] On the other hand, this document provides a method for treating viral infection in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library or a composition comprising a recombinant TCR library, wherein the recombinant TCR library is prepared by a method comprising transforming a cell population with a vector library comprising a plurality of vectors, each vector comprising (a) a vector backbone; and (b) a first polynucleotide encoding a TCRα polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or (b) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide; wherein the first polynucleotide and the second polynucleotide are a pair, and wherein the first polynucleotide and the second polynucleotide are derived from mRNA of a single lysed T cell captured in a compartment by an mRNA capture reagent. In some embodiments, the viral infection is caused by a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus.

[0177] In some embodiments, the method further includes activating a suicide switch to kill cells or cell populations containing a carrier with a suicide switch (e.g., i-cysteine ​​9), thereby reducing the risk of harm to the patient. In some embodiments, the suicide switch is triggered after significant improvement or apparent cure of the subject's cancer or infection in order to reduce the risk of long-term side effects.

[0178] In some embodiments, the method further includes pre-stimulating the cells in vitro prior to administration to achieve the desired TCR function or T cell identity in vivo, as described in the preparation methods above.

[0179] In some embodiments, the method further includes co-expressing one or more transcription factors in recombinant cells to influence the development of T cells into effective anticancer or antiviral phenotypes, or to prevent the development of immunosuppressive Tregs, as described in the preparation methods above.

[0180] The administration of cells, libraries, cell populations, or compositions can be performed as a single dose, continuously, or intermittently throughout the treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of treatment, and the subject being treated. Single or multiple administrations may be performed depending on the dose level and pattern selected by the treating physician. Suitable dosage formulations and methods of administering the reagents are known in the art. In another aspect, the cells and compositions of this disclosure can be administered in combination with other treatments. Cells and cell populations are administered to a host using methods known in the art and, for example, those described in PCT / US2011 / 064191. Such administration of the cells or compositions of this disclosure can be performed to generate animal models of desired diseases, conditions, or ailments for experimental and screening assays.

[0181] The compositions disclosed herein can be administered in a manner suitable for treating or preventing a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, although the appropriate dosage can be determined through clinical trials.

[0182] In some implementations, the effective dose of the recombinant TCR library contains approximately 50 to approximately 10 2 10 cells, approximately 10 2 From one cell to about 10 3 10 cells, approximately 10 2 From one cell to about 10 4 10 cells, approximately 10 3 From one cell to about 10 5 10 cells, approximately 10 4 From one cell to about 10 6 10 cells, approximately 10 5 From one cell to about 10 7 10 cells, approximately 10 6 From one cell to about 10 8 10 cells, approximately 10 7 From one cell to about 10 9 One cell or about 10 8 From one cell to about 10 10 Cells. In a specific implementation, the effective dose contains approximately 5 × 10⁶ cells. 5 From one cell to approximately 1.5 × 10⁻⁶ 6 One cell or approximately 1 × 10 4 From one cell to approximately 5 × 10 4 1 cell, approximately 5 × 10 4 From one cell to approximately 5 × 10 5 One cell or approximately 2.5 × 10 5 From one cell to approximately 7.5 × 10⁶ 5 Each cell.

[0183] In some embodiments, administration may be repeated and / or modified as needed in response to a subject’s specific response to the therapy. For example, repeated administration may be necessary when cancer cells or viruses reappear in subjects in need (e.g., tumor immune “evasion”). In some embodiments, repeated administration includes cells, libraries, cell populations, or compositions prepared by a method different from the initial administration (e.g., repeated doses include additional steps of pre-screening or pre-activating T cells). In specific embodiments, the treatment methods provided herein further include administration of second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth doses of a recombinant TCR library or composition.

[0184] In some implementations, the recombinant TCR library contains cells that are autologous or allogeneic to the subject being treated.

[0185] Immunoassay and imaging. In some respects, the recombinant TCR libraries disclosed herein can be used to determine the presence of target cells in biological samples isolated from a subject (e.g., human plasma). Target cells (e.g., cancer cells) can be detected by exposing the recombinant TCR library to the biological sample and measuring TCR activation and / or binding.

[0186] In addition to determining the presence of target cells, the recombinant TCR libraries disclosed herein can also be used for in vivo imaging. Detectable markers that can be incorporated into the recombinant TCR libraries include those detectable by X-ray radiography, NMR, or ESR. For X-ray radiography, suitable markers include radioactive isotopes (such as barium or cesium) that emit detectable radiation but do not cause significant harm to the subject. Suitable markers for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into the TCR library. The recombinant TCR library can be further enhanced with appropriate detectable imaging components (such as radioactive isotopes, e.g., 131 I, 112 In、 99 A TCR library labeled with mTc, radiopaque material, or a material detectable by MRI is introduced into the subject (e.g., parenteral, subcutaneous, or intraperitoneal). It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging fraction required to produce diagnostic images. In the case of a radioisotope fraction, for human subjects, the amount of radioactivity injected is typically between approximately 5 and 20 millicuries. 99 Within the range of mTc. Then, the recombinant TCR library will preferentially accumulate at the location of the target cells. For example, in vivo tumor imaging is described in SW Burchiel et al., Tumor Imaging: Radiochemical Detection of Cancer (…). Tumor Imaging: The Radiochemical Detection of Cancer ) 13 (1982)

[0187] Diagnostic usesThe recombinant TCR library disclosed herein can be used in diagnostic methods. Therefore, this disclosure provides a method for diagnosing cancer or viral infection in a subject using the recombinant TCR library disclosed herein. The diagnostic method includes contacting a biological sample isolated from the subject with the recombinant TCR library of this disclosure. The biological sample can be obtained from any tissue (including biopsy specimens), cells, or body fluids of the subject. The activity or binding of the recombinant TCR library is determined. If the recombinant TCR library is activated upon exposure to the biological sample, the subject's biological sample contains cells recognized by the TCR library. Therefore, the subject is diagnosed with cancer or a viral infection.

[0188] Prognostic uses The recombinant TCR library disclosed herein can be used in prognostic methods. Therefore, this disclosure provides a method for predicting the prognosis of a subject with cancer or a viral infection using the recombinant TCR library disclosed herein. The prognostic method involves contacting a biological sample isolated from the subject with the recombinant TCR library of this disclosure. The biological sample can be obtained from any tissue (including biopsy specimens), cells, or body fluids of the subject. The activity or binding of the recombinant TCR library is determined. If the recombinant TCR library is activated upon exposure to the biological sample, the subject's biological sample contains cells recognized by the TCR library. Therefore, the subject is identified as having cancer and / or a solid tumor or a viral infection, or is at risk of developing cancer and / or a solid tumor or a viral infection.

[0189] In some implementations, the subjects are humans, animals, non-human primates, dogs, cats, sheep, mice, horses, or cows. In a particular implementation, the subjects are humans.

[0190] IV. Reagent test kit As described herein, this disclosure provides methods for TCR library preparation, therapeutic methods, diagnostic methods, and prognostic methods. In one particular aspect, this disclosure provides kits for performing these methods, as well as instructions for performing the methods of this disclosure (such as collecting donor cells and / or performing screening and / or analyzing results).

[0191] In some embodiments, the kit comprises, or optionally consists substantially of, the recombinant TCR library disclosed herein and instructions for use. In some embodiments, the kit comprises, or optionally consists substantially of, the vectors disclosed herein and instructions for use. In some embodiments, the kit comprises, or optionally consists substantially of, the pharmaceutical composition disclosed herein and instructions for use.

[0192] In some respects, the kit may include: one or more vectors, cells, populations or recombinant TCR libraries as disclosed herein; a device for determining the amount of reactive antigens or cells in a biological sample or for measuring the activity of the TCR library; and a device for comparison with standards.

[0193] The kit components (e.g., reagents) may be packaged in suitable containers. For example, these suggested kit components may be provided as solutions or as liquid dispersions. The kit may also contain, for example, buffers, preservatives, or protein stabilizers. The kit may further contain components necessary for the detection of a detectable marker, such as enzymes or substrates. The kit may also contain a control sample or a series of control samples that can be measured and compared with the test sample. Each component of the kit may be sealed in a single container, and all the various containers may be in a single package along with instructions for interpreting the results of assays performed using the kit. The kits disclosed herein may contain a written product on or within the kit container. The written product describes how to use the reagents contained in the kit.

[0194] The embodiments of the present invention include the following items.

[0195] 1. A recombinant T-cell receptor (TCR) library vector, comprising: (a) Carrier skeleton; and (b)(i) the first polynucleotide encoding the TCRα polypeptide and the second polynucleotide encoding the TCRβ polypeptide; or (ii) The first polynucleotide encoding the TCRγ polypeptide and the second polynucleotide encoding the TCRδ polypeptide; The first polynucleotide and the second polynucleotide are a pair, and the first polynucleotide and the second polynucleotide are derived from mRNA isolated from a single lysed T cell present in a compartment.

[0196] 2. The vector according to Project 1, wherein the mRNA of the single lysed T cell is isolated using an mRNA capture reagent or reverse transcription-PCR (RT-PCR).

[0197] 3. The vector according to Project 2, wherein the first polynucleotide and the second polynucleotide are operatively linked, optionally operatively linked via a linker polynucleotide, and optionally wherein the first polynucleotide and the second polynucleotide are operatively linked via reverse transcription and PCR amplification of the T cell mRNA.

[0198] 4. The vector according to Project 1, wherein the first polynucleotide and the second polynucleotide have been cloned into the vector backbone by cleavage at a target restriction endonuclease site naturally found in the TCR gene.

[0199] 5. The vector according to Project 4, wherein the target restriction endonuclease site appears at a low frequency in the TCR gene.

[0200] 6. The vector according to Project 4, wherein the first polynucleotide and the second polynucleotide have been modified to incorporate at least one target restriction endonuclease site disclosed in Table 7 or 8.

[0201] 7. The vector according to Project 4, wherein the target restriction endonuclease site contains a silencing mutation.

[0202] 8. The vector according to Project 2, wherein the mRNA capture reagent is selected from the group consisting of: poly(dT) coated beads, oligonucleotide coated beads, hydrogel beads, and printed oligomers on the surface of microarray pores.

[0203] 9. The carrier according to Project 1, wherein the compartment is an emulsion droplet or a pore.

[0204] 10. The carrier according to item 9, wherein the pores are located in a printed polymer slide, plastic plate, microtiter plate or gel.

[0205] 11. The carrier according to Project 1, wherein the compartment has a volume of 5 nL or less.

[0206] 12. The vector according to Item 1, further comprising at least one polynucleotide encoding an expression control element, the polynucleotide encoding the expression control element being operatively linked to the first polynucleotide and / or the second polynucleotide.

[0207] 13. The vector according to item 12, wherein the expression control element is selected from the group consisting of: promoter, p2A sequence and IRES sequence.

[0208] 14. The vector according to item 13, wherein the promoter is an EF1α promoter or a CMV promoter.

[0209] 15. The vector according to item 12, wherein the polynucleotide encoding the expression control element is located between the first polynucleotide and the second polynucleotide.

[0210] 16. The carrier according to Project 1, wherein the carrier is circulated.

[0211] 17. The carrier according to item 12, wherein the carrier has been circularized before the expression control element is incorporated into the carrier.

[0212] 18. The carrier according to item 12, wherein the carrier has been circularized after the expression control element is incorporated into the carrier.

[0213] 19. The carrier according to item 12, wherein the expression control element has been incorporated near the prototype spacer adjacent motif (PAM).

[0214] 20. The vector according to item 12, wherein the expression control element has been incorporated into the vector using a DNA-modifying enzyme selected from restriction enzymes or TALENs.

[0215] 21. The vector according to Item 1, further comprising one or more polynucleotides encoding transposons, the polynucleotides encoding transposons being linked to at least one of the first polynucleotide and the second polynucleotide.

[0216] 22. The vector according to Project 1 further comprises a polynucleotide encoding a detectable biomarker.

[0217] 23. The vector according to Project 1, further comprising a polynucleotide encoding a selection marker.

