Universal chimeric antigen receptor T cell as well as preparation method and application thereof

Abstract

The invention discloses a universal chimeric antigen receptor T cell (CAR-T cell) as well as a preparation method and application thereof, and relates to the technical field of medicine and immunology. According to the preparation method of the universal CAR-T cells, disclosed by the invention, the preparation time of the CAR-T cells can be effectively shortened, the cost is reduced, and the CAR-T cells with small batch difference and complete functions are provided. The FAP-targeted CAR-T cell and the preparation method thereof disclosed by the invention can be used for clinically treating tumors, cardiovascular diseases and organ fibrosis, so that the survival time of a patient is prolonged, and the survival rate is increased. The invention further discloses a cell medicine / medicine composition. The universal CAR-T cell disclosed by the invention provides a new direction and idea for treatment of tumors, cardiovascular diseases, organ fibrosis and the like.

Description

Technical Field

[0001] This invention relates to the fields of medicine and immunology, and mainly to universal chimeric antigen receptor T cells (CAR-T cells), their preparation methods, and applications. Background Technology

[0002] Host-versus-graft disease (HvGD) occurs when the host's immune cells recognize and kill infused CAR-T cells as "non-self" cells, resulting in a shortened survival time and inability to function of the infused CAR-T cells. This is primarily because the human leukocyte antigen (HLA) on the infused CAR-T cells differs from that of the host, leading to recognition and destruction.

[0003] During the infusion of allogeneic CAR-T cells, graft-versus-host disease (GvHD) is a condition caused by the infused T cells using proteins in the host body as antigens, thereby non-specifically killing normal cells, resulting in tissue damage, organ failure, and other symptoms.

[0004] β2-microglobulin (β2M) plays a crucial role in the proper folding and transport of MHC I (major histocompatibility complex I) to the cell membrane surface. Knocking out this gene results in the absence of MHC I molecules on the cell membrane, thereby effectively reducing the risk of host recognition of MHC I and the resulting HvGD side effect.

[0005] Autologous CAR-T cells are derived from the patient's blood. CAR-T cells prepared from different donors exhibit varying expansion and cytotoxic abilities, potentially leading to difficulties in expansion and culture, gene editing, and low cytotoxicity. T cells from certain patients may not meet the criteria for CAR-T cell preparation, such as those undergoing radiotherapy or chemotherapy, or those with hematological malignancies. The cost of autologous CAR-T cells is high; currently, a single course of CAR-T cell therapy in China costs 1.2 million yuan, limiting its widespread adoption. The preparation cycle for CAR-T cells is also lengthy, requiring 2-3 weeks from blood sample collection, potentially delaying effective treatment. Furthermore, the preparation process is complex, and according to literature, there is a 2-10% risk of failure.

[0006] Therapies that involve extracting immune cells from the body, modifying and expanding them, and then reinfusing them into the patient are called adoptive cell transfer therapy (ACT), with CAR-T cell therapy being the most prominent in this field. As of September 2023, nine CAR-T therapies had been approved for marketing globally, with over 200 entering clinical trials, and this therapy is gradually expanding beyond cancer treatment.

[0007] Existing autologous CAR-T cell therapy has achieved positive results in tumor immunotherapy, especially in the treatment of hematologic malignancies. However, it still has problems such as long preparation cycle, unstable performance, and high cost. Especially for some diseases with short treatment windows, there is a greater need to develop effective technical means to improve cell stability and shorten the reinfusion cycle.

[0008] Cardiovascular disease accounts for approximately 31% of global deaths. Cardiac fibrosis is a common pathological phenomenon occurring during cardiac damage and aging, characterized by excessive accumulation of collagen fibers in myocardial tissue, significantly elevated collagen concentration, myocardial hardening, and decreased pumping capacity, seriously threatening the lives and health of cardiovascular disease patients. Cardiac fibrosis has complex underlying causes, such as tissue damage and inflammation, and occurs in almost all types of heart disease, including myocardial infarction, aortic coarctation, dilated cardiomyopathy, and hypertrophic cardiomyopathy. Developing novel treatments for myocardial fibrosis is of great significance for improving the survival rate and prolonging the survival time of cardiovascular disease patients. Cardiac fibroblasts play a central role in maintaining normal cardiac tissue structure and physiological function and are closely related to the occurrence and development of myocardial fibrosis. Fibroblasts in the heart are responsible for depositing collagen and other components in the extracellular matrix (ECM) and constructing the extracellular fiber network of connective tissue. Excessive and sustained activation of fibroblasts under pathological stimulation leads to excessive collagen deposition in the myocardium, resulting in myocardial fibrosis. Activated myocardial fibroblasts upregulate the expression of various proteins, such as fibroblast actiation protein-α (FAP), smooth muscle actin alpha (α-SMA), and vimentin. FAP is a membrane surface marker of activated myofibroblasts and can serve as a diagnostic marker for fibrosis and a target for immunotherapy.

[0009] In clinical practice, anti-inflammatory drugs and immunomodulators are traditionally used to alleviate symptoms of myocardial fibrosis. Although fibrosis is the final manifestation of many chronic diseases and seriously threatens patients' lives, currently only a few anti-fibrotic drugs, such as pirfenidone, have passed clinical drug approval. Therefore, actively developing and preparing universal CAR-T cells holds promise for application in the treatment of myocardial fibrosis, reducing the degree of fibrosis and providing an effective treatment method for clinical use. Summary of the Invention

[0010] In order to overcome the above-mentioned problems in the existing technology, the purpose of this invention is to provide a universal CAR-T cell and its preparation method, and it is expected that the method will further broaden the application scope of CAR-T and provide an effective treatment.

[0011] This invention provides a universal CAR-T cell that expresses a chimeric antigen receptor and knocks out the following combination of immune rejection-related genes such as TRAC, TRBC1, CD3E, and β2M in the T cells:

[0012] Combination 1: Simultaneous knockout of TRAC, TRBC1, and β2M.

[0013] Combination 2: Simultaneous knockout of CD3E and β2M.

[0014] The CAR includes: an anti-FAP single-chain antibody segment (MFP5 ScFv), a CD8 hinge region, a CD8 transmembrane region, 4-1BB / CD28, and CD3ζ (see details). Figure 1 In some structures, HLA-A and HLA-E molecules can be optionally expressed to avoid being killed by immune cells in the host body. Compared with autologous CAR-T cells prepared by existing technologies, a key feature is the knockout of immune rejection-related genes.

[0015] The nucleotide sequence of TRAC is shown in SEQ ID NO:1, the nucleotide sequence of TRBC1 is shown in SEQ ID NO:2, the nucleotide sequence of CD3E is shown in SEQ ID NO:3, and the nucleotide sequence of β2M is shown in SEQ ID NO:4.

[0016] Furthermore, the universal CAR-T cells include CAR-T cells that additionally express TGFR-wtFc, CAR-T cells that additionally express OX40, or CAR-T cells that additionally express HLA-A or HLA-E.

[0017] The present invention also provides a method for preparing universal CAR-T cells, the method comprising the following steps: obtaining T cells from peripheral blood, activating and culturing T cells in vitro, knocking out immune rejection-related genes by nuclear electroporation, transfecting CAR into T cells with lentivirus to obtain CAR-T cells, and activating CAR-T cells in vitro using cytokines and / or antibodies.

[0018] In this invention, the nuclear electroporation method transfers CRISPR-Cas9 protein and sgRNA of immune rejection-related genes into T cells;

[0019] The sgRNA targets the TRAC, TRBC1, CD3E, and β2M genes in the T cells, and the nucleotide sequences of these genes are shown in SEQ ID NO:1-SEQ ID NO:4; and / or,

[0020] The nucleotide sequences of the sgRNA are shown in SEQ ID NO:5-SEQ ID NO:8.

[0021] The method described in this invention uses anti-CD3 and anti-CD28 to activate and culture T cells. Specifically, a 96-well plate is pre-coated with 2 μg / ml anti-mouse IgG Fcγ (catalog number 115-005-071), and the isolated PBMCs (peripheral blood mononuclear cells) are cultured at a density of 2 × 10⁻⁶ cells / well. 6 The cells were seeded at a density of 1 cell / ml in 96-well plates and supplemented with 0.5 μg / ml anti-human CD3 (clone number: OKT3) and 1 μg / ml anti-human CD28 (clone number: CD28.2);

[0022] This invention utilizes nuclear transfection to transfect CRISPR-Cas9 protein and corresponding sgRNAs for knockout-related genes into T cells, thereby knocking out one or more of TRAC, TRBC1, CD3E, and β2M proteins, effectively reducing the side effects of GvHD and HvGD. 48-72 hours after cell activation, the preferred sgRNA (5' to 3') is co-transfected with CRISPR / Cas9 protein into the cell nucleus to knock out the target gene.

[0023] Furthermore, the knockout combinations are as follows:

[0024] Combination 1: Simultaneous knockout of TRAC, TRBC1, and β2M, i.e., simultaneous knockout of TRAC, TRBC1, and β2M using SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8.

[0025] Combination 2: Simultaneous knockout of CD3E and β2M; that is, simultaneous knockout of CD3E and β2M using SEQ ID NO:7 and SEQ ID NO:8.

[0026] In this invention, for in vitro activation and expansion, aAPCs (artificial antigen-presenting cells) expressing FAP (fibroblast activating protein) or methods based on anti-CD3, anti-CD28, and IL-2 are used.

