Vector and method for transfecting non-activated T cells in vitro

Abstract

The invention relates to the field of gene engineering, in particular to a vector and a method for transfecting non-activated T cells in vitro. The invention discloses a vector and a method for contacting a non-activated T cell and transfecting the non-activated T cell by using the vector. The surface of the vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof, wherein the antibodies or the antigen-binding fragments thereof and / or the ligands or the receptor-binding fragments thereof are specifically bound with an endocytosis receptor of the non-activated T cell; the vector can carry nucleic acid for coding the CAR, and enters and effectively transfects the non-activated T cells through the endocytosis caused by the endocytosis receptor specifically combined with the non-activated T cells, so that the technical problem that the non-activated T cells are difficult to effectively transfect in the process of preparing the CAR-T cells in vitro is effectively solved, and the CAR-T cells can be used for preparing the CAR-T cells. Further, the cost for preparing the CAR-T cells is reduced, and the preparation period is shortened.

Description

A vector and a method for in vitro transfection of non-activated T cells Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a vector and a method for in vitro transfection of non-activated T cells. Background Art

[0002] Lentiviral vectors or retroviral vectors are one of the commonly used gene vectors in genetic engineering because they can stably integrate exogenous genes, such as shuttle genes encoding CAR, into the genome of host cells.

[0003] By deleting HIV (Human Immunodeficiency Virus) virulence genes, such as env, vif, vpr, vpu and nef genes, and performing multiple attenuation, a self-inactivating, replication-defective, and biosafe lentiviral vector is constructed.

[0004] Vesicular Stomatitis Virus strains, such as the Indiana strain, are widely infectious because their envelope glycoprotein (VSV-G) can specifically bind to the low-density lipoprotein receptor (LDL-R) that is widely present on the surface of various cells.

[0005] Activated T cells express LDL-R. Therefore, artificially synthesized, biosafe lentiviral vectors or retroviral vectors usually use wild-type VSV-G to construct their envelope glycoprotein (VSV-G lentiviral vector or retroviral vector) to transfect activated T cells.

[0006] However, non-activated T cells lack the expression of LDL-R, so it is difficult to effectively infect non-activated T cells using VSV-G lentiviral vectors or retroviral vectors (Amirache F., Lévy C, et al., Mystery solved: VSV-G-LVs do not allow efficient gene transfer into unstimulated T cells, B cells, and HSCs because they lack the LDL receptor. Blood. 2014 Feb 27; 123(9): 1422-4.), which is also a major technical challenge in the field of adoptive cell therapy.

[0007] Chimeric Antigen Receptor (CAR) is an engineered receptor used to genetically engineer lymphocytes such as T cells for adoptive cellular immunotherapy (Pule, et al., Cytother, 5:3, 2003; Restifo, et al., Nat. Rev. Immunol., 12:269, 2012). CAR-T cells have become a highly regarded and widely used cell therapy product in the field of cell therapy.

[0008] Because VSV-G lentiviral vectors or retroviral vectors are difficult to infect non-activated T cells, the conventional preparation method of CAR-T cells in vitro usually requires the use of T cell activation molecules or co-stimulatory molecules, such as anti-CD3 antibodies and anti-CD28 antibodies. After activating and stimulating non-activated T cells, the activated T cells are genetically edited to express CAR molecules. The CAR-T cells are then expanded and cultured to a certain order of magnitude before being injected into the patient.

[0009] However, the step of activating and stimulating non-activated T cells is not only time-consuming, but also requires the use of expensive products such as anti-CD3 antibodies, anti-CD28 antibodies, and cytokines for in vitro cell culture. This not only increases the time cost for patients receiving CAR-T cell therapy, but also inevitably increases the financial cost for patients.

[0010] Generally speaking, the complete cycle of preparing CAR-T cells in vitro is about 14-16 days. Refer to relevant patent documents, such as Chinese invention patent number CN109652378A and Chinese invention patent number CN112292147A. It takes 2-4 days to separate non-activated T cells from the patient's PBMCs, activate and stimulate non-activated T cells using anti-CD3 antibodies and anti-CD28 antibodies (if there is a sorting step, the corresponding antibody magnetic beads are used) and perform cell culture; it takes 1-8 days to infect activated T cells with lentiviral vectors; and it takes 8-12 days to expand and culture the prepared CAR-T cells to reach the ideal order of magnitude for treatment. The above patent documents are incorporated herein by reference in their entirety.