[0218] 24. The vector according to Item 1, further comprising a polynucleotide encoding a switching mechanism for controlling the expression and / or activation of the first polynucleotide and the second polynucleotide.

[0219] 25. The vector according to Item 1 further comprises a polynucleotide encoding a Kozak common sequence or enhancer.

[0220] 26. The vector according to Project 1, wherein the vector backbone is selected from the group consisting of: retroviral vector backbone, lentiviral vector backbone, adenovirus vector backbone and adeno-associated virus vector backbone.

[0221] 27. The vector according to Item 1, wherein the vector encodes a TCR that has binding specificity to a target cell or disease antigen.

[0222] 28. The vector according to item 27, wherein the target cell is a cancer cell or a virus-infected cell, optionally wherein the target cell is isolated from the subject.

[0223] 29. The vector according to item 27, wherein the disease antigen is a viral antigen or a tumor antigen, optionally wherein the antigen is loaded into an antigen:MHC complex.

[0224] 30. The vector according to Item 29, wherein the disease antigen is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus.

[0225] 31. The vector according to Item 29, wherein the disease antigen is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, Lengsin, Meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tumor necrosis factor receptor superfamily member 10a (TRAILR1), receptor activator of nuclear factor κ-B ligand (RANKL), insulin-like growth factor 1 receptor (IGF1R), epithelial cell adhesion molecule (EpCAM), and carcinoembryonic antigen (CEA).

[0226] 32. A recombinant cell comprising a vector according to any one of items 1-31, wherein the recombinant cell is optionally a bacterial cell, a mammalian cell, or a yeast cell.

[0227] 33. A recombinant TCR vector library comprising a plurality of vectors according to any one of items 1-31.

[0228] 34. The recombinant TCR vector library according to item 33, wherein the plurality of vectors contain a TCR library.

[0229] 35. The recombinant TCR vector library according to Item 33, wherein each of the plurality of vectors has been selected based on one or more of the following characteristics: TCR clonal universality, TCR enrichment characteristics determined in vitro, TCR binding specificity, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motif and / or CDR3 gene motif.

[0230] 36. The recombinant TCR vector library according to Item 33, wherein the TCR vector library has been characterized by nucleic acid sequencing of the first polynucleotide and the second polynucleotide.

[0231] 37. An isolated immune cell comprising a vector according to any one of items 1-31.

[0232] 38. Isolated immune cells according to item 37, wherein the immune cells are hematopoietic stem cells, hematopoietic progenitor cells, T cells or natural killer (NK) cells.

[0233] 39. A cell population comprising a vector according to any one of items 1-31 or a vector library according to any one of items 33 to 36.

[0234] 40. The cell population according to item 39, wherein the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells and / or NK cells.

[0235] 41. A method for preparing a recombinant TCR library, the method comprising transforming a cell population with a vector library according to any one of items 33 to 36.

[0236] 42. The method according to item 41, wherein the cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a T cell, or an NK cell.

[0237] 43. The method according to item 41 further includes screening the library for specific binding to target cells.

[0238] 44. The method according to item 43, wherein the target cell is a cancer cell or a virus-infected cell, optionally wherein the cell is isolated from the subject.

[0239] 45. The method according to item 41, further comprising screening the library for specific binding to the antigen:MHC complex.

[0240] 46. ​​The method according to item 45, wherein the antigen of the MHC complex is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus.

[0241] 47. The method according to item 45, wherein the antigen of the MHC complex is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, Lengsin, Meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, EGFR, HER2, TRAILR1, RANKL, IGF1R, EpCAM, and CEA.

[0242] 48. The method according to item 41 further includes screening the library for T-cell phenotypic markers.

[0243] 49. The method of claim 41, further comprising screening the library for activity in a co-culture system, wherein the co-culture system comprises at least one of the following: (a) Cancer cell line; (b) Multiple cells infected by a known virus; (c) Multiple tumor cells isolated from a cancer patient; (d) Immortalized cell lines; or (e) Multiple cells derived from patient tissue biopsy samples.

[0244] 50. The method according to item 41 further includes in vitro activation of the transformed cell population.

[0245] 51. The method according to Item 50, wherein one or more of the following stimulants are used for in vitro activation: anti-CD3 antibody, anti-CD8 antibody, anti-CD27 antibody, IL-2, IL-4, IL-21, anti-PD1 antibody, anti-CTLA4 antibody, tumor cell lysate, cell co-culture with virus-infected cells, and tumor cell lines.

[0246] 52. The method according to item 41 further includes transforming the cell population with a polynucleotide encoding a transcription factor.

[0247] 53. The method according to Item 52, wherein the transcription factor is selected from the group consisting of: FOXP3, BLIMP-1, Ikaros, Helios, and TGF-β.

[0248] 54. The method according to item 41 further comprises selecting a single vector for inclusion in the recombinant TCR library based on one or more of the following characteristics: TCR clonal universality, TCR enrichment characteristics determined in vitro, TCR binding specificity, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motif and / or CDR3 gene motif.

[0249] 55. The method according to item 54, wherein the selection includes mixing individual vectors at defined ratios to generate a synthetically derived TCR library.

[0250] 56. A recombinant TCR library prepared by the method according to item 41.

[0251] 57. A composition comprising a recombinant TCR library and a vector as described in item 56.

[0252] 58. The composition according to item 57, wherein the carrier is a pharmaceutically acceptable carrier.

[0253] 59. A method of treating a subject in need, the method comprising administering to the subject an effective amount of the recombinant TCR library according to item 56 or the composition according to item 57 or 58.

[0254] 60. A method of treating cancer in a subject in need, the method comprising administering to the subject an effective amount of the recombinant TCR library according to item 56 or the composition according to item 57 or 58.

[0255] 61. The method described in Item 60, wherein the cancer is acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; AIDS-related cancer; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rod-shaped tumor; brain cancer; basal cell carcinoma of the skin; bile duct cancer; bladder cancer; bone cancer; breast cancer; bronchoma; Burkitt lymphoma; carcinoid tumor (gastrointestinal); germ cell tumor; primary CNS lymphoma; cervical cancer; bile duct cancer; chordoma; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); chronic myeloproliferative disorder. Sexual tumors; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; ductal carcinoma in situ (DCIS); endometrial cancer; ependymoma; esophageal cancer; neuroblastoma; extracranial germ cell tumor; gonadal germ cell tumor; ocular cancer; intraocular melanoma; retinoblastoma; fallopian tube cancer; malignant fibrous histiocytoma and osteosarcoma of bone; gallbladder cancer; gastric cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); germ cell tumor; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; cardiac tumors; hepatocellular carcinoma; Langerhans cell histiocytosis; Hodgkin's lymphoma; hypopharyngeal cancer; intraocular melanoma Pancreatic cancer; islet cell tumors, pancreatic neuroendocrine tumors; kidney cancer; laryngeal cancer; leukemia; lip and oral cancer; liver cancer; lung cancer; lymphoma; male breast cancer; malignant fibrous histiocytoma and osteosarcoma of bone; melanoma; Merkel cell carcinoma; mesothelioma; metastatic cancer; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides; myelodysplastic syndrome, chronic myeloproliferative neoplasm; nasopharyngeal carcinoma; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer; oral cancer, oropharyngeal cancer; osteosarcoma; ovarian cancer; pancreatic cancer; pancreatic neuroendocrine tumors; papillary tumors Tumors; paragangliomas; sinus carcinomas; parathyroid carcinomas; pharyngeal carcinomas; pheochromocytomas; pituitary adenomas; pleural pulmonary blastomas; prostate cancer; rectal cancer; recurrent cancers; renal cell carcinomas; retinoblastomas; rhabdomyosarcomas; salivary gland carcinomas; sarcomas; Ewing sarcomas; Kaposi's sarcoma; osteosarcomas; uterine sarcomas; Cezary syndrome; skin cancer; small cell lung cancer; small bowel cancer; soft tissue sarcomas; squamous cell carcinoma of the skin; squamous neck carcinoma; gastric cancer; T-cell lymphomas; testicular cancer; laryngeal cancer; nasopharyngeal carcinoma; hypopharyngeal carcinoma; thymic carcinoma; thyroid cancer; urethral cancer; uterine cancer; vaginal cancer; hemangiomas; vulvar cancer; or Wilms' tumor.

[0256] 62. A method for inhibiting tumor growth in a subject in need, the method comprising administering to the subject an effective amount of the recombinant TCR library according to item 56 or the composition according to item 57 or 58.

[0257] 63. The method according to item 62, wherein the tumor is a solid tumor.

[0258] 64. A method of treating a subject with a viral infection, the method comprising administering to the subject an effective amount of the recombinant TCR library according to item 56 or the composition according to item 57 or 58.

[0259] 65. The method according to item 64, wherein the viral infection is caused by a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71 (EV71), Ebola virus, hepatitis A (HAV), hepatitis B (HBV), cytomegalovirus (CMV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), human immunodeficiency virus (HIV), human papillomavirus (HPV) Herpes simplex virus (HSV), human T-lymphotropic virus (HTLV), influenza A virus, influenza B virus, Japanese encephalitis virus, leukemia virus, measles virus, molluscum contagiosum virus, sheep pox virus, parvovirus, rabies virus, respiratory syncytial virus, Rift Valley fever virus, rubella virus, rotavirus, tick-borne encephalitis virus (TBEV), simian immunodeficiency virus, tobacco etch virus (TEV), varicella-zoster virus, smallpox virus, West Nile virus, Zika virus, and chikungunya virus.

[0260] 66. The method according to any one of items 59 to 65, further comprising administering to the subject a second dose of the recombinant TCR library according to item 56 or the composition according to item 57 or 58.

[0261] 67. The method according to any one of items 59 to 66, wherein the recombinant TCR library comprises cells that are autologous or allogeneic to the treated subject.

[0262] 68. The method according to any one of items 59 to 67, wherein the subject is a human, animal, non-human primate, dog, cat, sheep, mouse, horse, or cow.

[0263] 69. The method according to item 68, wherein the subject is a human being.

[0264] Example The present technology is further illustrated by the following embodiments, which should not be construed as limiting in any way.

[0265] Example 1: Generation and cloning of paired natural TCRα:β amplicon into expression vector First, a human T cell population was isolated to collect paired TCRα:β amplicones as a source of naturally paired T cell receptor genes. Histopaque was used. ®Peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulated whole blood from healthy patients at -1077 g / cm³ by centrifugation at 400 × g for 30 min. After removing the supernatant plasma layer, plasma and histopaque were collected. ® Mononuclear cells with a concentration between -1077 and 1077 were collected and resuspended in phosphate-buffered saline. The cells were gently mixed and centrifuged at 250×g for 10 min to remove platelets. The supernatant was removed and the cell pellet was resuspended in PBMC medium (RPMI containing 10% fetal bovine serum) or cell cryopreservation medium (RPMI containing 10% fetal bovine serum and 10% DMSO).

[0266] PBMCs were then cultured in RPMI medium supplemented with 10% fetal bovine serum for 4 hours, followed by emulsion PCR to amplify the full-length T cell receptor α and β chains. Phlorizol 12-myristate 13-acetate (PMA) and ionomycin were added to the PBMC medium to a final concentration of 100 ng / mL for T cell stimulation. Using a custom-designed polymer flow nozzle, individual PBMC cells were isolated into emulsion microdroplets with cell lysis buffer and poly(dT) beads for mRNA capture (references 1-4). The nozzle design ensured that cells were not exposed to the lysis buffer before being isolated into individual microdroplets. Within the microwells, cells were co-encapsulated with lysis reagent to release mRNA, and poly(dT) beads were used for mRNA capture and purification. Emulsion was broken using ether, while the poly(dT) beads were recovered, first washed with high-salt hybridization buffer and then with PCR buffer. The beads were then re-emulsified in an overlap extension reverse transcription PCR OE-RT-PCR mixture, using primers specific to the TCRα and β chains modified from those by Boria et al. (reference number 5), and incorporating restriction enzyme sequences and T-cell receptor leader sequences for downstream cloning. Adapter sequences were also included to allow the TCRα and β chains to be physically ligated during TCR OE-RT-PCR. The SuperScript™ III one-step RT-PCR system with Platinum™ Taq DNA polymerase was used as the emulsion PCR reagent. OE-PCR thermal cycling conditions are provided in Table 3 and primer sequences are provided in Table 4.