[0027] In the aAPC method, Raji-hFAP cells overexpressing the FAP target are pre-cultured, irradiated with 100 Gy, and then aliquoted and cryopreserved as tool cells to stimulate the expansion of FAP-CAR-T cells. During CAR-T cell culture, each generation lasts five days. On the fifth day of each generation, aAPC is added at a ratio of 1 / 4 of the CAR-T cells, increasing the generation by one generation.

[0028] In this invention, the quality control of CAR-T cells includes: daily counting and detection of amplification during culture; flow cytometry analysis on the fourth day of each generation to detect the expression of TRAC, TRBC1, β2M, CD3E molecules, and CAR molecules; and co-culturing CAR-T cells from the fourth day of each generation with CFPAC1-hFAP cells or CFPAC1 cells (human pancreatic cancer cells) to detect the in vitro killing ability of CAR-T cells against target cells.

[0029] This invention constructs a chimeric antigen receptor targeting the FAP protein and applies it to the treatment of myocardial fibrosis. The fusion protein includes a CAR structure (for target recognition and mediating a killing response), and also includes proteins that simultaneously express CAR and TGFβ-wtFc (for blocking the TGFβ signaling pathway, reducing TGFβ-stimulated myofibroblast activation, and inhibiting T cell function). First, a DNA fragment is constructed and ligated into a plasmid vector. The sequence is verified by DNA sequencing. Then, plasmid extraction, lentiviral packaging, concentration, and titer determination are performed. CD8+ T cells are isolated and screened from PBMCs and activated in vitro. The activated T cells are then infected with lentivirus, and subsequent culture and periodic stimulation lead to large-scale cell proliferation.

[0030] The CAR-T cells produced by the method described in this invention have had genes such as TRAC, TRBC1, CD3E, and β2M knocked out that may lead to immune rejection, and are targeted at FAP. The CAR-T cells produced by this invention include a CAR-T cell that additionally expresses TGFR-wtFc, a CAR-T cell that additionally expresses OX40, and a CAR-T cell that additionally expresses HLA-A or HLA-E.

[0031] The present invention also provides an expression vector, characterized in that the expression vector is used to prepare universal chimeric antigen receptor T cells as described above.

[0032] Preferably, the expression vector is a recombinant lentiviral vector.

[0033] The present invention also provides a composition comprising any one or more of the universal chimeric antigen receptor T cells as described above, or the expression vectors as described above.

[0034] The present invention also provides a cell drug / pharmaceutical composition containing, as described above, universal CAR-T cells as an active ingredient, and / or a pharmaceutically acceptable carrier.

[0035] In an optional embodiment, the above-described cell drug / drug composition is used for the prevention and / or treatment of tumors.

[0036] In an optional implementation, the tumor is selected from solid tumors or non-solid tumors.

[0037] In an optional implementation, the tumor is selected from hematomas.

[0038] In an optional embodiment, the above-described cell drug / pharmaceutical composition is used for the prevention and / or treatment of cardiovascular diseases.

[0039] In an optional implementation, the cardiovascular disease mentioned above is selected from myocardial fibrosis.

[0040] In optional embodiments, the above-described cell drug / drug composition is used to prevent and / or treat organ fibrosis conditions such as pulmonary fibrosis and liver fibrosis.

[0041] In this invention, the cell drug / drug composition is used alone and / or in combination with cancer treatment drugs, such as small molecule targeted drugs, PD-1 / PD-L1 antibodies, CTLA-4 antibodies, etc.; and drugs that improve ventricular remodeling, such as beta-receptor antagonists, ACEI receptor antagonists, GLP-1 receptor agonists, SGLT2 inhibitors, aldosterone inhibitors, etc.

[0042] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0043] Preferably, the cell drug / drug composition may further contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0044] Preferably, the cell drug / drug composition comprises 0.01 to 99.99% CAR-T cells and 0.01 to 99.99% pharmaceutically acceptable carrier, wherein the percentages are percentages by mass of the drug composition.

[0045] In this invention, the cell drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0046] Preferably, the dosage form of the cell drug / drug composition includes injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field.

[0047] In this invention, the cell drug / drug composition is administered orally, by injection, by nasal administration, by transdermal administration, or by mucosal administration.

[0048] Preferably, the administration route of the cell drug / drug composition is parenteral administration, injection, or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The drug composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, gaseous, or liquid form, i.e., aqueous solution, non-aqueous solution, or suspension; more preferably, tablets, capsules, granules, injections, or infusions. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the drug composition can also be administered as an aerosol or coarse spray, i.e., nasal administration; or intrathecal, intramedullary, or intraventricular administration. More preferably, the cell drug / drug composition can also be administered transdermally, percutaneously, topically, intraenterically, intravaginally, sublingually, or rectally. The cell drug / drug composition of the present invention can be formulated into various dosage forms as needed, and the physician can determine the beneficial dosage for the patient based on factors such as patient type, age, weight, general disease condition, and administration method. Administration methods may include injection or other treatment methods.

[0049] The dosage level of the cell drug / pharmaceutical composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments thereof. The selected dosage level and regimen are subject to reasonable adjustment based on various factors including the activity and stability (i.e., half-life) of the cell drug / pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0050] The therapeutically effective dose of the cell-based drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0051] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, etc.

[0052] It should be noted that, based on the content of this invention, those skilled in the art will readily conceive of applying the universal CAR-T cells of this invention to the treatment of various tumors, not only solid tumors but also non-solid tumors. Regardless of the type of tumor, they all fall within the protection scope of this invention.

[0053] The present invention also provides a reagent / kit comprising the universal chimeric antigen receptor T cells as described above, or the expression vector as described above, or the composition as described above, or the cell drug / drug composition described above.

[0054] In an optional implementation, the reagent / kit is used to detect the universal CAR-T cells.

[0055] The present invention also provides the use of the universal CAR-T cells, the expression vector, the composition, the cell drug / drug composition, and the reagent / kit in the preparation of drugs for the prevention and / or treatment of tumors, cardiovascular diseases, and organ fibrosis.

[0056] In an optional implementation, the tumor is selected from solid tumors or non-solid tumors.

[0057] It should be noted that, based on the content of this invention, those skilled in the art will readily conceive of applying the universal CAR-T cells of this invention to the treatment of various tumors, not only solid tumors, but also non-solid tumors. Regardless of the type of tumor, they all fall within the protection scope of this invention.

[0058] In an optional embodiment, the solid tumor is selected from human pancreatic cancer cells CFPAC1, mouse melanoma cells B16-OVA, etc.

[0059] In an optional implementation, the cardiovascular disease mentioned above is selected from conditions such as myocardial fibrosis.

[0060] In an optional embodiment, the organ fibrosis mentioned above is selected from myocardial fibrosis, pulmonary fibrosis, liver fibrosis, etc.

[0061] The terminology involved in this invention is as follows:

[0062] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this invention are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0063] The amino acid three-letter codes and single-letter codes used in this invention are as known to those skilled in the art, or as described in J. Biol. Chem, 243, p3558 (1968).

[0064] In this invention, the terms "comprising" or "including" are intended to mean that a composition and method include the said elements but exclude other elements, but, depending on the context, also include the case of "composed of".

[0065] In this invention, the term "T cell" refers to a type of lymphocyte that participates in the human immune response and plays an important role in acquired immunity. Some T cells can directly recognize and kill antigens expressed by target cells, and can also secrete cytokines to enhance the killing effect or activate other types of immune cells. Antigens need to form a peptide-MHC complex to be recognized by the T cell receptor (TCR) complex of cytotoxic T cells. The TCR-CD3 complex is a key protein complex used by T cells to recognize antigens and activate signal transduction. It consists of TCRα, TCRβ, and CD3, where TCR is responsible for directly binding to the peptide-MHC complex, and CD3 is responsible for downstream intracellular signal transduction. "T cell" includes all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells (NKT cells), T regulatory cells (Treg cells), and γ-δ T cells, etc.

[0066] In this invention, the term "chimeric antigen receptor (CAR)" refers to an artificially constructed chimeric receptor composed of four parts: an extracellular antigen-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular domain. The intracellular domain includes the activation domain CD3ζ from the TCR, as well as co-stimulatory receptors (4-1BB and CD28 in this invention). Due to its structural and functional characteristics, the CAR can directly recognize and bind to antigens without relying on the peptide-MHC complex, thereby activating T cells through downstream intracellular signaling and achieving cytotoxic-mediated target cell killing. The CAR's structural design incorporates both the "first signal" (i.e., the TCR-CD3 pathway) and the "second signal" (also known as the co-stimulatory signal, i.e., the CD28 or 4-1BB pathway) for T cell activation. Previous studies have shown that introducing co-stimulatory signaling pathways such as OX40 can effectively enhance the CAR-T cell's expansion capacity and anti-apoptotic ability in vitro and in vivo.

[0067] In this invention, the term "universal CAR-T" (uCAR-T) refers to CAR-T cells prepared using T cells from a third-party donor, typically CAR-T cells prepared from T cells isolated from the blood of a healthy volunteer. The opposite is autologous CAR-T, which refers to CAR-T cells prepared from T cells isolated from the blood of a patient.

[0068] In this invention, the term "expression vector" refers to a composition comprising isolated nucleic acids and capable of delivering the isolated nucleic acids into cells. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses; therefore, the term "expression vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0069] In this invention, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved by various methods known in the art, including calcium phosphate-DNA coprecipitation, DEAE-glucan-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0070] In this invention, the term "immune cell" refers to a cell capable of inducing an immune response. "Immune cell" and its other grammatical forms can refer to immune cells of any origin. "Immune cells" include, for example, leukocytes (white blood cells), lymphocytes (T cells, B cells, natural killer (NK) cells), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells), derived from hematopoietic stem cells (HSCs) produced in the bone marrow. The term "immune cell" can also be human or non-human. For example, immune cells can be derived from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, and allogeneic NK cells, or they can be derived from cell lines, such as NK cells prepared using EBV infection, NK cells induced from embryonic stem cells and iPSCs, and the NK92 cell line, etc.