[0011] Currently, the existing technology for preparing CAR-T cells in vitro without activating and stimulating non-activated T cells during the preparation process includes serum starvation of non-activated T cells (Ghassemi, S., et al., Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118-128 (2022)). However, when serum starving non-activated T cells, non-activated T cells must be cultured, which places high demands on experimental equipment and operating environment, such as requiring cell culture under cGMP conditions, thereby increasing the time and money costs required for patients to receive cell therapy, and is still not conducive to broadening the access to cell therapy for patients.

[0012] Therefore, there is an unmet need for a method to rapidly prepare CAR-T cells in vitro to reduce the time and money costs required for patients.

[0013] Summary of the Invention

[0014] In view of this, in order to at least solve one of the technical problems in conventional methods for preparing CAR-T cells in vitro, such as the difficulty in infecting inactive T cells, delivering the target gene into the genome of inactive T cells, the long CAR-T cell preparation cycle and the high preparation cost, the first aspect of the present invention provides a vector, the surface of which contains one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to endocytic receptors of inactive T cells.

[0015] T cells are one of the important white blood cells in the human immune system and play an important role in acquired immune responses.

[0016] "Non-activated T cells" refer to T cells that are not proliferating, not differentiating, in a resting state, do not recognize antigens, and have not been activated or stimulated by co-stimulatory molecules, such as T cells in the G0 phase of the cell cycle, resting / quiescent T cells, or immature T cells. T cells.

[0017] Endocytosis refers to the process by which substances enter cells. During endocytosis, the substance to be taken in is surrounded by a region of the plasma membrane, which then buds into the cell to form a vesicle containing the taken in substance. Endocytosis can be divided into four categories: receptor-mediated endocytosis (also known as clathrin-mediated endocytosis), caveolae, pinocytosis, and phagocytosis (Marsh M, Endocytosis. Oxford University Press. p. vii., 2001).

[0018] "Endocytosis receptor" refers to a receptor that mediates endocytosis. Lymphocytes, such as non-activated T cells, have a variety of endocytosis receptors on their surfaces.

[0019] Therefore, by constructing at least one antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof that specifically binds to at least one endocytic receptor of non-activated T cells on the surface of the carrier, the carrier disclosed in the present invention can enter non-activated T cells through endocytosis.

[0020] In some embodiments of the present invention, the surface of the carrier comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to endocytic receptors of non-activated T cells, and the endocytic receptor is not LDL-R.

[0021] In some embodiments of the present invention, the endocytic receptor of the non-activated T cells is CD7.

[0022] In some embodiments of the present invention, the endocytic receptor of the non-activated T cells is CD3.

[0023] In some embodiments of the present invention, the endocytic receptors of the non-activated T cells are CD3 and CD28.

[0024] In some embodiments of the present invention, the endocytic receptor of the non-activated T cells is CD5.

[0025] In some embodiments of the present invention, the endocytic receptor of the non-activated T cells is TCRα.

[0026] In some embodiments of the present invention, the endocytic receptor of the non-activated T cells is TCRβ.

[0027] In some embodiments of the present invention, the surface of the carrier comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to the endocytic receptor of non-activated T cells; the endocytic receptor of non-activated T cells is CD7.

[0028] In some embodiments of the present invention, the surface of the carrier comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to the endocytic receptor of non-activated T cells; the endocytic receptor of non-activated T cells is CD3.

[0029] In some embodiments of the present invention, the surface of the carrier comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to endocytic receptors of non-activated T cells; the endocytic receptors of non-activated T cells are CD3 and CD28.

[0030] In some embodiments of the present invention, the vector is selected from the following vectors: lipid nanoparticles and viruses; the virus is a virus with a viral envelope; preferably, the virus with a viral envelope is a lentiviral vector or a retroviral vector.

[0031] In some embodiments of the present invention, the carrier is a lipid nanoparticle.