[0267] Table 3: Thermal cycling conditions for OE-PCR

[0268] Table 4: Primers used for OE-PCR.

[0269] After the OE-RT-PCR reaction, the PCR sample was purified using a DNA-cleaning kit and then subjected to DNA gel electrophoresis. Figure 1 As shown, the linked TCRα and β amplicon were identified at around 1000 bp, indicating a successful OE-RT-PCR reaction.

[0270] Following DNA gel electrophoresis, gel extraction was performed to obtain the TCRα:β amplicon. 5 ng of purified PCR product was used as a template for semi-nested PCR. The thermal cycling conditions for nested PCR are provided in Table 5.

[0271] Table 5: Thermal cycling conditions for nested PCR

[0272] DNA was cleaned and the PCR products purified using the Zymo DNA Clean & Concentrator Kit (Zymo Research). The purified PCR products were analyzed by DNA electrophoresis. The PCR results are shown below. Figure 2 The band of approximately 1000 bp indicates successful amplification of the TCRα:β amplicon.

[0273] Following DNA electrophoresis, the TCRα:β amplicons were excised and purified using the Zymoclean™ Gel DNA Recovery Kit (ZymoResearch). The purified amplicons were then subjected to zero-end cloning (Thermo Fisher Scientific) to analyze the TCRα:β amplicon sequences. Sequences were identified using the NCBI IGBLAST T cell receptor gene database (available at www.ncbi.nlm.nih.gov / igblast / ). Sequencing results for the eight TCRα:β amplicons and their respective TCRα and β strand genes are listed in Table 6.

[0274] Table 6: Gene sequencing results of TCRα:β amplicon cloned into plasmid vectors for single-colony sequencing.

[0275] The sequenced TCRα:β amplicons represent a diverse TCRα:β library from human PBMCs, supporting the conclusion that the TCR primer design described in this embodiment can successfully amplify the TCR library. Primers with restriction endonuclease target cleavage sites shown in Table 4 were incorporated into the amplicons, and OE-PCR and nested PCR were successfully performed. Restriction enzymes were selected to target cleavage sites that are rarely found (e.g., found in fewer than 5 segments) in the T cell receptor gene (containing variable, diversity, conjugation, and constant regions), thereby allowing expression of the original TCR amino acid sequence without loss of TCR library diversity and reducing TCR library bias due to cleavage during restriction enzyme digestion and subsequent cycles of expression plasmid processes. Silent and non-silent mutations were incorporated at several restriction sites to add additional restriction enzyme cleavage sites rarely found in the native TCR gene (Tables 7 and 8).

[0276] Table 7: Silent mutations used to clone T-cell receptors into lentiviral expression vectors without altering the amino acid sequence of the gene.

[0277] Table 8: Non-silent mutations that minimize modification of the native amino acid sequence for cloning T-cell receptors into lentiviral expression vectors.

[0278] The TCRα:β amplicon was subcloned into the lentiviral expression vector pLVX-EF1α-IRES-mCherry (Clontech). The lentiviral expression vector containing the TCR, along with the lentiviral packaging and envelope vectors, was co-transfected into HEK293T cells to generate lentiviral particles for TCR expression. HEK293T cells were transfected at the following ratio: 10 μg pLVX, 8 μg psPAX2 packaging plasmid, 3 μg pMD2.g envelope plasmid, and 100 μl Fugene transfection reagent. The recombinant lentivirus encoding the TCR was transduced into the J.RT3 T cell line for TCR expression. The endogenous TCRα and β genes of J.RT3 cells were knocked out, allowing transgenic expression of exogenous TCRα and β chains. The transduction protocol was optimized to achieve an MOI ≤ 0.2, ensuring that the vast majority of cells expressed only a single TCR-paired α:β amplicon. Finally, transduced cells were stained with anti-α chain antibody, anti-β chain antibody, anti-CD8 antibody, and anti-CD3 antibody for flow cytometry analysis to determine the fraction of mCherry-expressing cells displaying a fully assembled TCR complex for functional peptide-MHC binding analysis.

[0279] Figure 15This image shows the DNA electrophoretic analysis of T cell receptor (TCR) amplicon (α:β chain) from single-cell emulsion overlap extension RT-PCR. In short, T cells purified from PBMCs using a CD8 separation kit were isolated into oil emulsion microwells with lysis buffer and poly(dT) beads to capture mRNA for cell lysis and RNA capture. The poly(dT) beads containing T cell RNA were then re-emulsified for cDNA synthesis and overlap extension PCR (OE-PCR). Two semi-nested PCRs (first and second half-nested) were performed to increase the TCR α:β chain amplicon concentration. Mi-seq PCR was then performed to add next-generation sequence barcoding for high-throughput sequencing analysis.

[0280] After high-throughput sequencing, the TCR gene was quality filtered and annotated using the NCBI IgBLAST and CDR3 motif algorithms. The TCR gene was then compiled into a TCR library by pairing the α and β strands. Figure 16 Shows an overview of the distribution of the linked α:βT cell receptor genes.

[0281] Example 2: Evaluation of TCR clones containing silent or non-silent mutations To successfully clone the TCR gene using the restriction enzymes listed herein, the target cloning vector pLVX-EF1α-IRES-mCherry was modified to remove the following four cleavage sites via site-directed mutagenesis: AgeI (2415), SphI (2331), NheI (8192), and MluI (6669) cleavage sites (the numbers indicate the locations of these cleavage sites).

[0282] The previously identified TCR (anti-HIV-Nef-Rm9) was used as a model TCR to evaluate the TCR genes with different mutations listed in Tables 7 and 8. The wild-type anti-HIV-Nef-RM9 TCR gene fragment was subcloned into the lentiviral vector pLVX-EF1α-IRES-mCherry for expression of the active TCR. All anti-HIV-Nef-RM9 mutant clones were introduced via gene synthesis along with restriction enzyme digestion and ligation (Tables 7 and 8). Lentiviral expression vectors containing both wild-type and mutant anti-HIV-Nef-Rm9 TCRs were transfected separately into HEK293FT cells with envelope and packaging plasmids (psPAX2 and pMD2.G) to express lentivirus. The engineered Jurkat cell line JRT3 / CD8, modified to lack active T cell receptor expression, was used as the TCR expression platform. Lentivirals encoding wild-type and mutant anti-HIV-Nef-RM9 TCRs were individually transduced into JRT3 / CD8 cells for TCR expression. The binding affinity of wild-type and mutant anti-HIV-Nef-RM9 TCRs to the HIV peptide RPQVPLRPM (SEQ ID NO:1) conjugated to the major histocompatibility complex (MHC) was evaluated. The RPQVPLRPM-MHC complex (“RPQVPLRPM” disclosed as SEQ ID NO:1) was conjugated with streptavidin-allophycocyanin (SA-APC) to detect TCR binding to the peptide-MHC. In parallel, transduced JRT3 / CD8 cells were stained with an anti-TCR antibody conjugated to the BV421 fluorescent marker to evaluate T cell receptor expression of anti-HIV-Nef-RM9 with different mutations.

[0283] Figure 17 and Figure 18 It was confirmed that mutations in anti-HIV-Nef-Rm9 TCRs with different non-natural leader peptides led to varying levels of TCR expression. This was compared with wild-type anti-HIV-TCR expression levels and their binding affinity to pMHC. Figure 17 Compared to subplots C and D, TCRs containing mutations in either TRBV24-1 or TRAV40-1 leader peptides showed comparable TCR expression levels and pMHC binding affinity. Figure 17 Subplots G and H target TRBV24-1, while subplots K and L target TRAV40-1. TCRs with mutations in the TRBV30 leader peptide exhibited reduced TCR expression and pMHC binding ability. Figure 17 (Figures E and F). Anti-HIV TCRs with mutations in the TRAV17-1 leader peptide exhibit no pMHC binding tendency and low levels of TCR expression. Figure 17Subplots I and J). The expression levels of wild-type anti-HIV-TCR and its binding affinity to pMHC (…). Figure 17 Compared to subplots C and D, TCRs with silencing mutations (used for incorporation into SpeI restriction sites) exhibit comparable TCR expression levels and pMHC binding affinity. Figure 18 (See Figures A and B). TCRs with a single amino acid mutation, TRBCV30A, exhibit similar TCR expression and pMHC binding ability to those of wild-type anti-HIV TCRs. Figure 18 (See Figures C and D). Anti-HIV TCRs with a single amino acid mutation, TRBCP9L, exhibited no pMHC binding tendency and no TCR expression. Figure 18 (Subplots E and F).

[0284] Figure 19a This demonstrates the isolation of human effector T cells used for tumor-specific T cell analysis in humanized mouse models and cancer patients. Figure 19b The Vα:Vβ gene was shown in 31,718 isolated, RT-PCR amplified, and NGS-analyzed human CD8+ TCR clusters.

[0285] Example 3: Screening for natural TCRα:β based on binding to known peptide:MHC combinations In this embodiment, tetramer staining was performed using a recombinant human leukocyte antigen (HLA) tetramer complex loaded with a known Epstein-Barr virus (EBV) peptide. EBV-reactive T cells with TCRs on their cell surface will recognize the EBV peptide-HLA tetramer complex. The HLA complex is biotinylated and conjugated with streptavidin linked to a fluorophore, thereby allowing the sorting of EBV-reactive T cells. PBMCs were isolated from Epstein-Barr virus-positive donors with known HLA alleles as described in Example 1, and emulsion overlap reverse transcription extension PCR (OE-RT-PCR) was performed to isolate the donor's T cell receptor library. The TCR amplicons were inserted into the developed TCR expression lentiviral vector constructed in Example 1. After generating a vector for expressing a naturally paired T cell receptor library, the J.RT3 cell line was transduced with the lentiviral vector at an MOI of ≤0.2, thereby allowing the expression of TCRα and β chains on the in vitro cell library.

[0286] Several previously characterized EBV TCR-peptide-MHC interactions were evaluated (references 6-8). In particular, HLA-B8 loaded with the known immunodominant EBV peptide HLA-B8-restriction epitope FLRGRAYGL (SEQ ID NO: 79), derived from the potential EBV antigen protein EBNA3A (reference 9), was assessed. The interaction of transgenic J.RT3 cells with HLA-A from LMP2 was also tested. 02.01 Restriction Epitope Response: LMP is a known EBV oncogenic protein that is considered to have important potential protective effects against EBV, but includes more measured EBV responses (Reference No. 10). The biotinylated HLA tetramer was then conjugated with a streptavidin-allophycocyanin conjugate from Thermo Fisher Scientific (Catalogue No. S32362), wherein the molar ratio of MHC monomer to streptavidin as a cell sorting marker was 5:1. The streptavidin-labeled MHC complex was cultured with lentivirus-transduced J.RT3 cells possessing recombinant TCRs on the cell surface, and fluorescently activated cell sorting was performed as described by Altman et al. (Reference No. 11).

[0287] Sorted J.RT3 cells expressing the EBV-responsive T-cell receptor sequence (α:β chain) were sorted by flow cytometry and recovered overnight in RPMI with 10% FBS. The TCR amplicon sequences were then identified by next-generation sequencing analysis of the sorted T-cell receptor libraries, as previously described (references 12-16). Optionally, EBV-responsive J.RT3 cells were seeded at a density of 1 cell / well in 96-well plates to isolate single-cell clones. After single-cell proliferation, RNA was extracted and cDNA was synthesized by reverse transcriptase. PCR was performed to amplify the TCR α:β pairing sequence, as previously described (references 17-19). Plasmids were extracted and sequences were identified by Sanger sequencing.