[0071] In this invention, the term "any" means that the event or circumstance described subsequently may, but does not necessarily, occur; the description includes instances where the event or circumstance occurs or does not occur. The term "a" as used in this invention refers to one or more grammatical objects. Unless explicitly stated otherwise, the term "or" is used in this invention to mean and / or the term "and / or" and is used interchangeably with it. The methods and compositions disclosed in this invention cover nucleic acids having a specified sequence, a variant sequence, or a sequence substantially identical or similar thereto, for example, a sequence that is at least 85%, 90%, 95%, 99%, or more identical to the specified sequence.

[0072] Compared to existing technologies, the universal CAR-T cells proposed in this invention utilize T cells derived from healthy volunteers. After preparation, the universal CAR-T cells undergo in vitro and in vivo experiments to verify their function, activity, and efficacy. This allows for the acquisition and cryopreservation of a large number of qualified CAR-T cells, ready for immediate use. It eliminates the need to draw blood from each patient for preparation and testing, thus reducing preparation costs. Furthermore, the cryopreserved CAR-T cells can be thawed and reinfused into the patient in just 5-7 days, significantly shortening the waiting time and effectively improving treatment outcomes.

[0073] The universal CAR-T preparation method provided by this invention can effectively shorten the CAR-T preparation time, reduce costs, and provide CAR-T cells with small batch-to-batch variability and complete function. The FAP-targeting CAR-T cells and preparation method provided by this invention can be used clinically to treat myocardial fibrosis, prolonging patient survival time and improving survival rates. Attached Figure Description

[0074] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0075] Figure 1 This is a schematic diagram of the CAR molecule constructed in this invention.

[0076] Figure 2 This is a flowchart for patient treatment.

[0077] Figure 3 This shows the expression of CAR molecules after lentiviral infection of T cells.

[0078] Figure 4 FAP-CAR-T specifically kills CFPAC1-hFAP cells.

[0079] Figure 5FAP-CAR-T effectively reduced myocardial fibrosis and significantly increased left ventricular ejection fraction in a mouse model of acute myocardial infarction. Figure 5 A shows the results of mouse heart anatomy; the left ventricular wall thickness in the FAP-CAR-T treatment group was significantly greater than that in the control group. Figure 5 B and Figure 5 Echocardiography results in mice showed that the FAP-CAR-T treatment group significantly improved left ventricular systolic function, specifically by increasing ejection fraction (LVEF) and ventricular systolic velocity (LVFS).

[0080] Figure 6 FAP-CAR-T effectively reduced the degree of myocardial fibrosis in a mouse model of angiotensin II / adrenaline-induced myocardial fibrosis. Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0082] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0083] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0084] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0085] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0086] This invention discloses a universal chimeric antigen receptor T cell (CAR-T cell), its preparation method, and its applications, relating to the fields of medicine and immunology. The universal CAR-T cell preparation method disclosed in this invention can effectively shorten CAR-T cell preparation time, reduce costs, and provide CAR-T cells with minimal batch-to-batch variation and complete functionality. The FAP-targeting CAR-T cells and their preparation method disclosed in this invention can be used clinically to treat tumors, cardiovascular diseases, and organ fibrosis, prolonging patient survival time and improving survival rates. This invention also discloses a cell drug / drug composition. The universal CAR-T cells disclosed in this invention provide new directions and ideas for the treatment of tumors, cardiovascular diseases, organ fibrosis, etc.

[0087] Example 1: Preparation and application of FAP-CAR-T. The preparation steps are as follows:

[0088] Step 1: Collect and screen peripheral blood from volunteers, and add anticoagulants to prevent blood clotting.

[0089] Step 2: Centrifuge the collected peripheral blood to obtain plasma, dilute it with physiological saline, and use commercially available lymphocyte separation solution (such as Ficoll separation solution) to separate peripheral blood mononuclear cells (PBMCs).

[0090] Step 3: Use a commercially available T-cell screening kit to isolate and obtain CD8+ T cells.

[0091] Step 4: Activate the CAR gene using a suitable culture medium (e.g., Lonza x-vivo 15 medium) and stimulation conditions (e.g., simultaneous addition of anti-human CD3 and anti-human CD28) to facilitate its transfer and amplification.

[0092] Step 5: Nuclear protein transfection. Using conventional nuclear electroporation, CRISPR-Cas9 protein and sgRNA of immune rejection-related genes are transfected into T cells. Knockout T cell populations are obtained by flow cytometry detection and sorting.

[0093] Step 6: FAP-CAR-T transfection. Using conventional techniques, CAR lentiviral vectors are prepared and titers are detected. CAR is then transfected into T cells using lentivirus to obtain CAR-T cells. Lentiviral-mediated integration of the CAR gene into the T cell genome allows for stable long-term expression and theoretically has the potential to cause tumors. However, current clinical data have not yet observed tumorigenesis.

[0094] Step 7: Expand CAR-T cells in vitro using cytokines and activating stimulants to reach the required effective dose. The main expansion methods currently used are based on anti-CD3, anti-CD28, and IL-2. Antigen-specific aAPCs (artificial antigen-presenting cells) can also be used. CAR positivity and viability are then measured.

[0095] Step 8: Optional, cryopreserve large quantities of universal CAR-T cells from healthy volunteers for use in subsequent treatments.

[0096] When used for patient treatment, the following steps are required:

[0097] Step 9: The patient undergoes whole-body irradiation or chemotherapy to eliminate their own lymphocytes, creating an environment conducive to CAR-T amplification.

[0098] Step 10: Analyze the patient's HLA typing, select CAR-T cells with small HLA differences as the treatment agent, reinfuse the prepared effective dose of CAR-T cells into the patient's body, and monitor the expansion of CAR-T cells and the concentration of blood cytokines at regular intervals.

[0099] When used for establishing and treating myocardial infarction models in mice, the subsequent steps are as follows:

[0100] Step 9: Mouse Model Establishment. Scid-Beige mice underwent chest hair removal and fasting 12 hours prior to surgery. They were then anesthetized and placed in a supine position. The heart was extracted through the third or fourth intercostal space. A suture needle with 8-0 thread was used to ligate the left atrial appendage 1-2 mm below the left atrial appendage and 0.5 mm beside the pulmonary artery conus, passing through the left anterior descending coronary artery. The ligation was performed with appropriate tightness, controlling the needle depth (so that the needle is barely visible) and the suture width (approximately 2 mm). Successful ischemia was indicated when the outer surface of the left ventricular anterior wall turned pale. After ligation, the heart was gently returned to the thoracic cavity, and the pleural cavity was squeezed to expel air while simultaneously tightening the sutures at the ligation site. The control group underwent sham surgery, with sutures placed after chest opening.

[0101] Step 10: On the seventh day of modeling, human FAP-CAR-T cells were injected into some mice via tail vein injection, while others received a combination of tail vein injection and open-chest apical injection. The mice were then observed and weighed daily thereafter.

[0102] When used for the establishment and treatment of drug-induced myocardial fibrosis in mice, the subsequent steps are as follows:

[0103] Step 9: Mouse Model Establishment. Angiotensin II and epinephrine were prepared to standard dosage concentrations and infused into a miniature pump via a perfusion needle. The matching flow regulator was inserted into the miniature osmotic pump. Mice were anesthetized, and the skin was incised at the scapula. The skin and connective tissue were bluntly dissected, and the miniature pump was subcutaneously implanted. The wound was secured with wound clips, and the anesthesia was removed. The mice were allowed to awaken. Using the Alzet 2004 pump, angiotensin II was released at 1.5 μg / (g·d), and epinephrine at 50 μg / (g·d).

[0104] Step 10: On the seventh day of modeling, human FAP-CAR-T cells were injected into some mice via tail vein injection, while others received a combination of tail vein injection and open-chest apical injection. The mice were then observed and weighed daily thereafter.

[0105] Technical Effects: CAR is an artificially constructed chimeric receptor composed of four parts: an extracellular antigen-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular domain. The intracellular domain includes the activation domain CD3ζ from the T cell receptor complex (TCR), as well as the co-stimulatory receptor (4-1BB in this invention). Due to its structural and functional characteristics, CAR can directly recognize and bind to antigens without relying on the peptide-MHC complex, thereby activating T cells through downstream intracellular signaling and completing cytotoxic-mediated target cell killing.

[0106] The CAR-T cells produced by the method described in this invention can be detected by flow cytometry, and the proportion of CAR-positive cells increases with increasing culture time and stimulation frequency. See details... Figure 3 .

[0107] The CAR-T cell targeting FAP provided by this invention can effectively recognize and activate FAP in target cells, thereby killing the target cells. It can be activated by CFPAC1-hFAP cells expressing the antigen and secrete downstream IFNγ molecules, as detailed below. Figure 4 .