[0032] In some embodiments of the present invention, the vector is a virus with a viral envelope, and the virus with a viral envelope is a lentiviral vector or a retroviral vector, and the envelope glycoprotein of the lentiviral vector or retroviral vector is selected from the following envelope glycoproteins and variants thereof: envelope glycoproteins of vesicular stomatitis virus strains and variants thereof, envelope glycoproteins of baboon endogenous retrovirus BaEV and variants thereof, envelope glycoproteins of feline endogenous retrovirus RD114 and variants thereof, and envelope glycoproteins of gibbon ape leukemia virus GALV and variants thereof;

[0033] The envelope glycoprotein of the vesicular stomatitis virus strain and its variants are selected from the following envelope glycoproteins and their variants: envelope glycoprotein of Indiana strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Cocal strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Maraba strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Morreton strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Alagoas strain of vesicular stomatitis virus and its variants, envelope glycoprotein of New The envelope glycoprotein of Jersey strain and its variants, the envelope glycoprotein of Carajas strain and its variants, the envelope glycoprotein of Chandipura strain and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

[0034] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0035] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 2.

[0036] In some embodiments of the present invention, the vector is a lentiviral vector or a retroviral vector, and the surface of the lentiviral vector or retroviral vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to an endocytic receptor of a non-activated T cell; the endocytic receptor of the non-activated T cell is CD7;

[0037] Preferably, the envelope glycoprotein of the lentiviral vector or retroviral vector is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 1;

[0038] Preferably, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 2.

[0039] In some embodiments of the present invention, the vector is a lentiviral vector or a retroviral vector, and the surface of the lentiviral vector or retroviral vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to an endocytic receptor of a non-activated T cell; the endocytic receptor of the non-activated T cell is CD3;

[0040] Preferably, the envelope glycoprotein of the lentiviral vector or retroviral vector is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 1;

[0041] Preferably, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 2.

[0042] In some embodiments of the present invention, the vector is a lentiviral vector or a retroviral vector, and the surface of the lentiviral vector or retroviral vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to endocytic receptors of non-activated T cells; the endocytic receptors of non-activated T cells are CD3 and CD28;

[0043] Preferably, the envelope glycoprotein of the lentiviral vector or retroviral vector is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 1;

[0044] Preferably, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus, and the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 2.

[0045] In some embodiments of the present invention, the envelope glycoprotein undergoes a first mutation, which weakens or loses the ability of the envelope glycoprotein to specifically recognize a receptor relative to before the first mutation.

[0046] By causing a first mutation in a viral envelope glycoprotein, such as VSV-G or Cocal-G, the ability of the viral envelope glycoprotein to bind to a glycoprotein receptor, such as LDL-R, is weakened (the mutated viral envelope glycoprotein retains membrane fusion ability to mediate lysosomal escape), thereby further improving the targeting ability of a lentiviral vector or retroviral vector containing the mutated viral envelope glycoprotein to bind to endocytic receptors of non-activated T cells, such as CD7, or CD3 and CD28, through antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof contained in its viral envelope in unsorted cells such as PBMCs.

[0047] In some embodiments of the present invention, the envelope glycoprotein undergoes a first mutation, which weakens or loses the ability of the envelope glycoprotein to specifically recognize receptors relative to before the first mutation and retains the ability of membrane fusion to mediate endosomal / lysosomal escape.

[0048] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus or a variant thereof, and the receptor is the low-density lipoprotein receptor (LDL-R); the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 1;

[0049] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0050] (a) substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, or deletion of amino acids 345-353 of SEQ ID NO: 1;

[0051] (b) After optimal global alignment with SEQ ID NO: 1, the position corresponding to SEQ ID NO: 1 substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353;

[0052] More preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0053] substitution of K47, deletion of K47, and substitution of R354;

[0054] More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0055] The amino acid at position 47 was replaced by lysine K to glutamine Q, the amino acid at position 354 was replaced by arginine R to glutamine Q, and the amino acid at position 47 was deleted;

[0056] Most preferably, the first mutation comprises a mutation in the amino acid sequence comprising the following amino acid: deletion of K47.

[0057] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the receptor is the low-density lipoprotein receptor (LDL-R); the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 2;

[0058] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0059] (a) substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 2;

[0060] (b) after optimal global alignment with SEQ ID NO: 2, substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 2;

[0061] More preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution of K47, deletion of K47, substitution of R354;

[0062] More preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution of amino acid 47 from lysine K to glutamine Q, substitution of amino acid 354 from arginine R to glutamine Q, and deletion of amino acid 47 from lysine;

[0063] Most preferably, the first mutation comprises a mutation in the amino acid sequence comprising the following amino acid: deletion of K47.