[0288] Example 4: Screening for natural TCRα:β to kill EBV-infected cells in vitro and in cell lines Immature T cells were isolated from PBMCs of healthy human donors using the EasySep™ Human Immature CD8+ T Cell Isolation Kit (Stemcell Technology). The TCRα:β gene sequence recovered as described in Examples 1 and 2 was delivered into the isolated immature T cells using the Sleeping Beauty transposon / transposase system. The TCR sequence library, along with the EF1α promoter, was subcloned in the pLVX vector (for gene expression) from Example 2 into the Sleeping Beauty transposon cloning vector pT2 / BH (reference number 20) containing the SB transposase. Immature T cells were transfected with the pT2 / BH vector and pCMV(CAT)T7-SB100 (Addgene plasmid number 34879). pCMV(CAT)T7-SB100 expresses a highly active Sleeping Beauty transposase, thereby allowing expression of the full-length TCR. Following the manufacturer's instructions (Lonza, Cologne, Germany), a gene delivery transposon plasmid containing EBV TCRs and an SB100X transposase plasmid were transfected into a T cell population using a 4DNucleofector. In parallel, naive T cells were transfected with non-EBV-responsive TCRs identified as a negative control in Example 2. The transduced T cell library was identified by staining for TCR surface markers (anti-α, anti-β, anti-CD8, anti-CD3) indicating stable TCR complex assembly and analyzed by flow cytometry. The efficiency of productive TCR display on the transduced cells was analyzed.

[0289] Next, the in vitro function of the transfected TCR library was analyzed by co-culturing it with EBV-infected B cells (Raji cells, a cancer cell line that has been detected as EBV-positive), and its activity against EBV-infected autologous donor B cells was also analyzed. T cells were seeded with EBV-positive B cells at a ratio of 10:1 and co-cultured for 24 hours. After 24 hours of in vitro co-culture, the T cells were recovered, stained, and sorted for T cell markers and upregulated CD107 / membrane TNF-α expression (references 21-23). ​​The TCRs encoded by these cells were analyzed using NextGen sequence analysis to reveal EBV-targeted tumor-infiltrating lymphocyte (TIL) sequences in the library.

[0290] The monoclonal paired TCRα:β cDNA was then recovered and transduced into naive human T cells as monoclonal TCRs. The ability of these TCRs to target EBV peptides was validated by co-culturing with an in vitro population of identical EBV-infected B cells. Co-culture assays were repeated, and pro-inflammatory cytokines were measured using ELISA kits for IFNγ, TNF-α, perforin, and granzyme. Naive T cells expressing EBV-specific TCRs showed increased levels of all pro-inflammatory cytokines compared to naive T cells transfected with negative control TCRs.

[0291] In vitro cytotoxicity assays were also performed to evaluate the activity of transformed naive T cells against EBV-infected B cells or Laji cells. Laji cells and autologous B cells were individually diluted to 5 × 10⁻⁶. 6 The cells were incubated at a concentration of 1 / mL with 0.25 μM fluorescein diacetate succinimide (CFSE) in a 37°C CO2 incubator for 30 min to covalently bind the CFSE dye to all free amines from the cells. The CFSE dye allowed for the evaluation of B cell viability and proliferation. The stained B cells were washed three times with RPMI medium. Then, naive T cells expressing EBV-responsive TCRs alone, as well as control TCR libraries, were co-cultured with CFSE-stained B cells or Laji cells at a range of effector cell to target cell ratios. Cell cytotoxicity was evaluated by flow cytometry at excitation and emission wavelengths of 492 nm and 517 nm, respectively. Compared to naive T cells in the control TCR assay (TCRs without EBV peptide affinity), EBV-responsive TCRs transduced into a healthy naive T cell population exhibited superior cytotoxicity against EBV-infected autologous B cells or Laji cells.

[0292] Example 5: Gene transfer of a natural TCRα:β library for cancer treatment Animal models were used to demonstrate the isolation, recovery, and subsequent application of cancer-specific TCRs, as well as the recovered TCR library, as a cell-based therapeutic agent. Human PBMC-transplanted CD34-NSG-modified mice were implanted with human RKO colon cancer xenografts (Charles River Labs). First, CD34-NSG was transplanted into human PBMCs via intravenous (iv) injection, followed by subcutaneous injection into 50% Martigel-modified TCR libraries. ® 1×10 in the Matrix (Corning) 7RKO tumor cells were collected. Mice were then given two checkpoint inhibitors (pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4)) to enhance the anti-tumor TCR response. Mice were given 100 μg pembrolizumab and 100 μg ipilimumab every two weeks. On day 15, spleen and tumor tissue were removed from mice to isolate spleen and tumor-infiltrating T cells. Central memory (TCM) and transitional memory (TTM) T cells were obtained by FACS analysis targeting the expression of CD3+CD8+CD45RACCR7. The TCM and TTM populations were then amplified and subjected to emulsion OE-PCR and nested PCR as described in Examples 1 and 2 to obtain a TCR library, which was cloned into the Sleeping Beauty transposon / transposase transduction plasmid as described in Example 3.

[0293] The Illumina MiSeq 2×300 end-pairing read platform was used to precisely define and determine the molecular characteristics of TCR libraries. Raw Illumina sequences were quality-filtered, mapped to the V, D, and J genes, and CDR3 was extracted using the International Immunogenetics Information System (IMGT, reference 24). Sequence data were filtered for in-frame V(D)J junctions, and productive TCRα and TCRβ sequences were paired using Illumina read IDs. Accurate CDR3 nucleotides and V(D)J gene matching were then used for compilation. CDR-β3 nucleotide sequences were extracted and clustered to 96% nt identity, ignoring terminal gaps (USEARCH v5.2.32, reference 25), and the resulting Vα:Vβ pairs with ≥2 reads constituted a list of Vα:Vβ clusters.

[0294] Optionally, after emulsion RT-PCR and nested PCR, the TCR α:β amplicon library can be ligated into the Sleeping Beauty transposon vector (pT2 / BH) and transformed into highly competent *E. coli* cells (such as XL-Gold (Agilent ID 200314)) to clone a highly diverse TCR library. The paired αβ TCRs are then sequenced individually using bacterial colony Sanger sequencing. Approximately 100 colonies are combined with known TCR sequences to generate a precisely defined paired αβ TCR library for delivery into naive T cells. A TCR library obtained from TCM and TTM T cells derived from humanized mice that have not been transferred into the RKO cancer cell line is used as a negative control.

[0295] Next, as reported in Example 3, two TCR libraries (derived from RKO xenograft TCRs or derived from mice without RKO xenograft TCRs) were expressed in naive T cells using the Sleeping Beauty transposon / transposase system. The transformed TCR libraries were administered individually to different RKO xenograft mice via intravenous injection. Tumor volume and body weight changes in individual mice were measured daily to track tumor size and disease progression. RKO xenograft mice treated with T cells transduced from TCR libraries derived from the control group (i.e., mice from human PBMC CD34-transplanted mice without RKO xenografts) exhibited delayed tumor growth compared to mice treated with T cells transduced from TILs derived from other RKO xenograft mice. This demonstrates the suitability of precisely defined TCR libraries as cell-based therapeutic agents that effectively reduce tumor progression.

[0296] Example 6: Gene transfer of a preselected natural TCRα:β library for cancer treatment targeting anticancer activity In this embodiment, a similar procedure and the same mouse model (CD34-NSG humanized mice with human PBMCs transplanted into an RKO colon cancer xenograft model) were used as in Example 4, but an additional T cell preselection step was performed to isolate anti-cancer TCRs via in vitro selection before use as a cell-based therapeutic agent. Splenic cells and tumor-infiltrating lymphocyte populations were isolated after RKO colon cancer transplantation. TCMs and TTMs were isolated using the markers CD3+CD8+CD45RA and CCR7 via FACS. As described in Example 4, a TCR library was isolated using emulsion OE-PCR and nested PCR, then subcloned into a Sleeping Beauty transposon vector and re-delivered to naive T cells isolated from PBMCs. Transgenic naive T cells were stimulated by co-culturing with irradiated RKO colon cancer cell lines as described in Example 3, and isolated using flow cytometry targeting CD107 and membrane TNF-α.

[0297] Optionally, dendritic cells (DCs) were isolated from humanized mice using the Blood Dendritic Cell Isolation Kit II (Miltenyi Biotec) and cultured in RPMI containing 10% FBS supplemented with 50 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF). The DCs were pulsed with RKO cancer cell lysate or human colon cancer cell lysate for two hours. Naïve T cells transduced with a TCR library cloned into the Sleeping Beauty transposon / transposase vector were then co-cultured with DCs at a 10:1 ratio for 4 days. Stimulated anti-cancer cytotoxic T cells were gated by membrane-bound CD107, membrane-bound TNF-α expression, or CD137 (4-1BB) expression. CD107a is a marker for degranulation of activated CD8+ T cells (References 26-33). CD137 belongs to the TNFR family and is associated with T cell proliferation and survival (References 34-35). As described in Example 4, sorted T cells (anti-cancer T cells) were delivered to an RKO colon cancer mouse model. Mice treated with naive T cells transduced with TCRs targeting RKO, isolated through in vitro functional screening, exhibited higher survival rates and reduced tumor growth compared to mice treated with naive T cells transduced with TCRs from non-RKO xenograft mice. Activation of T cells in the anti-cancer co-culture assay was selected, and enrichment was performed against cancer-specific TCRs to enhance the anti-cancer efficacy of the final cell-based therapeutic agent.

[0298] Example 7: Pre-selection and in vitro pre-activation of natural anti-cancer activity to enhance anti-tumor cell killing after tumor cell killing Gene transfer from TCRα:β library The experimental procedures of Example 6 were performed as described in Example 5, followed by an additional T cell activation step after transducing the naturally paired TCR library into naive T cells. This activation step pre-sensitized the T cells and activated them to enhance cancer cell killing. Briefly, after OE-PCR and initial nested PCR, the TCR library was cloned into a Sleeping Beauty transposon vector for transgenic TCR expression. Next-generation sequencing or Sanger sequencing of individual plasmids was performed to define the size and diversity of the TCR library. The pooled TCR transposon plasmid was co-transfected with the Sleeping Beauty transposase expression vector SB100 to allow TCR expression in naive T cells. Transgenic T cells were first stimulated with RKO cell lines or T cells treated with cancer antigen pulses, and anti-cancer T cells expressing CD137 / CD107 / TFN-α were sorted by FACS. These cells were then activated in vitro and subsequently delivered as a cell-based therapeutic agent to an RKO tumor mouse model. Gibco™ Dynabeads™ human T activator CD3 / CD28 (Thermo Fisher Scientific) was added to TCR-transduced naive T cell cultures at a final bead-to-cell ratio of 1:1 and cultured in a 37°C CO2 incubator for 3 days. Dynabeads were conjugated with anti-CD3 and anti-CD28 antibodies to mimic the in vivo interaction between T cells and antigen-presenting cells (APCs), allowing for T cell clonal expansion and differentiation (Reference No. 36). Cell growth and viability were monitored daily after activation.

[0299] The expression of IFN-γ and TNF-α was examined by ELISA, indicating T cell differentiation. On day 3 after bead addition, 20 U / mL of recombinant human interleukin-2 (IL2) was added to the culture medium to induce further T cell expansion. On day 7, the expanded T cells were counted and transfused via tail vein. iv The cells were delivered via injection into RKO xenograft mice. Tumor growth and individual mouse weight were evaluated and compared with those obtained in Examples 4 and 5. RKO-transplanted mice treated with transgenic RKO tumor-specific, stimulated, and activated T cells in this example demonstrated superior anticancer efficacy compared to those from Example 4 (without tumor selection or activation of transgenic T cells) and Example 5 (without in vitro pre-activated T cells selected for responsiveness to RKO). The additional activation step described in this example allows effector cytotoxic T cells to differentiate prior to therapeutic delivery, enhancing the speed and intensity of tumor immunosuppression in the mouse model.

[0300] Example 8: Gene transfer of a natural TCRα:β library for inducing antigen-specific immune tolerance It is known that CD34+ human PBMC transplantation into a mouse model induces graft-versus-host disease (GvHD) in mice. To alleviate this problem, regulatory T cells with TCRs isolated from mice suffering from GvHD were induced and used as therapeutic agents to induce immune tolerance.