[0108] In a mouse model, FAP-CAR-T cells effectively slowed the progression of myocardial fibrosis. In the combination of myocardial injection and tail vein injection, compared to HBSS injection, mice injected with FAP-CAR-T cells showed a significant reduction in cardiac fibrosis, with statistically significant differences. Figure 5 A). Simultaneously, the left ventricular systolic function of mice in the FAP-CAR-T injection group was significantly improved ( Figure 5 (B, C). In a drug-induced myocardial fibrosis model, FAP-CAR-T also effectively reduced collagen aggregation and decreased the degree of myocardial fibrosis in mice.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0110] The nucleotide sequences of SEQ ID NO:1-NO:8 are shown below:

[0111] SEQ ID NO:1(TRAC):>NG_001332.3:925603-930229Homo sapiens T cellreceptor alpha delta locus(TCRA / TCRD)on chromosome 14

[0112] ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCA

[0113] GTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTC

[0114] ACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACAT

[0115] GAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAAT

[0116] CTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACA

[0117] CCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTT

[0118] TCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGT

[0119] CTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCC

[0120] TCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACA

[0121] CGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCC

[0122] AGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTG

[0123] TTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTG

[0124] CCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGT

[0125] CAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCAC

[0126] ATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGG

[0127] TGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGG

[0128] GAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGA

[0129] AAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATG

[0130] CTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAG

[0131] GCCTGGGACAGGAGCTCAATGAGAAAGGAGAAGAGCAGCAGGCATGAGT

[0132] TGAATGAAGGAGGCAGGGCCGGGTCACAGGGCCTTCTAGGCCATGAGAGG

[0133] GTAGACAGTATTCTAAGGACGCCAGAAAGCTGTTGATCGGCTTCAAGCAGG

[0134] GGAGGGACACCTAATTTGCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

[0135] GAGATGGAGTTTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGTGCATCTT

[0136] GGCTCACTGCAACCTCCGCCTCCCAGGTTCAAGTGATTCTCCTGCCTCAGC

[0137] CTCCCGAGTAGCTGAGATTACAGGCACCCGCCACCATGCCTGGCTAATTTTT

[0138] TGTATTTTTAGTAGAGACAGGGTTTCACTATGTTGGCCAGGCTGGTCTCGAA

[0139] CTCCTGACCTCAGGTGATCCACCCGCTTCAGCCTCCCAAAGTGCTGGGATT

[0140] ACAGGCGTGAGCCACCACACCCGGCCTGCTTTTCTTAAAGATCAATCTGAG

[0141] TGCTGTACGGAGAGTGGGTTGTAAGCCAAGAGTAGAAGCAGAAAGGGAG

[0142] CAGTTGCAGCAGAGAGATGATGGAGGCCTGGGCAGGGTGGTGGCAGGGA

[0143] GGTAACCAACACCATTCAGGTTTCAAAGGTAGAACCATGCAGGGATGAGA

[0144] AAGCAAAGAGGGGATCAAGGAAGGCAGCTGGATTTTGGCCTGAGCAGCTG

[0145] AGTCAATGATAGTGCCGTTTACTAAGAAGAAACCAAGGAAAAAATTTGGG

[0146] GTGCAGGGATCAAAACTTTTTGGAACATATGAAAGTACGTGTTTATACTCTT

[0147] TATGGCCCTTGTCACTATGTATGCCTCGCTGCCTCCATTGGACTCTAGAATG

[0148] AAGCCAGGCAAGAGCAGGGTCTATGTGTGATGGCACATGTGGCCAGGGTC

[0149] ATGCAACATGTACTTTGTACAAACAGTGTATATTGAGTAAATAGAAATGGTG

[0150] TCCAGGAGCCGAGGTATCGGTCCTGCCAGGGCCAGGGGCTCTCCCTAGCA

[0151] GGTGCTCATATGCTGTAAGTTCCCTCCAGATCTCTCCACAAGGAGGCATGG

[0152] AAAGGCTGTAGTTGTTCACCTGCCCAAGAACTAGGAGGTCTGGGGTGGGA

[0153] GAGTCAGCCTGCTCTGGATGCTGAAAGAATGTCTGTTTTTCCTTTTAGAAA

[0154] GTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGGTAAGACA

[0155] GGGGTCTAGCCTGGGTTTGCACAGGATTGCGGAAGTGATGAACCCGCAATA

[0156] ACCCTGCCTGGATGAGGGAGTGGGAAGAAATTAGTAGATGTGGGAATGAA

[0157] TGATGAGGAATGGAAACAGCGGTTCAAGACCTGCCCAGAGCTGGGTGGGG

[0158] TCTCTCCTGAATCCCTCTCACCATCTCTGACTTTCCATTCTAAGCACTTTGA

[0159] GGATGAGTTTCTAGCTTCAATAGACCAAGGACTCTCTCCTAGGCCTCTGTAT

[0160] TCCTTTCAACAGCTCCACTGTCAAGAGAGCCAGAGAGAGCTTCTGGGTGG

[0161] CCCAGCTGTGAAATTTCTGAGTCCCTTAGGGATAGCCCTAAACGAACCAGA

[0162] TCATCCTGAGGACAGCCAAGAGGTTTTGCCTTCTTTCAAGACAAGCAACA

[0163] GTACTCACATAGGCTGTGGGCAATGGTCCTGTCTCTCAAGAATCCCCTGCC

[0164] ACTCCTCACACCCACCCTGGGCCCATATTCATTTCCATTTGAGTTGTTCTTAT

[0165] TGAGTCATCCTTCCTGTGGTAGCGGAACTCACTAAGGGGCCCATCTGGACC

[0166] CGAGGTATTGTGATGATAAATTCTGAGCACCTACCCCATCCCCAGAAGGGC

[0167] TCAGAAATAAAATAAGAGCCAAGTCTAGTCGGTGTTTCCTGTCTTGAAACA

[0168] CAATACTGTTGGCCCTGGAAGAATGCACAGAATCTGTTTGTAAGGGGATAT

[0169] GCACAGAAGCTGCAAGGGACAGGAGGTGCAGGAGCTGCAGGCCTCCCCC

[0170] ACCCAGCCTGCTCTGCCTTGGGGAAAACCGTGGGTGTGTCCTGCAGGCCA

[0171] TGCAGGCCTGGGACATGCAAGCCCATAACCGCTGTGGCCTCTTGGTTTTAC

[0172] AGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCT

[0173] CCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGT

[0174] CCAGCTGAGGTGAGGGGCCTTGAAGCTGGGAGTGGGGTTTAGGGACGCGG

[0175] GTCTCTGGGTGCATCCTAAGCTCTGAGAGCAAACCTCCCTGCAGGGTCTTG

[0176] CTTTTAAGTCCAAAGCCTGAGCCCACCAAACTCTCCTACTTCTTCCTGTTAC

[0177] AAATTCCTCTTGTGCAATAATAATGGCCTGAAACGCTGTAAAATATCCTCAT

[0178] TTCAGCCGCCTCAGTTGCACTTCTCCCCTATGAGGTAGGAAGAACAGTTGT

[0179] TTAGAAACGAAGAAACTGAGGCCCCACAGCTAATGAGTGGAGGAAGAGA

[0180] GACACTTGTGTACACCACATGCCTTGTGTTGTACTTCTCTCACCGTGTAACC

[0181] TCCTCATGTCCTCTCTCCCCAGTACGGCTCTCTTAGCTCAGTAGAAAGAAG

[0182] ACATTACACTCATATTACACCCCAATCCTGGCTAGAGTCTCCGCACCCTCCT

[0183] CCCCCAGGGTCCCCAGTCGTCTTGCTGACAACTGCATCCTGTTCCATCACC

[0184] ATCAAAAAAAAACTCCAGGCTGGGTGCGGGGGCTCACACCTGTAATCCC

[0185] AGCACTTTGGGAGGCAGAGGCAGGAGGAGCACAGGAGCTGGAGACCAGC

[0186] CTGGGCAACACAGGGAGACCCCGCCTCTACAAAAAGTGAAAAAATTAACC

[0187] AGGTGTGGTGCTGCACACCTGTAGTCCCAGCTACTTAAGAGGCTGAGATGG

[0188] GAGGATCGCTTGAGCCCTGGAATGTTGAGGCTACAATGAGCTGTGATTGCG

[0189] TCACTGCACTCCAGCCTGGAAGACAAAGCAAGATCCTGTCTCAAATAATAA

[0190] AAAAAATAAGAACTCCAGGGTACATTTGCTCCTAGAACTCTACCACATAGC

[0191] CCCAAACAGAGCCATCACCATCACATCCCTAACAGTCCTGGGTCTTCCTCA

[0192] GTGTCCAGCCTGACTTCTGTTCTTCCTCATTCCAGATCTGCAAGATTGTAAG

[0193] ACAGCCTGTGCTCCCTCGCTCCTTCCTCTGCATTGCCCCTCTTCTCCCTCTC

[0194] CAAACAGAGGGAACTCTCCTACCCCCAAGGAGGTGAAAGCTGCTACCACC

[0195] TCTGTGCCCCCCCGGCAATGCCACCAACTGGATCCTACCCGAATTTATGATT

[0196] AAGATTGCTGAAGAGCTGCCAAACACTGCTGCCACCCCCTCTGTTCCCTTA

[0197] TTGCTGCTTGTCACTGCCTGACATTCACGGCAGAGGCAAGGCTGCTGCAGC

[0198] CTCCCCTGGCTGTGCACATTCCCTCCTGCTCCCCAGAGACTGCCTCCGCCA

[0199] TCCCACAGATGATGGATCTTCAGTGGGTTCTCTTGGGCTCTAGGTCCTGCA

[0200] GAATGTTGTGAGGGGTTTATTTTTTTTTAATAGTGTTCATAAAGAAATACATA

[0201] GTATTCTTCTTCTCAAGACGTGGGGGGAAATTATCTCATTATCGAGGCCCTG

[0202] CTATGCTGTGTATCTGGGCGTGTTGTATGTCCTGCTGCCGATGCCTTC

[0203] SEQ ID NO:2(TRBC1):>NG_001333.2:645749-647196 Homo sapiens Tcellreceptor beta locus(TRB)on chromosome 7