[0064] In some embodiments of the present invention, the envelope glycoprotein undergoes a second mutation, which enhances the ability of the envelope glycoprotein to antagonize inactivation by complement relative to before the second mutation or prevents the envelope glycoprotein from being inactivated by complement.

[0065] The complement system is composed of a series of proteins and is part of the innate immune system. Complement (C) is present in the serum, tissue fluid, and cell membrane surfaces of normal humans and animals. After activation, it has enzymatic activity and can undergo complex cascade reactions. The complement system is activated through a series of enzymes (enzymes) that cut each other, ultimately forming a membrane attack complex that resembles a hole on the target microorganism, causing the microorganism to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, and clear immune complexes through lysis, conditioning, phagocytosis, and mediating inflammatory responses, demonstrating corresponding biological functions. Complement is widely involved in the body's defense response against microbial infection and immune regulation, and also mediates immunopathological damage reactions. It is an effector system and effector amplification system with important biological functions in the body.

[0066] In some embodiments of the present invention, the envelope glycoprotein undergoes a second mutation, which enhances the ability of the envelope glycoprotein to antagonize inactivation by complement relative to before the second mutation, or prevents the envelope glycoprotein from being inactivated by complement and retains the ability to mediate endosomal / lysosomal escape by membrane fusion.

[0067] In some embodiments of the present invention, the envelope glycoprotein whose ability to antagonize inactivation by complement is enhanced compared to before the second mutation or is not inactivated by complement is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof;

[0068] the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO: 1;

[0069] Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0070] (a) amino acid position 214 of SEQ ID NO: 1;

[0071] (b) after optimal global alignment with SEQ ID NO: 1, is located at amino acid position 214 corresponding to SEQ ID NO: 1;

[0072] (c) amino acid position 352 of SEQ ID NO: 1;

[0073] (d) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to SEQ ID NO: 1 at position 352;

[0074] (e) amino acid position 50 of SEQ ID NO: 1;

[0075] (f) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to 50 of SEQ ID NO: 1;

[0076] (g) amino acid position 146 of SEQ ID NO: 1;

[0077] (h) after optimal global alignment with SEQ ID NO: 1, located at amino acid position 146 corresponding to SEQ ID NO: 1;

[0078] More preferably, the amino acid mutation includes amino acid deletion, insertion or substitution;

[0079] More preferably, the amino acid sequence comprises at least one substitution of the following amino acids:

[0080] (a) amino acid position 214 of SEQ ID NO: 1;

[0081] (b) after optimal global alignment with SEQ ID NO: 1, is located at amino acid position 214 corresponding to SEQ ID NO: 1;

[0082] (c) amino acid position 352 of SEQ ID NO: 1;

[0083] (d) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to SEQ ID NO: 1 at position 352;

[0084] (e) amino acid position 50 of SEQ ID NO: 1;

[0085] (f) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to 50 of SEQ ID NO: 1;

[0086] (g) amino acid position 146 of SEQ ID NO: 1;

[0087] (h) After optimal global alignment with SEQ ID NO: 1, it is located at the amino acid position corresponding to SEQ ID NO: 146.

[0088] In some embodiments of the present invention, the second mutation includes one or more of the following site mutations in the amino acid sequence: substitution of T214, substitution of T352, substitution of K50, substitution of S146;

[0089] Preferably, the amino acid sequence comprises one or more of the following site mutations: amino acid 214 is substituted from threonine T to asparagine N, amino acid 352 is substituted from threonine T to alanine A, amino acid 50 is substituted from lysine K to threonine T, and amino acid 146 is substituted from serine S to threonine T;

[0090] Preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0091] (a) Replacement of T214 and T352;

[0092] (b) Replacement of T214, T352, K50 and S146;

[0093] More preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0094] (a) amino acid 214 is substituted from threonine T to asparagine N and amino acid 352 is substituted from threonine T to alanine A;

[0095] (b) The amino acid at position 214 was replaced by threonine T to asparagine N, the amino acid at position 352 was replaced by threonine T to alanine A, the amino acid at position 50 was replaced by lysine K to threonine T, and the amino acid at position 146 was replaced by serine S to threonine T.