[0301] T cells were isolated from mouse spleens and PBMCs prepared as described in Example 4. T cells were isolated 30 days post-transplantation, at the onset of GvHD episodes. Overlap extension reverse transcriptase PCR was performed using the TCR cloning primer set from Example 1 to obtain TCR libraries from both Treg cells (CD4+CD25+) and conventional T cells (CD4+CD25-). The two TCR libraries were then subcloned into the pT2 / BH Sleeping Beauty transposon vector. Next-generation sequencing was performed to analyze the two sets of T cell receptor libraries and characterize T cell receptor gene usage.

[0302] The following sorting markers were used to isolate naive T cells from PBMCs: CD25 - CD44 低 CD62L 高 Cells were transfected with a TCR donor transposon plasmid containing the two libraries described above, along with the Sleeping Beauty transposase plasmid (SB100), to facilitate TCR expression using a TCR sequence library isolated from GvHD mice. Transgenic naive T cells were then induced into T reg cells using Dynabeads™ human T activator CD3 / CD28 along with 5 ng / mL TGF-β1. TGF-β (shown to be a major regulator for inducing Foxp3 expression) allows naive T cells to differentiate into regulatory T cells (references 37-38). Five days after induction, rIL-2 was supplied to the induced T regulatory cells (iTregs), enabling T cell proliferation for treatment of GvHD relapse mice.

[0303] Optionally, as previously described (references 39-40), T cells were directly transduced using the Foxp3 sequence. Three days after expansion, iTreg cells with and without transgenic TCRs were individually intravenously injected via tail vein into GvHD-stricken mice (n=5); these mice had been transfected with CD34+ human PBMCs from the same donor and sampling time point as the earlier GvHD mice. Mice treated with iTregs possessing CD4+ CD25+ transgenic TCRs exhibited delayed GvHD disease onset compared to mice derived from iTreg cells with transgenic CD4+ CD25-TCRs and mice derived from iTreg cells without transgenic TCRs.

[0304] Body weight changes and GvHD scores were analyzed among three groups (non-transgenic TCR, CD4+CD25+TCR, and CD4+CD25-TCR). Mice injected with T cells expressing CD4+CD25+TCR showed the lowest body weight changes and GvHD scores compared to mice infused with T cells only and mice infused with T cells expressing CD4+CD25-TCR. Tolerogenic activity and T cell phenotype were monitored. Twenty days after T cell infusion, spleen cells were isolated by euthanizing humanized mice, harvesting spleens, and digesting with erythrocyte lysis buffer. The resulting leukocytes were stained with anti-CD25, anti-CD4+, anti-CD3, and Foxp3, followed by flow cytometry analysis to evaluate tolerogenic activity. Leukocytes from mice with transgenic CD4+CD25+ TCR showed the strongest Foxp3 and CD25 signals as analyzed by flow cytometry staining compared to leukocytes from the other two groups (non-transgenic TCR and CD4+CD25-TCR).

[0305] Example 9: Application of natural TCRα:β library gene transfer in tumor therapy in mammalian or human patients In this embodiment, a mammalian patient, such as a human cancer patient, is treated. In some embodiments, the cancer type is lung cancer, melanoma, renal cell carcinoma, breast cancer (including triple-negative breast cancer), colon cancer, or prostate cancer. The treatment procedure can be similar to that described in Example 4. Simply put, T cells from a mammalian or human patient are recovered, which may be derived from PBMCs, tumor-infiltrating lymphocytes, spleen tissue, affected organs, or other tissues from the patient. The TCR gene is then recovered and cloned into an expression vector. In some embodiments, a T cell library is first pre-screened for anti-tumor activity. Screening can identify TCRs that are reactive to tumor peptide neoantigens, or optionally TCRs that are reactive to whole tumor cells in vitro. In some embodiments, the tumor cells may be derived from a cancer patient.

[0306] Next, the selected T-cell library can be used as a cell-based therapeutic agent. In some embodiments, high-throughput sequencing is used to analyze the library to precisely define the molecular composition of the cell-based TCR therapeutic agent. In other embodiments, the library is selected by secondary sampling of numerous individual plasmid colonies in bacteria (ranging from 3 to 100,000 colonies derived from the library) and sequencing each plasmid colony individually after micro-preparation. These individual plasmids are then mixed at defined ratios to regenerate a precisely defined molecular library. These precisely defined molecular libraries reduce the presence of PCR error variants that may occur when the TCR library is initially generated via RT-PCR.

[0307] In some embodiments, the mouse TCR constant region gene will be used to prevent the TCR transgene from associating with the natural human T cell receptor gene in the gene recipient T cells. The library will be used to transfer the TCR and any other plasmid genes into human T cells. In some embodiments, these T cells are derived from a patient. In some embodiments, these T cells will be naive T cells derived from a patient. In other embodiments, these T cells may be derived from other individuals or from cell lines. In some embodiments, as described in Example 6, an activation step may be present to pre-sensitize the T cells and activate them to enhance cancer cell killing.

[0308] In some embodiments, as described in Example 7, transcription factors can be used to influence cell fate. Transformed T cells expressing the TCR transgene can be expanded in vitro to generate a cell bank, or they can be administered directly to a patient. In some embodiments, T cells are injected directly into the tumor. In other embodiments, T cells are administered intravenously or intrathecally.

[0309] The plasmid gene library can be reused at any time to regenerate a T-cell transgenic population for subsequent repeated administrations; in other embodiments, the expanded cell library can be used for repeated therapeutic administrations. The entire treatment process can be repeated in cases of ongoing cancer progression and / or tumor growth or inadequate treatment of the tumor in any way. In some embodiments, this may include recapturing the patient's T-cell genes and repeating the screening and library generation process. In other embodiments, repeated therapy may include rescreening the initially captured TCR gene library against a resistant tumor cell population to identify TCRs targeting evolving cancer cells. In other embodiments, repeated therapy may include additional activation of the TCR gene library. In some embodiments, the in vitro cell activation process may be altered over multiple treatment administrations; for example, as cancer progresses, a more effective activation step may be used for in vitro cell-based therapeutic agents prior to therapeutic administration.

[0310] In some implementations, the i-cysteine ​​gene or other inducible suicide switch genes may be included in the transgenic vector to control the fate of cell-based therapies after therapeutic administration.

[0311] Example 10: Application of natural TCRα:β library gene transfer in the treatment of viral infections Post-transplant lymphoproliferative disease (PTLD) is a serious complication of solid organ transplantation. Primary Epstein-Barr virus (EBV) infection is a major risk factor, and approximately 60–80% of PTLD cases are EBV seropositive. T-cell responses to EBV peptides are crucial for suppressing malignancies (Llaurador, G. et al.). Curr. Opin. Pediatr. 29, 34-40 (2017)).

[0312] The TCRα:β library disclosed in this paper will be used to treat or prevent PTLD in mouse models that serve as models for antiviral TCR therapy (Ahmed, EH and Baiocchi, R. A, ILAR J. 57, 55-62 (2016); Ricciardelli, I. et al., Blood 124, 2514-2522 (2014). In short, anti-EBV TCRs will be identified by single-cell isolation of paired α and β genes, cloned into a TCR display vector, and cell-based screening for anti-EBV peptide binding will be performed via FACS. In other embodiments, anti-EBV TCRs can be identified by identifying the recognition, amplification, and / or in vitro killing activity of T cells transformed with transgenic TCRs. In other embodiments, T-cell receptors that target other viruses for different antiviral therapies will be identified, including but not limited to human cytomegalovirus (HCMV), herpes simplex virus 1 or 2 (HSV-1 / HSV-2), and yellow fever virus (YFV).

[0313] After isolating antiviral T-cell receptor polynucleotides, they are cloned into a DNA vector and used in autologous gene therapy to treat PTLD. The transformed T cells can be used to treat other viral infections or for allogeneic gene therapy. In other embodiments, they can be used as prophylactic cell therapy to prevent PTLD or other viral infections. In other embodiments, the vector can be used as a virus-associated prophylactic cancer vaccine in gene therapy.

[0314] Lentiviral gene transduction will be used to transform human pan-T cells with antiviral TCRs to evaluate the in vitro killing efficacy of virus-infected cells. Transformed T cells can be co-cultured with virus-infected cells under any of the following stimulation conditions: none, IL-2, anti-CD3 / anti-CD28 magnetic beads, or IL-2 / anti-CD3 / anti-CD28. In vitro killing of virus-infected cells will be assessed using the IncuCyte viability assay, which measures the loss of fluorescently labeled tumor cells due to cell killing (Single, A. et al.). J. Biomol. Screen. 20, 1286-1293 (2015)).

[0315] Next, human pan-T cells will be transduced with antiviral TCRs under optimized conditions and used to treat viral infections in mouse models. Mouse models infected with EBV and PTLD (Johannessen, I. et al.) will be used. J. Med. Virol.83, 1585-1596 (2011). In other embodiments, mice can be infected with the virus and treated with T cells specific to the viral proteins. Transduced T cells are isolated by flow cytometry for transgenic TCR expression to obtain a pure transduced TCR library. The transformed cells are then stimulated using a T cell stimulation protocol (which may include the conditions described above), and the stimulated T cells are delivered to mice via intravenous tail vein injection (Kurtz, A. Mesenchymal Stem Cell Delivery Routes and Fate). Int. J. Stem Cells 1, 1-7 (2008)).

[0316] Mice will be tracked for 30 days to record weight loss and PTLD tumor volume. Treated mice will be compared to untreated control mice and other controls treated with antiviral antibodies and antiviral small molecule inhibitors. Any delay in PTLD tumor growth will be quantified as the primary study endpoint. Mice treated with transformed cells incorporating anti-EBV TCR, including this technology, are expected to show delayed PTLD growth, delayed viral growth kinetics, and / or faster disease recovery after viral infection compared to untreated controls. These results will confirm the applicability of the compositions of this technology in methods for treating or preventing viral infections and virus-related cancers in subjects of need.

[0317] Example 11: Evaluation of antigen-specific TCRα:β in cancer treatment in a PDX mouse model Patient-derived xenograft (PDX) mouse models from ovarian cancer patients will be established as a regenerative cell source for discovering anti-tumor T cells. Figure 22 ).

[0318] Human pan-T cells were transduced with a multiplicity of infection (MOI) of less than 0.2 (i.e., one TCR transgene per cell), and the transduced library was screened in vitro for TCR activation against co-cultured PDX tumor cells. Amplification and activator gene expression (CD69 / CD107 / membrane TNF-α) were quantified as markers of anti-tumor TCR recognition. TCR library diversity was analyzed using Illumina MiSeq 2×300 end-paired sequencing, and Vβ and Vα genes were characterized at each step of the cloning and selection process (i.e., cDNA infusion, post-lentiviral particle generation, and mCherry+J.RT3 cells with surface-displayed TCRs). 2×300 sequencing allowed for complete coverage of the CDRα3 and CDRβ3 regions. High-throughput sequencing will validate that the TCR library maintains high diversity and will generate ≥10 TCRs displayed on mammalian T cells. 5 A natural TCRα:β. NGS will also be used to identify PDX neoantigens, and TCR molecular modeling will be performed to determine neoantigen targets for anti-PDX TCRs.

[0319] After identifying anti-PDX TCRs through screening, autologous patient T cells will be transduced with those anti-tumor TCRs, and their ability to kill PDX tumor cells in vitro will be tested. Human T cells will be transfected with the anti-tumor TCR α:β gene via lentiviral gene transduction. Figure 20 Patient T cells were transduced using the natural human TCR constant region gene, including cysteine ​​modifications to promote preferential expression of the transduced TCR. The transduced T cells were pre-activated with a T cell stimulant (e.g., anti-CD3 / anti-CD28 magnetic beads / IL-2) and cultured with IncuCyte-labeled tumor cells to assess TCR-based tumor cell killing in vitro. Autologous T cells transduced with a non-tumor cell-targeted TCRα:β gene were used as a negative control. ANOVA was used for statistical analysis of tumor cell killing compared to the negative control TCR gene.