[0204] AGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCA

[0205] GAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCAC

[0206] AGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG

[0207] AGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCC

[0208] CGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGC

[0209] CACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTA

[0210] CGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTC

[0211] ACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGGTGAGTGGGGCCT

[0212] GGGGAGATGCCTGGAGGAGATTAGGTGAGACCAGCTACCAGGGAAAATGG

[0213] AAAGATCCAGGTAGCAGACAAGACTAGATCCAAAAAGAAAGGAACCAGC

[0214] GCACACCATGAAGGAGAATTGGGCACCTGTGGTTCATTCTTCTCCCAGATT

[0215] CTCAGCCCAACAGAGCCAAGCAGCTGGGTCCCCTTTCTATGTGGCCTGTGT

[0216] AACTCTCATCTGGGTGGTGCCCCCCATCCCCCTCAGTGCTGCCACATGCCAT

[0217] GGATTGCAAGGACAATGTGGCTGACATCTGCATGGCAGAAGAAAGGAGGT

[0218] GCTGGGCTGTCAGAGGAAGCTGGTCTGGGCCTGGGAGTCTGTGCCAACTG

[0219] CAAATCTGACTTTACTTTTAATTGCCTATGAAAATAAGGTCTCTCATTTATTT

[0220] TCCTCTCCCTGCTTTCTTTCAGACTGTGGCTTTACCTCGGGTAAGTAAGCCC

[0221] TTCCTTTTCCTCTCCCTCTCTCATGGTTCTTGACCTAGAACCAAGGCATGAA

[0222] GAACTCACAGACACTGGAGGGTGGAGGGTGGGAGAGACCAGAGCTACCT

[0223] GTGCACAGGTACCCACCTGTCCTTCCTCCGTGCCAACAGTGTCCTACCAGC

[0224] AAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCA

[0225] CCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTAAGCA

[0226] GGAGGGCAGGATGGGGCCAGCAGGCTGGAGGTGACACACTGACACCAAG

[0227] CACCCAGAAGTATAGAGTCCCTGCCAGGATTGGAGCTGGGCAGTAGGGAG

[0228] GGAAGAGATTTCATTCAGGTGCCTCAGAAGATAACTTGCACCTCTGTAGGA

[0229] TCACAGTGGAAGGGTCATGCTGGGAAGGAGAAGCTGGAGTCACCAGAAA

[0230] ACCCAATGGATGTTGTGATGAGCCTTACTATTTGTGTGGTCAATGGGCCCTA

[0231] CTACTTTCTCTCAATCCTCACAACTCCTGGCTCTTAATAACCCCCAAAACTT

[0232] TCTCTTCTGCAGGTCAAGAGAAAGGATTTCTGA

[0233] SEQ ID NO:3(CD3E):>NG_007383.1:5001-16596 Homo sapiens CD3epsilonsubunit ofT-cell receptor complex(CD3E),RefSeqGene(LRG_38)onchromosome11