[0096] In some embodiments of the present invention, the envelope glycoprotein whose ability to antagonize inactivation by complement is enhanced compared to before the second mutation or is not inactivated by complement is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus or a variant thereof;

[0097] the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO: 2;

[0098] Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0099] (a) amino acid position 214 of SEQ ID NO: 2;

[0100] (b) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 2;

[0101] (c) amino acid position 352 of SEQ ID NO: 2;

[0102] (d) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position equivalent to SEQ ID NO: 2 at position 352;

[0103] (e) amino acid position 50 of SEQ ID NO: 2;

[0104] (f) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position equivalent to SEQ ID NO: 2;

[0105] (g) amino acid position 146 of SEQ ID NO: 2;

[0106] (h) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position 146 corresponding to SEQ ID NO: 2;

[0107] More preferably, the amino acid mutation includes amino acid deletion, insertion or substitution;

[0108] More preferably, the amino acid sequence comprises at least one substitution of the following amino acids:

[0109] (a) amino acid position 214 of SEQ ID NO: 2;

[0110] (b) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 2;

[0111] (c) amino acid position 352 of SEQ ID NO: 2;

[0112] (d) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position equivalent to SEQ ID NO: 2 at position 352;

[0113] (e) amino acid position 50 of SEQ ID NO: 2;

[0114] (f) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position equivalent to SEQ ID NO: 2;

[0115] (g) amino acid position 146 of SEQ ID NO: 2;

[0116] (h) After optimal global alignment with SEQ ID NO: 2, it is located at the amino acid position corresponding to SEQ ID NO: 2 at position 146.

[0117] In some embodiments of the present invention, the second mutation includes one or more of the following site mutations in the amino acid sequence: substitution of K214, substitution of T352, substitution of K50, substitution of S146;

[0118] Preferably, the amino acid sequence comprises one or more of the following site mutations: amino acid 214 is substituted from lysine K to asparagine N, amino acid 352 is substituted from threonine T to alanine A, amino acid 50 is substituted from lysine K to threonine T, and amino acid 146 is substituted from serine S to threonine T;

[0119] Preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0120] (a) Replacement of K214 and T352;

[0121] (b) Replacement of K214, T352, K50 and S146;

[0122] More preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0123] (a) amino acid 214 was substituted from lysine K to asparagine N and amino acid 352 was substituted from threonine T to alanine A;

[0124] (b) The amino acid at position 214 was replaced by lysine K to asparagine N, the amino acid at position 352 was replaced by threonine T to alanine A, the amino acid at position 50 was replaced by lysine K to threonine T, and the amino acid at position 146 was replaced by serine S to threonine T.

[0125] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 3; relative to SEQ ID NO: 1, SEQ ID NO: 3 comprises a K47 deletion.

[0126] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 4; relative to SEQ ID NO: 1, SEQ ID NO: 4 comprises R354Q.

[0127] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 5; relative to SEQ ID NO: 1, SEQ ID NO: 5 comprises T214N and T352A.

[0128] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 6; relative to SEQ ID NO: 1, SEQ ID NO: 6 comprises a K47 deletion, T214N and T352A.

[0129] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 7; relative to SEQ ID NO: 1, SEQ ID NO: 7 comprises R354Q, T214N and T352A.

[0130] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 28, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 28; relative to SEQ ID NO: 2, SEQ ID NO: 28 comprises a K47 deletion.

[0131] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof that specifically binds to any one of the endocytic receptors of the non-activated T cells is directly or indirectly linked to the transmembrane region and is included on the surface of any one of the aforementioned carriers;

[0132] Preferably, the transmembrane region is selected from the transmembrane regions of the following proteins:

[0133] CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70;

[0134] More preferably, the transmembrane region includes the transmembrane region of CD8α;

[0135] Preferably, the antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof is indirectly connected to the transmembrane region via a linker domain;

[0136] More preferably, the linker domain is selected from:

[0137] (i) an immunoglobulin hinge region selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0138] (ii) a hinge region selected from the wild-type or modified hinge region of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0139] (iii) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and

[0140] (iv) a stem region of a type II C-lectin, wherein the type II C-lectin is selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0141] More preferably, the connecting domain includes the hinge region of CD8α.