[0320] Next, transgenic anti-PDX T cells will be delivered to PDX mice for antigen-specific tumor immunotherapy. Transduced T cells will be isolated by flow cytometry to obtain a purified transduced TCR library. Mouse pan-T cells will be transduced with anti-PDX TCRs and pre-stimulated using conditions defined in a cell killing assay; the stimulated T cells will be delivered to mice via intravenous tail vein injection. Mice will be tracked for 30 days, and treated mice will be compared with control groups and animals treated with checkpoint inhibitors. Delayed tumor growth will be quantified as the primary endpoint, and statistical significance will be validated using a Kaplan-Meier survival analysis. Animals treated with T cells containing anti-PDX TCRs are expected to match or exceed the treatment responses observed in animals treated with checkpoint immunotherapy. These results will confirm the applicability of this technology's TCR library for personalized antigen-specific therapy in human patients.

[0321] Example 12: Evaluation of antigen-specific TCRα:β in cancer treatment in a PDX mouse model Renal cell carcinoma (RCC) is sensitive to immunotherapy and is often clinically silent until the tumor is locally advanced or metastatic. Therefore, late-stage RCC is commonly diagnosed, and large tumors with extensive T-cell infiltration are surgically resected for laboratory studies. Advanced RCC is also an FDA-approved indication for checkpoint inhibition and recombinant IL-2 therapy, and RCC can be highly immunogenic with large T-cell infiltration that can be reactivated by immunotherapy. High-throughput screening of TIL TCRs responding to RCC will be used to identify neoantigen-specific TCRs that can be used for precision therapies.

[0322] First, tumor cells (TILs) and cancer cells were isolated from tumors using flow cytometry; the tumor cells were cryopreserved, while the TILs served as the input for TCR library isolation and cloning. Then, the TCR library was transformed into naive T cells, and the resulting display library was co-cultured with autologous patient tumor cells. After 24 hours of co-culture, activated T cells were sorted for CD69 / CD107 / membrane TNF-α upregulation using FACS, and the encoded TCRs were analyzed by NextGen sequencing to reveal tumor-targeting TCRs within the library. Finally, monoclonal paired TCRα:β cDNAs were recovered, transduced into human pan-T cells, and assayed via tumor cell co-culture to identify anti-RCC TCRs. These results will confirm that the TCR library of this technique can be used for personalized antigen-specific therapy in human patients.

[0323] Example 13: Application of paired TCRα:β gene therapy in the treatment of colon cancer, ovarian cancer, and B-cell cancer colon cancer A paired TCRα:β gene library targeting RKO cells (human colon cancer cell line) was cloned into mouse T cells. In short, pan-T cells were isolated from mouse spleens using magnetic beads, and TCR gene delivery was performed based on lentivirus or transposase.

[0324] according to Figure 20 The lentiviral strategy illustrated uses a human TCR constant region gene to prevent cross-dimerization with the native mouse TCR. T cells are then stained with antibodies to evaluate the surface expression of transgenic human α and β constant region proteins, and TCR expression is quantified by flow cytometry. Lentiviral gene transduction is used to transform mouse pan-T cells with anti-RKO TCR to evaluate RKO killing efficacy. Transduced T cells are isolated by flow cytometry for human TCR expression to obtain a purified transduced TCR library. Transformed cells can be co-cultured with RKO tumor cells under any of the following stimulation conditions: none, IL-2, anti-CD3 / anti-CD28 magnetic beads, or IL-2 / anti-CD3 / anti-CD28. In vitro RKO cell killing is assessed using the IncuCyte viability assay, which measures the loss of fluorescently labeled tumor cells due to cell killing.

[0325] For in vivo studies, stimulated T cells were delivered to mice via intravenous tail vein injection. Mice were tracked for 30 days to record weight loss and tumor volume. The treated mice were compared with untreated control mice (groups 1 and 4). Figure 21B ) and historical controls treated with currently approved antibody-based immunotherapies ( Figure 21A The tumor growth delay was compared with that observed in animals treated with T-cell therapy containing anti-RKO TCR. The delay in tumor growth was quantified as the primary endpoint. Animals treated with T-cell therapy containing anti-RKO TCR were expected to match or exceed the delay in tumor growth observed in animals treated with antibody-based immunotherapy.

[0326] Ovarian cancer. Human clinical PBMCs and TILs were collected from excised tumors and / or patient blood. A TCRα:β library was isolated from ovarian cancer patients. Clinical PBMCs and / or TILs were obtained after surgical resection and / or blood collection. TCRα:β cloning was performed using a green fluorescent protein (GFP) reporter system for retroviral transduction of TCRs into J.RT3 T cells. J.RT3 cells do not express endogenous TCRs and provide stabilizing proteins and co-expression factors (including CD3 and CD8) for TCR expression, making J.RT3 an ideal mammalian host for TCR display. Naturally paired α:β variable region genes were cloned into the pLVX lentiviral expression vector using a restriction site at the end of the amplicon. The vector library was amplified in *E. coli*, and the TCRα:β expression library, along with packaging and envelope vectors (psPax2 and pMD2.g), was co-transfected into HEK293T cells to generate lentiviral TCRα:β transduction particles. Figure 20 ).

[0327] B-cell cancer. B-cell lineage cancers, including lymphomas and multiple myeloma, express antibody genes as tumor-specific neoantigens targeted by TCRs. Paired TCRα:β gene libraries were isolated from B-cell lineage cancer patients. Paired TCRα:β clones were created using a green fluorescent protein (GFP) reporter system for retroviral transduction of TCR expression plasmids into J.RT3 T cells. J.RT3 cells do not express endogenous TCRs and provide stabilizing proteins and co-expression factors (including CD3 and CD8) required for stable TCR expression, making J.RT3 a suitable mammalian host for TCR library display. Naturally paired α:β variable region genes were cloned into pLVX lentiviral expression vectors (e.g., using a restriction site at the end of the ligation amplicons) using a restriction site at the end of the amplicons. Figure 20 (as shown in the image).

[0328] Use strains designed for stable lentiviral plasmid replication (e.g., Stbl3). 12The vector library was amplified in *E. coli*, and then the TCRα:β expression library, along with packaging and envelope vectors (psPax2 and pMD2.g), was co-transfected into HEK293T cells to generate lentiviral TCRα:β transduction particles. In parallel, tumor-specific BCRs were sequenced from clinical samples from PBMCs. J.RT3 T cells were transduced at a multiplicity of infection (MOI) of less than 0.2 (i.e., one TCR transgene per cell) to determine TCR binding to BCR neoantigens. Transduced cells were screened using overlapping peptide-MHC targeting neoantigens derived from tumor-encoding antibody genes sequenced from patients (CDR-H3, CDR-L3, and somatic mutation-derived neoantigens).

[0329] Illumina MiSeq 2×300 end-paired NGS system was used to analyze library diversity and characterize Vβ and Vα libraries at each step of the cloning process (i.e., cDNA input, post-lentiviral particle generation, and mCherry+J.RT3 / CD8 cells with surface-displayed TCRs). 2×300 sequencing allowed for complete coverage of both CDRα3 and CDRβ3 regions. High-throughput sequencing validated that the TCR libraries maintained high diversity throughout the cloning process and generated ≥10% of the libraries displayed on mammalian cells. 6 One natural TCRα:β. The affinity of TCRα:β discovered through peptide:MHC neoantigen screening was tested using surface plasmon resonance (SPR). These results will confirm that the TCR library of this technology can be used for personalized antigen-specific therapy in human patients.

[0330] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety by reference as if they were individually incorporated by reference. In case of any discrepancy, this specification (including definitions) shall prevail.

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Boswell, KL, Paris, R., Boritz, E., Ambrozak, D., Yamamoto, T., Darko, S., Wloka, K., Wheatley, A., Narpala, S., McDermott, A., Roederer, M., Haubrich, R., Connors, M., Ake, J., Douek, DC, Kim, J., Petrovas, C., and Koup, RA. Loss of circulating CD4 T cells with B cell helper function during chronic HIV infection. PLoS Pathog 10, e1003853 (2014) 30. Isolation of T-Cell Receptors Specifically Reactive with Mutated Tumor-Associated Antigens from Tumor-Infiltrating Lymphocytes Based on CD137 Expression. Maria Parkhurst, Alena Gros, Anna Pasetto, Todd Prickett, Jessica S. Crystal, Paul Robbins, and Steven A. Rosenberg 31. Activation-induced expression of CD137 allows for the detection, isolation, and expansion of the full repertoire of CD8+ T cells responding to antigen without requiring knowledge of epitope specificities. Matthias Wolfl,1 Jürgen Kuball,1 William Y. Ho,1 Hieu Nguyen,1 Thomas J. Manley,1 Marie Bleakley,1 and Philip D. Greenberg1,2 32. 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Ex vivoexpansion protocol for human tumor-specific T cells for adoptive T cell therapy. Journal of Immunological Methods Volume 355, Issues 1-2, April 15, 2010, pp. 52-60 37. Am J Transplant. October 2004; 4(10):1614-27 TGF-β induces Foxp3+ T-regulatory cells from CD4+CD25- precursors. Fu S 1 , Zhang N, Yopp AC, Chen D, Mao M, Chen D, Zhang H, Ding Y, Bromberg JS. 38. Science. February 14, 2003; 299(5609):1057-61. Published electronically on January 9, 2003. Control of regulatory T cell development by the transcription factor Foxp3. Hori S1, Nomura T, Sakaguchi S. 39. Wang, Y. et al. J Am Soc Nephrol. March 2006; 17(3):697-706. 40. Dons, E. et al. Hum Immunol April 2012; 73(4):328-34. 41. Nat Immunol . November 2014;15(11):1070-8. doi: 10.1038 / ni.3004. Electronic publication on September 28, 2014. Continuous requirement for the TCR in regulatory T cell function. Levine AG1, Arvey A1, Jin W1, Rudensky AY2. 42. Nat Immunol . November 2014;15(11):1070-8. doi: 10.1038 / ni.3004. Electronic publication on September 28, 2014. The persistent need for TCR in regulatory T cell function. Levine AG1, Arvey A1, Jin W1, Rudensky AY2. 43. Boyd, D., Florent, G., Kim, P., and Brattain, M. Determination of the Levels of Urokinase and Its Receptor in Human Colon Carcinoma Cell Lines. Cancer Res. 48, 3112-3116 (1988). 44. Lorz, A., Botesteanu, D.-A., and Levy, D. Universal response in the RKO colon cancer cell line to distinct antimitotic therapies. Sci. Rep. 8,8979 (2018). 45. June, CH, O'Connor, RS, Kawalekar, OU, Ghassemi, S. and Milone, MC for CAR T cell immunotherapy for human cancer. Science 359, 1361-1365 (2018). 46. ​​Kebriaei, P. et al. Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J. Clin. Invest. 126, 3363-3376 (2016). 47. Levine, BL, Miskin, J., Wonnacott, K., and Keir, C. Global Manufacturing of CAR T Cell Therapy. Mol. Ther. - Methods Clin. Dev. 4, 92-101 (2017). 48. Wang, X. and Rivière, I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol. Ther. - Oncolytics 3, 16015 (2016). 49. Single, A., Beetham, H., Telford, BJ, Guilford, P., and Chen, A. A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation. J. Biomol. Screen. 20, 1286-1293 (2015). 50. Kurtz, A. Mesenchymal stem cell delivery routes and fate. Int. J. Stem Cells 1, 1-7 (2008). 51. Antigen presentation profiling by Khodadoust, MS, et al. reveals the recognition of lymphoma immunoglobulin neoantigens. Nature 543, 723-727 (2017). 52. González, S., Volkova, N., Beer, P., and Gerstung, M. Immuno-oncology from the perspective of somatic evolution. Semin. Cancer Biol. (2017). doi:10.1016 / j.semcancer.2017.12.001 53.J.RT3-T3.5 ATCC® TIB-153 TM Human peripheral blood ac. Available at: https: / / www.atcc.org / Products / All / TIB-153.aspx. (Accessed: April 3, 2017) 54. Remarkable stability of an instability-prone lentiviral vector plasmidin Escherichia coli Stbl3 by Al-Allaf, FA, Tolmachov, OE, Zambetti, LP, Tchetchelnitski, V. and Mehmet, H. 3 Biotech 3, 61-70 (2013). 55. Descours, B. et al. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T cells. Retrovirology 9, 87 (2012). 56. High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire. (DeKosky, BJ, et al.) Nat Biotech 31, 166-169 (2013). 57. DeKosky, BJ et al.: In-depth identification and analysis of paired heavy and light chain antibody libraries. Nat. Med. 21, 86-91 (2015). 58. McDaniel, JR, DeKosky, BJ, Tanno, H., Ellington, AD, and Georgiou, G. Ultra-high-throughput sequencing of an immune receptor repertoire of millions of lymphocytes. Nat. Protoc. 11,429-42 (2016). 59. Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires. DeKosky, BJ et al. Proc. Natl. Acad. 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Cohen, CJ, et al. engineered T cells to express T-cell receptors with a second disulfide bond, resulting in enhanced antitumor activity of T cells. Cancer Res. 67, 3898-3903 (2007). 66. Dudley, 1, WN, Wickham, 2, R., and COOMBS, 3, N. An Introduction to Survival Statistics: Kaplan-Meier Analysis. J. Adv. Pract. Oncol. 7, 91-100 (2016). 67. Birnbaum, ME et al. Deconstructing the Peptide-MHC Specificity of T Cell Recognition. Cell 157, 1073-1087 (2014). 68. Glanville, J. et al. identified specificity groups in the T cell receptor repertoire. Nature advance onlinepublication, (2017). 69. Han, A., Glanville, J., Hansmann, L., and Davis, MM, linked T-cell receptor sequence to functional phenotype at the single-cell level. Nat. Biotechnol. 32, 684-692 (2014). 70. Atkins, M. Clinical manifestations, evaluation, and staging of renal cell carcinoma. UpToDate 18, (2017). 71. Betts, MR, et al. used flow cytometry for degranulation to sensitively and viably identify antigen-specific CD8+ T cells. J. Immunol. Methods 281, 65-78 (2003). 72. Relationship between CD107a expression and cytotoxic activity in Aktas, E., Kucuksezer, UC, Bilgic, S., Erten, G. and Deniz, G. Cell. Immunol. 254, 149-154 (2009). 73. Haney, D. et al. Isolation of live antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression. J. Immunol. Methods 369, 33-41 (2011). 74. Royal, KT, et al. used combined antigen-sensing circuits to perform precise tumor recognition via T cells. Cell 164, 770-779 (2016). Equivalent scheme This technology is not limited in its specific embodiments described herein, which are intended as a single illustration of a single aspect of the technology. Many modifications and variations of the technology may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. In addition to those listed herein, functionally equivalent methods and apparatus within the scope of this technology will also be apparent to those skilled in the art based on the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This technology is defined only by the terminology of the appended claims, together with the full scope of equivalents conferred by such claims. It should be understood that the technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0332] Furthermore, when describing features or aspects of this disclosure in terms of the Markush group, those skilled in the art will recognize that this disclosure is also being described in terms of any single member or subgroup of the Markush group.