[0234] TATTGTCAGAGTCCTCTTGTTTGGCCTTCTAGGAAGGCTGTGGGACCCAGC

[0235] TTTCTTCAACCAGTCCAGGTGGAGGCCTCTGCCTTGAACGTTTCCAAGTGA

[0236] GGTAAAACCCGCAGGCCCAGAGGCCTCTCTACTTCCTGTGTGGGGTTCAG

[0237] AAACCCTCCTCCCCTCCCAGCCTCAGGTGCCTGCTTCAGAAAATGGTGAGT

[0238] CTCTCTCTTATAAAGCCCTCCTTTTTCATCCTAGCATTGGGAACAATGGCCC

[0239] CAGGGTCCTTATCTCTAGCAGATGTTTTGAAAAAGTCATCTGTTTTGCTTTT

[0240] TTTCCAGAAGTAGTAAGTCTGCTGGCCTCCGCCATCTTAGTAAAGTAACAG

[0241] TCCCATGAAACAAAGATGCAGTCGGGCACTCACTGGAGAGTTCTGGGCCT

[0242] CTGCCTCTTATCAGGTGAGTAGGATGGAGTGGAAAGGGTGGTGTGTCTCCA

[0243] GACCGCTGGAAGGCTTACAGCCTTACCTGGCACTGCCTAGTGGCACCAAG

[0244] GAGCCTCATTTACCAGATGTAAGGAACTGTTTGTGCTATGTTAGGGTGAGG

[0245] GATTAGAGCTGGGGACTAAAGAAAAAGATAGGCCACGGGTGCCTGGGAGA

[0246] GCGTTCGGGGAGCAGGCAAAGAAGAGCAGTTGGGGTGATCATAGCTATTG

[0247] TGAGCAGAGAGGTCTCGCTACCTCTAAGTACGAGCTCATTCCAACTTACCC

[0248] AGCCCTCCAGAACTAACCCAAAAGAGACTGGAAGAGCGAAGCTCCACTCC

[0249] TTGTTTTGAAGAGACCAGATACTTGCGTCCAAACTCTGCACAGGGCATATAT

[0250] AGCAATTCACTATCTTTGAGACCATAAAACGCCTCGTAATTTTTAGTCCTTT

[0251] TCAAGTGACCAACAACTTTCAGTTTATTTCATTTTTTTGAAGCAAGATGGAT

[0252] TATGAATTGATAAATAACCAAGAGCATTTCTGTATCTCATATGAGATAAATAA

[0253] TACCAAAAAAAGTGCCATTTATTGTCAGATACTGTGTAAAGAAAAAATTAT

[0254] TTAGACGTGTTAACTGGTTTAATCCTACTTCTGCCTAGGAAGGAAGGTGTTA

[0255] TATCCCTTTTTTTAAAATTCTTTTTAATTTTGACTATATAAACTGATAAGTCCTC

[0256] TCTACTTCACAGATTAAGAAATTGATACTCAAAAAAAGTTAAATAACTTGTTT

[0257] TAAACCACATAGTAAGTGCCGAAGCCAATCTGTGAGACCAGGACTGTTTGT

[0258] ACTCTAAATGGCTGCACCACATGAGGCAATTGGCTCGTGATGGTTTATTT

[0259] CAAAGACCTAGAAAACACTATCACAGCTGGTGCTCCCGTCTCAGACCCAC

[0260] AGCAACGATGTCTCCCACTTCCTGCTTCATCTTGGGTTTTCTCACGTCTTGAAT

[0261] GTGCACACAAATCACCTGGGGATCTTGTTAAAATGCAGCTTCTGTTTCAA

[0262] AAGACCCAGACTGGAAATGGAGATTCCGCATGTCTAGTACGCTCTCAAGTT

[0263] TATTAATCTGCTGCTGGTCCTAGGAACATATTGAGTAGCGAGGGGCAGGATG

[0264] TGACTCCTGTAAGGAGTGGCCAGGCATTTTCTTAGAGACCTGTGTTATAAA

[0265] GTATGCTTTTCCTTAAAAAAAAGAAGAAGGAGGAGGAGGAGAAGGGCCA

[0266] GGTGCAGTGGCTTACGCCTGTAAGCCCAGCATTTTGGGGGACCAAAGTGG

[0267] GAAGATCACTTGAGCTCAGGAGTTCAAGACCAGCCTGGGCAAAGTAGTGA

[0268] AACCCCATTTCTACAAAAAATTAAAAATTAGCCAGGCAAGGTGGCACACAC

[0269] CTATAGTTCCAGCTACTTGGGAGGCTGAGGTGGAAGGATCACTTGAGCCCA

[0270] GGAGGTCGAGGCTGCAGTAAGCCATGATCAAGCCACTGCACTCCAGCCTG

[0271] GAGTGTCTCAAAATAGATAAATAAATAAATAAATAAATAAATAAATAAATAA

[0272] ATAAAAGAGACAGTATCAAAGACCCAATCACCTCTAGACATCTGGCATCAT

[0273] AGGAATGTGCCCAGTCTGCTCTGGGGATAGGAAAGTGGGGATCCTGTCTCC

[0274] CCCTGTGTAGAGGTTTCAGTAAAAGAAAGGCCTAGGTGTGCAGAAAGCTT

[0275] TCAGGCAATGCCAGGGAAACTGATCATTGTAATGAATCCAGGGTATTGCTG

[0276] AGTGAGGGCATCCTGGAGGGCCCGGTGGAAATGTGGTCAGGCTCTTCAAT

[0277] GCACAGGCCCTAGTTGATGAGTAATCAGGGTTTCAAATATTTCCATCTCTGT

[0278] CTCAAGCAGAAAACAAATGGAAAAACTGAACCACCAGAAAAGCAGAGCC

[0279] AGAGATGGAACAAGAATCCCAGTGTTTGTACCCAACCAAGAGCGTGTTTTT

[0280] CTTCCACAGACACCAATGTTCAAAATGGAGGCTTGGGGGCAAAATTCTTTT

[0281] GCTATGTCTCTAGTCGTCCAAAAAATGGTCCTAACTTTTTCTGACTCCTGC

[0282] TTGTCAAAAATTGTGGGCTCATAGTTAATGCTAGATGCTTCCTTCCTCTATTT

[0283] CCCCCCAAATTTCCTGGGAACCCCTGGTCAATACCAGCAGTAAGTTCCACT

[0284] GTTCTAGGGTGTAGAAATGGCTGTGACCCAGCAGCAAGAGGGAAGGACAT

[0285] CAGATGTCATCAGTGGTCATACTGCAACACAGCCCTTTTTCTGTTTAGGAAT

[0286] GCAGGTACCCACAACATTTACTAACACTTTTTTTTTCTTATTTATTTTCTAGT

[0287] TGGCGTTTGGGGGCAAGATGGTGAGATATGCTTTCTTTCTTTCTTTTTTATG

[0288] AAATCACCCCATCATTCTTTGTAGTTATGAATGGAGCTTTCTCTTAGGCCTCC

[0289] CACAGAACTTCCACAGAGGTCAGGAAAAGGAGTTTCTGCCATCTACCCCTT

[0290] TGACTTTCCTCACAAGTCTGGAGATATTTCTAGCCCAGAAGAGGGAAGCAA

[0291] CAGAGGCAGGAAATAATGAGTCTTAACCATACAAAAGAAAAATTGAGACTT

[0292] AAATGAAGTTGAAAGCACTAACAGTTTTCATTTGTTTGCATTTCATATTTGA

[0293] TGTGAGATTCTGCAGAGGAGACGTAGCCAGAATGCATGCACAGGGTTACTC

[0294] TGGATAAGCTGCTGGGGCAACATTTGGATGTGTGTTCAGAATCACATGTCT

[0295] GAATACTCTGAATATATGTGTGTACATGTGTATTTATGCAAGTGCACATGCAT

[0296] ATGAGTGTGCCCGGCCTGAACTTACTCTCTCAACCACAGCGGTAGAGTCAG

[0297] GAGTGTTCCAACATTGGAAGCCCCTCTATTCAATCAGCTCTTCCAAACTGA

[0298] GTGAACCAATGTTGTATTTAATGGCAACCATGGCTGGACACCATGGCTCAC

[0299] ACCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACTTGAGG

[0300] CCAGGAGTTCGAGACCAGCCTGGCCAACATGGCGAAACCCCGTCTCTACT

[0301] AAAAATACCAAAATCAGCCAGACATGGTGGTGTACGCCTGTAGTCCTAGCT

[0302] ACTCGAGAAGCTGAGGCAGGAGAATCGCTTGAACCTGGGAGGCAGAGGT

[0303] TGTAGTGAGCCGAGATCACACCACTGCACTCTAGCCTGGGTGACACATCGA

[0304] GACTGTCTCAAAAATAAAATAAAGACAACCATTATGCCAGCCTAGATTCCG

[0305] CCATGCTGCCTAATTTGTAGTGTCCTTAGGAGCCATTTTTGTAAATAGTCATC

[0306] AGATAAGATGTAAGGCCCATAACAGCTTTTTCTATGCAGCTGAGGGAATTG

[0307] GAAGATCCATTGTTTCCTAAGAGTTGAGGGAAGAGTCCCAACCCACGGGA

[0308] GCAGGGTCTGATCTTCATTGCCGATAGAAACATTACTAATGGCTTCTTACTG

[0309] TTTCCTTTTCAGGTAATGAAGAAATGGGTAAGAAGATTTCCACTCTATCTAG

[0310] CAAAAGTTTTCAAATATGGAATGAAATGCTCATAGAGTACAATCACAGTAA

[0311] CAAACCCTGAGAACTAAAACTATTAAAGGGAAAATACAAGTATCTTTCAAT

[0312] GGGATCCGTATGAAACTTGCCTGTATTTGTTGCTAGCTGTCATGTCAGATTAT

[0313] AGCTGTGCATATATGTATCTCTGATCATACACATATGGATGTGGGTTTGGAGCT

[0314] ACCATGTGTTTTTGTATAAGCCATGAAATCTTTGAAGGCAGACAGAGACAG

[0315] TGTCTCATTTACCTAGCCCAGTGTCTGGCACATAGTAGGTGCTCAATGAATA

[0316] TTTTTTGAATGAATAAATGAACAAACATATGAACACATTGCTAATTACCTCC

[0317] CCTCAAGAAGCTGATGGTCTTGTGTGAGAGACAAATAATTGAAAATATAGT

[0318] GAGTTGCATGTTATAATATGGGTAGATACAGAGTAAAATGAAGTATAAAGAG

[0319] GGGAGTGGTCAACTCTACTGAGTGTCGTTGGGAAAGGTTCCCTGGGGGAG

[0320] GTGGTCCTTGAGCTGAATTTTAAAGGATAAGCTTATGTTTTAGGGAAGAAA

[0321] AATATTTTATGCAGAAGAGATAAAGCTGTATAGTATGAGGATAAAGAGTCTAA

[0322] CTGAGCTAGATCAGAATGTTTGAATCTTGGCTCAACTCTCTACTTGCTGGGT

[0323] GTGTTTGAGTAATTTACCTAACTTTTCTGTGCCACAGCATCATCATGGTACA

[0324] ATGGAAATAATAGTGCTACCTAACTTGTAGGGTTATTATGAGGACCAAATGA

[0325] GTAATTCATTTAAGGCACTTAGAACATTATCTGACATAAAAGGCAGTAGGAG

[0326] GGCCGGGCATGGTGGCTCACACCTGTAATCCCAGCACTTTGAGAAGCCGA

[0327] GGTGGGAGGATCACCTGAGGTCAGGAGTTCGAGACCGGCCTGGCCAACAT

[0328] GGTGAAACTCCATCTCTGCTAAAAATACAAAAATTAGCCAGGCATGGTGGC

[0329] AGGTGCCTGTAATCTCAGCTACTCAGGAGGCTGAGGTAGGAGAATTGCTTG

[0330] AACCTGGGAGGCGGAGGTTGGAGTGAGCTGAGATTGTGCCATTGCACTCC

[0331] AGCCTGGGCGACAGAGCAAGACTCTGTCTCAAAACAAACAAACAAACAA

[0332] ACAGACAGTAGGTGAATTTTAGCTATTAATACATGGAAAGCATGCTGACTAT

[0333] AGATGATAAGCATTAAAGTTTACTGAGCATGTATGTTTTAGGCATTGCTCTA

[0334] AATATTTTACTTGAATTTCCTCATTTAATTCTTCCAACACCCCTACTGTACAG

[0335] TTAAGGAAACAAAGCCTCAAATAAATACAGAAATAAACAAAAATAAGTAA

[0336] ACAATCCAGTCCTGGGGATATAAATGCAGATTTAGGCCAAGTGCCATGGTTC

[0337] ATGCCTATAATCCCAACACTTTGGGAGGCCAAGGCAGGAGGCTCGCTTGAG

[0338] CTCAGAAGGTTGAGGCTGCATTGAGCAAAGATTGTGCCACTGTACTCGAGC

[0339] CTCTGTGGCAGAGAAAGACCCTGTCTCTGAAAAAATTAATAAATAGAAATT

[0340] TAAAAATAAAAAAAATTTTAATGCAGATTTATATGATACCGAAGTTCATTTTCT

[0341] CAACCATTATGAAATACTGTTTCTGGATATGTATAAAATCTTTGTGAGCACAC

[0342] ATATCTTTTTTTAACTTAACTTTCATTTTAAATTCAGGGGTACATGTGCAGGT

[0343] TTGTTATATAGGTAAACTTGTGCCATGGGGGTTTGTTGTATAGATTATTTCAT

[0344] CACCCAGGCATTAAGCCTGGTACCTGTTAGTTATTTTTCCTGATCCTCTCCCT

[0345] CCTCCCACCCTCCACCTTCTGAGAGGTCCCAGTGTGTGTCATTTCCCTCTGT

[0346] GTTCATGTGTTCTCATCATTTAGCTCCCACTTCTAAATGAGAACATGTGGTAT

[0347] TTGGTTTTCTGTCACTGTGTTAGTTTGCTAAAGATAATGGCCTCCAGTCCCA

[0348] TCCATGTTCCTGCAAAGGACGTGATCTCATTCTTTTTTATGGCTGCGTAGTAT

[0349] TCCATGGTGTATATGCAGCACATTTTTTTATCCAGTCTACCACTGACAGGCAT

[0350] TTAGGTTGATTCCATGTCTTTGCTATTGTGAATAGTGCACAATGAACATACG

[0351] TGGAGCACATTTCTGTCTAAGCACAGACATCTAGACCCTTGTGTGAGCATG

[0352] AGTTAAGTCTAAGCTCTGCTACTGAATTTGTGCCAATAAAAGTTGTGAGCA

[0353] ATTTTCTTTACATTTTTTTCAAACAAACACACCCAGCAGAGTATAATGTCT

[0354] ATGTACTTTATTTATGATTTCTAGTTCATTTAACATGTCTAAGAAACATCCGT

[0355] GTTGAAAAATTATTTATAAATTAAAATAATATAAACTATCTACTGTCCTTATAC

[0356] TCAACTCCCAATTATAAGCAGGTGGAAAAACCTGGAGAATGTTTTGTTTAC

[0357] ATTCTGTGCAGTCTTTGTCAGAGGGCTGCCTGAGCAACTGGGTCAGAGTTT

[0358] AGTTCTGCTCTGGGAGTAGCAGGACCTCAAGAAGGAAAGGAGGAAAGGA

[0359] AGTAACTTTTTCTTGAGCACCTGCTATGTGTCATTCACTTTCACCTTCATAAT

[0360] CCATTTAATTTTCGCAAAAACTTTGTGAGGTTGGTGTTTTATCTCCATTTCCC

[0361] TGATAAAGAAGTTGAGGTTCAGCAAAGTTAAATGACTTGCCCTCAGTCACA

[0362] CAGACTAGGGCAGATCCAGGATTCAAACTCAGGGCTTCTGACTCTTGAGTC

[0363] CAGAGCTCTGTCCCTGACAGCAGCAGCACTGCCTCTCCTCTCTTCCAGCTG

[0364] TTATGTCCAGACTGTAGCAGAACCCAGTGTTCCAGCCACAAGTTTTCCAGG

[0365] AAATAATAAAGGACTCCTAGCTCCACCTCCCAGGGCAAAAATGGCTGCTGT

[0366] GGGAAACACAGGCTGGACCTACGAATGGCATTAGTGGTTTATTAGTTGATT

[0367] TCAGTTGTCCACACTAATAGGCCTCCCTCTAACAAAAATAATTGAGAGCTG

[0368] ATTATGCTCAGATATAATGTAAAGTGAAGCCACTTTTTATTGGAAGAAG

[0369] CATTCCCTCAAAACGTGTAGAGTATTTCACATTATTTAAAGGCAAATAGAGA

[0370] GAAAATTATATGGAATAAGAACAAAGATGTTTCTTCTCTATTATGAGGGACT

[0371] CAGTTCTGAGAAAGGATTTTAAATTGTAAGAAATAGGTAAGTCCACGAATC

[0372] AGTGATTCAGTGGTGTGGAGAGCTTTATTTCTGAGAAGGCCAGTAGCGCTC

[0373] CCTTCTGACAAGCAAATCTAAGACCTGGATGACAGATGACTTCCTGCATTT

[0374] GGTTGGTTCTTTTGTCATTCATATCTATCTGTAATACAGTTCTGGCTAATTTA

[0375] AGAGGATAAGCTTGAAGACCTCTGGAATTTTTCGGCTTTAGGACTTTAAGG

[0376] CTTTCTGAGCTTCAGTAGATCTAGATCTAGGAGCTCATGCTGGTATATTCTG

[0377] AATCCGATGTATCTGAGTTACATCTATGAGCTACTTAATAAATATATCTATGA

[0378] GCTAAATCTCATAGGCTAAGCATGAACCTCACCTCCAAGACTCGGGGTTCC

[0379] TAAATGGATGAGACCCTCTTTGGGAAGTCTTGTGGGCAGTGTCTAATTCCA

[0380] CTAGAAAAGTTTTACCTACAATTTAAACTTAAACCATGATATTTTCTTACTGC

[0381] TGTTTCCTTTTTTCATTTTCAGGTGGTATTACACAGACACGTGAGTTTATTGG

[0382] TCTTTTATTTATGCCCTGTCTGAGGATGCAGATTGGTGGGTAGATGAGAAGG

[0383] AACTGATTGAGAGAGATTAACCCCAAGAACTGATATCTTCCCAGCATTGCA

[0384] TTCTCAACTCCATTTTAGAAAGGTTCCAAATAGGGACTTCTGTGGGTTTTTC

[0385] TTTACATCCATCTTACCCTTCCCAAGTCCCCATGTCCCTGCGTAAACCCTAA

[0386] AGCCACCTCTCAAAAGGTTCTCTAGTTCCCTTCAAGGTTCTCTAGTTCCCTT

[0387] CATTCCACATATCTCCTCTTCCACACCCTCTAGCCAGTAGAGCTCCCTTCTG

[0388] ACAAGCAAGTCTAAGATCTAGATGACAGATGACTTCCTGCATTTGGGTGGT

[0389] TCTTTTGTCACTAATTTGCCTTTTCTAAAATTGTCCTGGTTTCTTCTGCCAAT

[0390] TTCCCTTCTTTCTCCCCAGCATATAAAGTCTCCATCTCTGGAACCACAGTAA

[0391] TATTGACATGCCCTCAGTATCCTGGATCTGAAATACTATGGCAACACAATGA

[0392] TAAAAACATAGGCGGTGATGAGGATGATAAAAACATAGGCAGTGATGAGGA

[0393] TCACCTGTCACTGAAGGAATTTTCAGAATTGGAGCAAAGTGGTTATTATGT

[0394] CTGCTACCCCAGAGGAAGCAAACCAGAAGATGCGAACTTTTATCTCTACCT

[0395] GAGGGCAAGAGGTAATCCAGGTCTCCAGAACAGGTACCACCGGCTCTTTA

[0396] GGGAGGACCATTCAAAGGGCATTCTCAGTGATTTTCCCTAACCCAGCTCCA

[0397] CAGTGCCCAGGCGTCTTTGCGCTTCCTCCCACACTCAATCCTGGGACTCTC

[0398] TGGTACCACACGGCATCAGTGTTTTCTGGAATATAGATTAAACACCAATATG

[0399] AGGCTTCTGGGTAACCCCAGTCTGTGCGAGATCTAAAATAGCAACTCCCTA

[0400] AGAGACAGGACTGGGTCATTTGCACCGCATCACACCCAGGTTCATAGCACA

[0401] CCAACATGAGTTTATCTAATGCTTCCTCCAGAGATAAATTTTTCAGAAAGGT

[0402] TTGCAAAAAACACTCAAGGCCACTATAGTAAAATGGCATAAGCTAAGGTAT

[0403] AATAATAAAATAATAACAATACTTAACATTTATTGAGTGCTTATTAAGTCTCA

[0404] AGCACTGTCTGTACCCAACACTTATCAAGGATTCTTTTTCATGTAATCCTCT

[0405] CAACAACTATATGGGTTAAGTATCATTTTATTCCCATGAGTAAAGGGATGAG

[0406] GAAACAGAGGGTTTGTGAGTTGAAAACACATTTCACGCTTCTCACAGCTA

[0407] GTGAGTAATAAAGCTGGGACTCAAACCCAGGGCTGTTTGACTCCAGTGCCT

[0408] CTACCCACGGCCACCACTCTTTGCTTGTCAATGTTGTTCTAAACATATTGAA

[0409] GGGGGGGCTCTGACCGTGGCAAGCGTGTGAGTAGTAAGGGGAGAATGGCC

[0410] TTCATGCACTCCCTCCTCACCTCCAGCGCCTTGTGTTTTCCTTGCTTAGTGA

[0411] TTTCCCCTCTCCCCACCCCACCCCCCACAGTGTGTGAGAACTGCATGGAGA

[0412] TGGATGTGATGTCGGTGGCCACAATTGTCATAGTGGACATCTGCATCACTGG

[0413] GGGCTTGCTGCTGCTGGTTTACTACTGGAGCAAGAATAGAAAGGCCAAGG

[0414] CCAAGCCTGTGACACGAGGAGCGGGTGCTGGCGGCAGGCAAAGGGGTAA

[0415] GGCTGTGGAGTCCAGTCAGAGGAGATTCCTGCCAAGGGGGACGACCAGCC

[0416] TGGGCCAGGGTGGGTGGCAAGTCCACAGCTAGGTCAGAACAGCTTCTCTA

[0417] GAGCTTCTATGCACAGCTTCTATTACTGTGATGACAAGATCTCAACAGACG

[0418] GTTTCAAATCTCACATCACTCCCCTCCTTCCCATCCTAGAAAAGTGCAAAA

[0419] AAGTTTATGAAAGTGATGGGCTTCCTCACATACCTGTCAATGCCTGCAGTCA

[0420] TCCGATTCCGCCCCTAAGCTGTGGGAAGAGAGACTTTGATATATTAGCTCCT

[0421] GCCTTTTCCTTTCCCTTCCCCTATGGAGAGAAACAATGGGAGGATCTTGAG

[0422] CTGAGGAAAGTCACAAAATGATGAGAAGAGTGTAGGGTCCTTAGAGATGA

[0423] ATGAAAGAAAAAAAAAGAGAAAGGACGTCTGAACAGAAAAGGGAGCGGT

[0424] AGAGGAGAGAACAATGGGGTTTGCCATTCTCTATCTGGGTCTCACTGGCAC

[0425] AGACAGTGCTGCAAGATTGGTTCCCTCATGGGAATGAAATGTTTCCCCTCC

[0426] TTCCTCCGCAGGACAAAACAAGGAGAGGCCACCACCTGTTCCCAACCCAG

[0427] ACTATGAGGTAACGTGGGATAGAAATGGGCCAGGACGCTGGAGGGGATGT

[0428] CCCTCCAGGGGGGAAGGAAACAGATGGGATGGCCCATCTTGTCTGCCAGA

[0429] TGCCTCAAAGCCCCTCACTCAGGGCTTCCATTACAACCCTCTATGTGCCAC

[0430] CTCTGCGTCCTTCATGGTAAAACAGGACTGTCTCAAAGGCTGCATGGCTTC

[0431] CACAACCATGGAGAGGTGGAAGCTTGCAGGAGACATACTCCTCTTTCTCTG

[0432] GCTTATTCATTGACTGGGATACAGCCATGGAGAATATTATATATGCAAATTCT

[0433] AACACAATAAATTCTGGGCTGATATTCCACCAGCATGCACCAGTATAGCGA

[0434] GTTATTGAAATATTAAAATTATATAAATATTATATAAAAGTTATTGAAATATTA

[0435] AAATACTCATTGGGAAATAGCCCCAAACTTTGCTCACCCCAACCCACCCTT

[0436] ACACACACACATACACACACACACACACACACACACACACACACACACAC

[0437] ACGTGACCAGACATCCCAGTCCCTCCCCTACCGGGCTGCCTCTTGAGTTGG

[0438] GGTAACAAAGAGTTAATGCCTGGCATGGCAGAGGATCACCAGGATTGTTCT

[0439] AGTTGATTGGTATGTGTGCACTCCTAGTTGTTAAATATTTTCACTATCACACC

[0440] TGGATATACTCAACAAATATTTGTTGAGCCAAATACTCAACACCAGCCAAA

[0441] CACGTAGTATTTACTTTAGCTTAAGCGAATTATTTAGCCCTGACAGAAGCCC

[0442] TGGAATGTGGGTCTTTAAGTTCCTATTTTTGAGATGGGAAAGCTGAGGCTC

[0443] ACGGAAGGAGGTGACCAGCTCAAGTCTCCTACCGTCCATGCCAAATTAGAA

[0444] TTCCAGCCTGCCTCCTGACTTCAAGTCCAAAGTTCTTCCCACGCACTAAAG

[0445] CTAGCTCTTCAGTGTCCTTTCTTAGGAGGTACTTCCTCCCGCACCACTGACC

[0446] GCCCCCTCTCTATTTCACCCCCAGCCCATCCGGAAAGGCCAGCGGGACCTG

[0447] TATTCTGGCCTGAATCAGAGACGCATCTGACCCTCTGGAGAACACTGCCTC