[0333] As those skilled in the art will understand, for any and all purposes, particularly in providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope may be readily considered sufficiently descriptive and such that the same scope can be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein may be readily divided into a lower third, a middle third, and an upper third, etc. Those skilled in the art will also understand that all terms such as “up to,” “at least,” “greater than,” “less than,” etc., include the cited numbers and refer to a scope that can subsequently be divided into subscopes as described above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 units means a group having 1, 2, or 3 units. Similarly, a group having 1-5 units means a group having 1, 2, 3, 4, or 5 units, and so on.

[0334] All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated in their entirety by reference, including all figures and tables, to the extent that they are not inconsistent with the explicit teachings of this specification.

[0335] This technology may include, but is not limited to, the features and combinations thereof described in the following lettered paragraphs. It should be understood that the following paragraphs should not be construed as limiting the scope of the appended claims or requiring that all such features be included in such claims: A. A recombinant T-cell receptor (TCR) library vector comprising: (a) Carrier skeleton; and (b)(i) the first polynucleotide encoding the TCRα polypeptide and the second polynucleotide encoding the TCRβ polypeptide; or (ii) The first polynucleotide encoding the TCRγ polypeptide and the second polynucleotide encoding the TCRδ polypeptide; The first polynucleotide and the second polynucleotide are a pair, and the first polynucleotide and the second polynucleotide are derived from mRNA isolated from a single lysed T cell present in a compartment.

[0336] B. The vector described in paragraph A, wherein the mRNA of the single lysed T cell is isolated using an mRNA capture reagent or reverse transcription-PCR (RT-PCR).

[0337] C. The vector according to paragraph A or paragraph B, wherein the first polynucleotide and the second polynucleotide are operatively linked, optionally operatively linked via a linker polynucleotide, and optionally wherein the first polynucleotide and the second polynucleotide are operatively linked via reverse transcription and PCR amplification of the T cell mRNA.

[0338] D. According to any of the preceding paragraphs, the first polynucleotide and the second polynucleotide are cloned into the vector backbone by cleavage at a target restriction endonuclease site naturally found in the TCR gene.

[0339] E. The vector according to paragraph D, wherein the target restriction endonuclease site appears at a low frequency in the TCR gene.

[0340] F. The vector according to paragraph D or paragraph E, wherein the first polynucleotide and the second polynucleotide have been modified to incorporate at least one target restriction endonuclease site disclosed in Table 7 or 8.

[0341] G. The vector according to any paragraph DF, wherein the target restriction endonuclease site contains a silencing mutation.

[0342] H. The vector according to any paragraph of paragraph DG, wherein the mRNA capture agent is selected from the group consisting of: poly(dT) coated beads, oligonucleotide coated beads, hydrogel beads, and printed oligomers on the surface of microarray pores.

[0343] I. The carrier according to any of the preceding paragraphs, wherein the compartment is an emulsion droplet or a pore.

[0344] J. The carrier according to paragraph I, wherein the pores are located in a printed polymer slide, plastic plate, microtiter plate or gel.

[0345] K. The carrier according to any of the preceding paragraphs, wherein the compartment has a volume of 5 nL or less.

[0346] L. The vector according to any of the preceding paragraphs further comprises at least one polynucleotide encoding an expression control element, the polynucleotide encoding the expression control element being operatively linked to the first polynucleotide and / or the second polynucleotide.

[0347] M. According to the vector described in paragraph L, the expression control element is selected from the group consisting of: promoter, p2A sequence and IRES sequence.

[0348] N. The vector according to paragraph M, wherein the promoter is an EF1α promoter or a CMV promoter.

[0349] O. The vector according to any paragraph LN, wherein the polynucleotide encoding the expression control element is located between the first polynucleotide and the second polynucleotide.

[0350] P. The carrier described in any of the preceding paragraphs, wherein the carrier is circularized.

[0351] Q. The carrier described in any paragraph LP, wherein the carrier has been circularized before the expression control element is incorporated into the carrier.

[0352] R. The carrier described in any paragraph LP, wherein the carrier has been circularized after the expression control element has been incorporated into the carrier.

[0353] S. The vector according to any paragraph in LR, wherein the expression control element has been incorporated into the vicinity of the prototype spacer adjacent motif (PAM).

[0354] T. The vector described in any of paragraphs LR, wherein the expression control element has been incorporated into the vector using a DNA-modifying enzyme selected from restriction enzymes or TALENs.

[0355] U. The vector according to any of the preceding paragraphs further comprises one or more polynucleotides encoding transposons, said polynucleotide being linked to at least one of the first polynucleotide and the second polynucleotide.

[0356] V. The vector according to any of the preceding paragraphs further comprises a polynucleotide encoding a detectable marker.

[0357] W. The vector according to any of the preceding paragraphs further comprises a polynucleotide encoding a selection marker.

[0358] X. The vector according to any of the preceding paragraphs further comprises a polynucleotide encoding a switching mechanism for controlling the expression and / or activation of the first polynucleotide and the second polynucleotide.

[0359] Y. The vector described in any of the preceding paragraphs further comprises a polynucleotide encoding a Kozak common sequence or enhancer.

[0360] Z. The vector according to any of the preceding paragraphs, wherein the vector backbone is selected from the group consisting of: retroviral vector backbone, lentiviral vector backbone, adenovirus vector backbone and adeno-associated virus vector backbone.

[0361] AA. A vector according to any of the preceding paragraphs, wherein the vector encodes a TCR that has binding specificity to a target cell or disease antigen.

[0362] BB. The vector according to paragraph AA, wherein the target cell is a cancer cell or a virus-infected cell, optionally wherein the target cell is isolated from the subject.

[0363] CC. According to the vector described in paragraph AA, wherein the disease antigen is a viral antigen or a tumor antigen, optionally wherein the antigen is loaded into an antigen:MHC complex.

[0364] DD. According to the vector described in paragraph CC, the disease antigen is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus.

[0365] EE. According to the vector described in paragraph CC, the disease antigen is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, lensin, meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tumor necrosis factor receptor superfamily member 10a (TRAILR1), receptor activator of nuclear factor κ-B ligand (RANKL), insulin-like growth factor 1 receptor (IGF1R), epithelial cell adhesion molecule (EpCAM), and carcinoembryonic antigen (CEA).

[0366] FF. A recombinant cell comprising the vector described in any of the preceding paragraphs, optionally wherein the recombinant cell is a bacterial cell, a mammalian cell, or a yeast cell.

[0367] GG. A recombinant TCR vector library comprising multiple vectors as described in any one of paragraphs A-EE.

[0368] HH. The recombinant TCR vector library described in paragraph GG, wherein the plurality of vectors contain a TCR library.

[0369] II. The recombinant TCR vector library according to paragraph GG or paragraph HH, wherein each of the plurality of vectors has been selected based on one or more of the following characteristics: TCR clonal universality, TCR enrichment characteristics determined in vitro, TCR binding specificity, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motif and / or CDR3 gene motif.

[0370] JJ. The recombinant TCR vector library described in any paragraph GG-II, wherein the TCR vector library has been characterized by nucleic acid sequencing of the first polynucleotide and the second polynucleotide.

[0371] KK. An isolated immune cell comprising a vector as described in any of paragraphs A-EE.

[0372] LL. Isolated immune cells as described in paragraph KK, wherein the immune cells are hematopoietic stem cells, hematopoietic progenitor cells, T cells, or natural killer (NK) cells.

[0373] MM. A cell population comprising a vector library as described in any of paragraphs A-EE or any of paragraphs GG-JJ.

[0374] NN. The cell population described in paragraph MM, wherein the population comprises hematopoietic stem cells, hematopoietic progenitor cells, T cells and / or NK cells.

[0375] OO. A method for preparing a recombinant TCR library, the method comprising transforming a cell population with a vector library according to any one of paragraphs GG-JJ.

[0376] PP. According to the method described in paragraph OO, the cell is a hematopoietic stem cell, hematopoietic progenitor cell, T cell, or NK cell.

[0377] QQ. The method described in paragraph OO or paragraph PP further includes screening the library for specific binding to target cells.

[0378] RR. The method described in paragraph QQ, wherein the target cell is a cancer cell or a virus-infected cell, optionally wherein the cell is isolated from the subject.

[0379] SS. The method described in paragraph OO or paragraph PP further includes screening the library for specific binding to antigen:MHC.

[0380] TT. According to the method described in paragraph SS, the antigen of the MHC complex is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71. (EV71), Ebola virus, Hepatitis A (HAV), Hepatitis B (HBV), Cytomegalovirus (CMV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Human T-lymphotropic Virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis Virus, Leukemia Virus, Measles Virus, Molluscum Contagiosum Virus, Sheep Pox Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Tick-borne Encephalitis Virus (TBEV), Simian Immunodeficiency Virus, Tobacco Etching Virus (TEV), Varicella-zoster Virus, Smallpox Virus, West Nile Virus, Zika Virus, and Chikungunya Virus.