[0448] CCGCTGGCCCAGGTCTCCTCTCCAGTCCCCCTGCGACTCCCTGTTTCCTGG

[0449] GCTAGTCTTGGACCCCACGAGAGAGAATCGTTCCTCAGCCTCATGGTGAAC

[0450] TCGCGCCCTCCAGCCTGATCCCCCGCTCCCTCCTCCCTGCCTTCTCTGCTGG

[0451] TACCCAGTCCTAAAATATTGCTGCTTCCTCTTCCTTTGAAGCATCATCAGTA

[0452] GTCACACCCTCACAGCTGGCCTGCCCTCTTGCCAGGATATTTATTTGTGCTA

[0453] TTCACTCCCTTCCCTTTGGATGTAACTTCTCCGTTCAGTTCCCTCCTTTTCTT

[0454] GCATGTAAGTTGTCCCCCATCCCAAAGTATTCCATCTACTTTTCTATCGCCGT

[0455] CCCCTTTTGCAGCCCTCTCTGGGGATGGACTGGGTAAATGTTGACAGAGGC

[0456] CCTGCCCCGTTCACAGATCCTGGCCCTGAGCCAGCCCTGTGCTCCTCCCTC

[0457] CCCCAACACTCCCTACCAACCCCCTAATCCCCTACTCCCTCCACCCCCCCTC

[0458] CACTGTAGGCCACTGGATGGTCATTTGCATCTCCGTAAATGTGCTCTGCTCC

[0459] TCAGCTGAGAGAGAAAAAAATAAACTGTATTTGGCTGCAAGA

[0460]

[0461] TTGTTCCACAAGTTAAAATAAATCATAAAACTTGATGTGTTATCTCTTATATCT

[0462] CACTCCCACTATTACCCCTTTATTTCAAACAGGGAAACAGTCTTCAAGTTC

[0463] CACTTGGTAAAAAATGTGAACCCCTTGTATATAGAGTTTGGCTCACAGTGTA

[0464] AAGGGCCTCAGTGATTCACATTTTCCAGATTAGGAATCTGATGCTCAAAGA

[0465] AGTTAAATGGCATAGTTGGGGTGACACAGCTGTCTAGTGGGAGGCCAGCCT

[0466] TCTATATTTTAGCCAGCGTTCTTTCCTGCGGGCCAGGTCATGAGGAGTATGC

[0467] AGACTCTAAGAGGGAGCAAAAGTATCTGAAGGATTTAATATTTTAGCAAGG

[0468] AATAGATATACAATCATCCCTTGGTCTCCCCTGGGGATTGGTTTCAGGACCC

[0469] CTTCTTGGACACCAAATCTATGGATATTTAAGTCCCTTCTATAAAATGGTATA

[0470] GTATTTGCATATAACCTATCCCACATCCTCCTGTATACTTTAAATCATTTCTAGA

[0471] TTACTTGTAATACCTAATACAATGTAAATGCTATGCAAATAGTTGTTATTGTTT

[0472] AAGGAATAATGACAAGAAAAAAAAGTCTGTACATGCTCAGTAAAGACACA

[0473] ACCATCCCTTTTTTTCCCCAGTGTTTTTGATCCATGGTTTGCTGAATCCACA

[0474] GATGTGGAGCCCCTGGATACGGAAGGCCCGCTGTACTTTGAATGACAAATA

[0475] ACAGATTTAAAATTTTCAAGGCATAGT

[0476] TTTATACCTGA

[0477] SEQ ID NO:5(TRAC sgRNA):TCTCTCAGCTGGTACACGGC

[0478] SEQ ID NO:6(TRBC1 sgRNA):CAAACACAGCGACCTCGGGT

[0479] SEQ ID NO:7(CD3E sgRNA):GGGCACTCACTGGAGAGTTC

[0480] SEQ ID NO:8(β2MsgRNA):CGTGAGTAAACCTGAATCTT

Claims

1. A universal chimeric antigen receptor T cell, characterized in that, The universal chimeric antigen receptor T cell expresses a chimeric antigen receptor and knocks out the following combination of immune rejection related genes in the T cell: combination 1: TRAC, TRBC1, β2M is knocked out at the same time, Combination 2: CD3E, β2M is knocked out at the same time; The chimeric antigen receptor comprises: an anti-FAP single-chain antibody segment, a CD8 hinge region, a CD8 transmembrane region, 4-1BB / CD28 and CD3ζ.

2. The universal chimeric antigen receptor T cell of claim 1, wherein, The nucleotide sequence of TRAC is shown as SEQ ID NO: 1, the nucleotide sequence of TRBC1 is shown as SEQ ID NO: 2, the nucleotide sequence of CD3E is shown as SEQ ID NO: 3, and the nucleotide sequence of β2M is shown as SEQ ID NO:

4.

3. The universal chimeric antigen receptor T cell as described in claim 1, characterized in that, The universal chimeric antigen receptor T cell further comprises a chimeric antigen receptor T cell additionally expressing TGFR-wtFc, a chimeric antigen receptor T cell additionally expressing OX40, and a chimeric antigen receptor T cell additionally expressing HLA-A or HLA-E.

4. A method for preparing universal chimeric antigen receptor T cells as described in claim 1, characterized in that, The preparation method comprises the following steps: obtaining T cells from peripheral blood, activating and culturing T cells in vitro, knocking out immune rejection related genes by nuclear electroporation, transfecting a chimeric antigen receptor into T cells using a lentivirus to obtain the universal chimeric antigen receptor T cell, and activating the chimeric antigen receptor T cell using a cytokine and / or an antibody for in vitro expansion.

5. The production method according to claim 4, wherein The nuclear electroporation method transfects CRISPR-Cas9 protein and sgRNA of immune rejection related genes into T cells; The sgRNA targets TRAC, TRBC1, CD3E, and β2M genes in the T cell, and the nucleotide sequences of the genes are shown as SEQ ID NO: 1-SEQ ID NO: 4; and / or, the nucleotide sequence of the sgRNA is shown as SEQ ID NO: 5-SEQ ID NO: 8; And / or, the in vitro expansion method is an anti-CD3, anti-CD28 and IL-2 based method; and / or, an antigen-specific artificial antigen presenting cell is used.

6. An expression vector, characterized by, The expression vector is used for preparing the universal chimeric antigen receptor T cell of claim 1.

7. The expression vector of claim 6, wherein, The expression vector is a recombinant lentivirus vector.

8. A composition characterized in that, The composition comprises any one or more of the universal chimeric antigen receptor T cell of claim 1, or the expression vector of claim 6.

9. A cell pharmaceutical / drug composition, characterized by, The cell drug / drug composition contains the universal chimeric antigen receptor T cell of claim 1 as an active ingredient, and / or a pharmaceutically acceptable carrier.

10. The cell pharmaceutical / drug composition of claim 9, wherein The cell drug / drug composition is used alone, and / or in combination with a cancer treatment drug, a drug for improving ventricular remodeling; and / or, the cell drug / drug composition comprises a liquid dosage form, a gas dosage form, a solid dosage form and a semi-solid dosage form; and / or, the cell drug / drug composition is administered orally, by injection, nasally, transdermally or mucosally.

11. The cell pharmaceutical / drug composition of claim 10, wherein The cancer treatment drugs include small molecule targeted drugs, PD-1 / PD-L1 antibodies, CTLA-4 antibodies; and / or, The drugs for improving ventricular remodeling include betacellulin receptor antagonists, ACEI receptor antagonists, GLP-1 receptor agonists, SGLT2 inhibitors, aldosterone inhibitors.

12. A reagent / kit characterized in that, The reagent / kit comprises the universal chimeric antigen receptor T cell of claim 1, or the expression vector of claim 6, or the composition of claim 8, or the cell drug / drug composition of claim 9.

13. Use of the universal chimeric antigen receptor T cell of claim 1, or the expression vector of claim 6, or the composition of claim 8, or the cell drug / drug composition of claim 9, or the reagent / kit of claim 12 in the preparation of a drug for preventing and / or treating tumors, cardiovascular diseases, organ fibrosis; preferably, the tumors include solid tumors and non-solid tumors.

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