[0381] UU. According to the method described in paragraph SS, the antigen of the MHC complex is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, Lengsin, Meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, EGFR, HER2, TRAILR1, RANKL, IGF1R, EpCAM, and CEA.

[0382] VV. The method described in any of paragraphs OO-UU further includes screening the library for T-cell phenotypic markers.

[0383] WW. The method according to any one of paragraphs OO-VV further includes screening the library for activity in a co-culture system, wherein the co-culture system comprises at least one of the following: (a) Cancer cell line; (b) Multiple cells infected with a known virus; (c) Multiple tumor cells isolated from a cancer patient; (d) Immortalized cell lines; or (e) Multiple cells derived from patient tissue biopsy samples.

[0384] XX. The method described in any of paragraphs OO-WW further includes in vitro activation of the transformed cell population.

[0385] YY. According to the method described in paragraph XX, in vitro activation is performed using one or more of the following stimulants: anti-CD3 antibody, anti-CD8 antibody, anti-CD27 antibody, IL-2, IL-4, IL-21, anti-PD1 antibody, anti-CTLA4 antibody, tumor cell lysate, cell co-culture with virus-infected cells, and tumor cell lines.

[0386] ZZ. The method described in any of paragraphs OO-YY further includes transforming the cell population with a polynucleotide encoding a transcription factor.

[0387] AAA. According to the method described in paragraph ZZ, the transcription factors are selected from the group consisting of: FOXP3, BLIMP-1, Ikaros, Helios, and TGF-β.

[0388] BBB. The method described in any of paragraphs OO-AAA further includes selecting a single vector for inclusion in the recombinant TCR library based on one or more of the following characteristics: TCR clonal universality, TCR enrichment characteristics determined in vitro, TCR binding specificity, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motif, and / or CDR3 gene motif.

[0389] CCC. According to the method described in paragraph BBB, the selection includes mixing individual vectors at defined ratios to generate a synthetically derived TCR library.

[0390] DDD. A recombinant TCR library prepared by means of the method described in any of the paragraphs OO-CCC.

[0391] EEE. A composition comprising a recombinant TCR library and a vector as described in paragraph DDD.

[0392] FFF. The composition according to paragraph EEE, wherein the carrier is a pharmaceutically acceptable carrier.

[0393] GGG. A method of treating a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library as described in paragraph DDD or a composition as described in paragraph EEE or paragraph FFF.

[0394] HHH. A method of treating cancer in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library as described in paragraph DDD or a composition as described in paragraph EEE or paragraph FFF.

[0395] III. According to the method described in paragraph HHH, the cancers mentioned are: Acute lymphoblastic leukemia (ALL); Acute myeloid leukemia (AML); Adrenocortical carcinoma; AIDS-related cancers; Anal cancer; Appendicitis cancer; Astrocytoma; Atypical teratoid / rod-shaped tumors; Brain cancer; Basal cell carcinoma of the skin; Bile duct cancer; Bladder cancer; Bone cancer; Breast cancer; Bronchoma; Burkitt lymphoma; Carcinoid tumor (gastrointestinal); Germ cell tumor; Primary CNS lymphoma; Cervical cancer; Bile duct cancer; Chordoma; Chronic lymphocytic leukemia (CLL); Chronic myeloid leukemia (CML); Chronic osteomyelitis. Myeloproliferative neoplasms; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; ductal carcinoma in situ (DCIS); endometrial cancer; ependymoma; esophageal cancer; neuroblastoma; extracranial germ cell tumor; gonadal germ cell tumor; ocular cancer; intraocular melanoma; retinoblastoma; fallopian tube cancer; malignant fibrous histiocytoma and osteosarcoma of bone; gallbladder cancer; gastric cancer; gastrointestinal carcinoid tumors; gastrointestinal stromal tumors (GIST); germ cell tumors; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; cardiac tumors; hepatocellular carcinoma; Langerhans cell histiocytosis; Hodgkin's lymphoma; hypopharyngeal cancer; intraocular cancer. Melanoma; Islet cell tumor, pancreatic neuroendocrine tumor; Kidney cancer; Laryngeal cancer; Leukemia; Lip and oral cavity cancer; Liver cancer; Lung cancer; Lymphoma; Male breast cancer; Malignant fibrous histiocytoma and osteosarcoma of bone; Melanoma; Merkel cell carcinoma; Mesothelioma; Metastatic cancer; Oral cancer; Multiple endocrine neoplasia syndrome; Multiple myeloma / plasma cell tumor; Mycosis fungoides; Myelodysplastic syndrome, chronic myeloproliferative neoplasm; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral cavity cancer, oropharyngeal cancer; Osteosarcoma; Ovarian cancer; Pancreatic cancer; Pancreatic neuroendocrine tumor; Nipples Pharyngeal carcinoma; paraganglioma; sinus carcinoma; parathyroid carcinoma; pharyngeal carcinoma; pheochromocytoma; pituitary adenoma; pleural pulmonary blastoma; prostate cancer; rectal cancer; recurrent cancer; renal cell carcinoma; retinoblastoma; rhabdomyosarcoma; salivary gland carcinoma; sarcoma; Ewing sarcoma; Kaposi's sarcoma; osteosarcoma; uterine sarcoma; Cezary syndrome; skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; squamous neck carcinoma; gastric cancer; T-cell lymphoma; testicular cancer; laryngeal cancer; nasopharyngeal carcinoma; hypopharyngeal carcinoma; thymic carcinoma; thyroid cancer; urethral cancer; uterine cancer; vaginal cancer; hemangioma; vulvar cancer; or Wilms' tumor.

[0396] JJJ. A method for inhibiting tumor growth in a subject in need, the method comprising administering to the subject an effective amount of a recombinant TCR library as described in paragraph DDD or a composition as described in paragraph EEE or paragraph FFF.

[0397] KKK. According to the method described in paragraph JJJ, the tumor is a solid tumor.

[0398] LLL. A method of treating a subject with a viral infection, the method comprising administering to the subject an effective amount of a recombinant TCR library as described in paragraph DDD or a composition as described in paragraph EEE or paragraph FFF.

[0399] According to the method described in paragraph LLL, the viral infection is caused by a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71 (EV71), Ebola virus, hepatitis A (HAV), hepatitis B (HBV), cytomegalovirus (CMV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), human immunodeficiency virus (HIV), and human papillomavirus (HPV). The following viruses are included: HPV, herpes simplex virus (HSV), human T-lymphotropic virus (HTLV), influenza A virus, influenza B virus, Japanese encephalitis virus, leukemia virus, measles virus, molluscum contagiosum virus, sheep pox virus, parvovirus, rabies virus, respiratory syncytial virus, Rift Valley fever virus, rubella virus, rotavirus, tick-borne encephalitis virus (TBEV), simian immunodeficiency virus, tobacco etch virus (TEV), varicella-zoster virus, smallpox virus, West Nile virus, Zika virus, and chikungunya virus.

[0400] NNN. The method according to any of paragraphs GGG-MMM further includes administering a second dose of the recombinant TCR library according to paragraph DDD or the composition according to paragraph EEE or paragraph FFF to the subject.

[0401] OOO. The method described in any of the paragraphs GGG-NNN, wherein the recombinant TCR library contains cells that are autologous or allogeneic to the treated subject.

[0402] PPP. The method described in any of paragraphs GGG-OOO, wherein the subject is a human, animal, non-human primate, dog, cat, sheep, mouse, horse, or cow.

[0403] QQQ. According to the method described in paragraph PPP, the subject is a human being.

[0404] Other embodiments are set forth in the appended claims.

Claims

1. A method for preparing a recombinant TCR library, the method comprising: (a) Lysing single T cells in compartments with a volume of 5 nL or less. (b) Using an mRNA capture reagent, the mRNA of the lysed individual T cells in the compartment is isolated; (c) The mRNA is reverse transcribed into cDNA in the compartment by overlap extension reverse transcription PCR (OE-RT-PCR) to generate a ligated cDNA product, wherein the ligated cDNA product comprises (i) The first polynucleotide encoding the T cell receptor (TCR) α polypeptide and the second polynucleotide encoding the TCR β polypeptide; or (ii) The first polynucleotide encoding the TCRγ polypeptide and the second polynucleotide encoding the TCRδ polypeptide. The first and second polynucleotides are associated pairs and are operatively linked via linker polynucleotides; (d) Amplify the ligated cDNA product using nested PCR to generate TCR amplicon; (e) A recombinant TCR library is generated by introducing the TCR amplicon into an expression vector or cell population.

2. The method of claim 1, further comprising screening the library for specific binding to target cells, optionally wherein... The target cells are cancer cells or cells infected by a virus, or The cells were isolated from the subject.

3. The method of claim 1, further comprising screening the library for specific binding to the antigen:MHC complex, optionally wherein... The antigen of the MHC complex is a viral antigen derived from a virus selected from the group consisting of: adenovirus, CMV, coronavirus, Coxsackievirus, dengue virus, Epstein-Barr virus (EBV), enterovirus 71 (EV71), Ebola virus, hepatitis A (HAV), hepatitis B (HBV), cytomegalovirus (CMV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), human immunodeficiency virus (HIV), and human papillomavirus (HPV). Viruses (HPV), herpes simplex virus (HSV), human T-lymphotropic virus (HTLV), influenza A virus, influenza B virus, Japanese encephalitis virus, leukemia virus, measles virus, molluscum contagiosum virus, sheep pox virus, parvovirus, rabies virus, respiratory syncytial virus, Rift Valley fever virus, rubella virus, rotavirus, tick-borne encephalitis virus (TBEV), simian immunodeficiency virus, tobacco etch virus (TEV), varicella-zoster virus, smallpox virus, West Nile virus, Zika virus, and chikungunya virus, or The antigen of the MHC complex is a tumor antigen selected from the group consisting of: CD45, phosphatidylinositol proteoglycan-3, IGF2B3, kallikrein 4, KIF20A, Lengsin, Meloe, mucin 5AC (MUC5AC), susceptin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b / GAGED2A, CD19, CD20, CD22, CD52, EGFR, HER2, TRAILR1, RANKL, IGF1R, EpCAM, and CEA.

4. The method according to claim 1, further comprising: The library was screened for T-cell phenotypic markers; or The library is screened for activity in the co-culture system. The co-culture system comprises at least one of the following: (a) Cancer cell line; (b) Multiple cells infected by a known virus; (c) Multiple tumor cells isolated from a cancer patient; (d) Immortalized cell lines; or (e) Multiple cells derived from patient tissue biopsy; or The transformed cell population is activated in vitro, optionally using one or more of the following stimulants: anti-CD3 antibody, anti-CD8 antibody, anti-CD27 antibody, IL-2, IL-4, IL-21, anti-PD1 antibody, anti-CTLA4 antibody, tumor cell lysate, cell co-culture with virus-infected cells, and tumor cell lines; or The cell population was transformed with a polynucleotide encoding a transcription factor, optionally wherein the transcription factor was selected from the group consisting of: FOXP3, BLIMP-1, Ikaros, Helios, and TGF-β; or Individual vectors for inclusion in the recombinant TCR library are selected based on one or more of the following characteristics: TCR clonal universality, TCR enrichment characteristics determined in vitro, TCR binding specificity, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motif and / or CDR3 gene motif, optionally including the mixing of individual vectors at defined ratios to generate a synthetically derived TCR library.

5. The method of claim 1, wherein the first polynucleotide and the second polynucleotide have been altered to include at least one target restriction endonuclease site selected from the group consisting of: Age I, SpeI, XhoI, NheI, BsiWI, BstZI7I, BstBI, and SphI, or wherein the target restriction endonuclease site comprises a silencing mutation; or Step (e) is performed by cleavage at the target restriction endonuclease site, optionally wherein the target restriction endonuclease site is naturally found in the TCR gene at a low frequency; or The cell population mentioned therein is hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells.

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