[0142] In some embodiments of the present invention, any of the aforementioned vectors carries one or more exogenous substances, and the exogenous substances are nucleic acids;

[0143] Preferably, the vector is any one of the lentiviral vectors or retroviral vectors.

[0144] In some embodiments of the present invention, the nucleic acid encodes a chimeric antigen receptor;

[0145] Preferably, the chimeric antigen receptor comprises an antigen binding region, a transmembrane region and an intracellular signaling domain;

[0146] More preferably, the antigen binding region can bind to at least one antigen selected from the group consisting of TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra , IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AmL, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, mL-IAP, TMPRSS2ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D;.

[0147] More preferably, the chimeric antigen receptor further comprises a hinge region and a costimulatory signaling domain consisting of one or more costimulatory molecules.

[0148] In some embodiments of the present invention, the antigen binding region of the CAR specifically binds to at least one of CD19, HER2, CD20, CD33, BCMA, CD79B, CD79A, CEA and Claudin18.2.

[0149] In some embodiments of the present invention, the antigen binding region of the CAR specifically binds to CD19.

[0150] In some embodiments of the present invention, the antigen binding region of the CAR specifically binds to CD33.

[0151] In some embodiments of the present invention, the antigen binding region of the CAR specifically binds to CEA.

[0152] In some embodiments of the present invention, the antigen binding region of the CAR specifically binds to HER2.

[0153] In some embodiments of the present invention, the antigen binding region of the CAR comprises an antibody or antigen binding fragment thereof and / or a ligand or receptor binding fragment thereof that specifically binds to the antigen, and the antibody or antigen binding fragment thereof is at least one of a full-length antibody, a half antibody, Fab, Fab', F(ab')2, Fv, a VHH domain, and a scFv.

[0154] In some embodiments of the present invention, the hinge region of the CAR sequentially connects the antigen binding region and the transmembrane region of the CAR.

[0155] In some embodiments of the present invention, the hinge region of the CAR is selected from the hinge region of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.

[0156] In some embodiments of the present invention, the hinge region of the CAR is the hinge region of CD8α.

[0157] In some embodiments of the present invention, the transmembrane region of the CAR is selected from the transmembrane region of the following proteins: CD2, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX 40. ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

[0158] In some embodiments of the present invention, the transmembrane region of the CAR is the transmembrane region of CD8α.

[0159] In some embodiments of the present invention, the costimulatory signaling domain of the CAR comprises one or more costimulatory signaling domains of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAmL, CD244, CD100, ICOS, CD40 and MyD88.

[0160] In some embodiments of the present invention, the costimulatory signaling domain of the CAR comprises the costimulatory signaling domain of 4-1BB and / or CD28.

[0161] In some embodiments of the present invention, the costimulatory signaling domain of the CAR is the costimulatory signaling domain of 4-1BB.

[0162] In some embodiments of the present invention, the intracellular signaling domain of the CAR is selected from the intracellular signaling domains of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FceRly, FceRβ, FcyRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12 and other proteins containing at least one ITAM intracellular signaling domain.

[0163] In some embodiments of the present invention, the intracellular signaling domain of the CAR is the intracellular signaling domain of CD3ζ.

[0164] In some embodiments of the present invention, the antigen binding region of the CAR specifically binds to an antigen associated with a disease, pathogen or cancer.

[0165] In some embodiments of the present invention, the disease, pathogen or cancer is selected from at least one of the following diseases, pathogens or cancers: acute myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal cancer, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic Myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, common germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin's lymphoma, Hodgkin's disease, hypopharyngeal cancer, invasive ductal carcinoma, Invasive lobular carcinoma, inflammatory breast cancer, colorectal cancer, intrahepatic bile duct cancer, invasive / invasive breast cancer, pancreatic islet cell carcinoma, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, interstitial carcinoma Glioblastoma, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, bone Primary sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary cancer, sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary tumor, primary central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, Rhabdomyosarcoma, salivary gland cancer, sarcoma, sarcoma, bone, sarcoma, soft tissue, sarcoma, uterus, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, vertebral cancer, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, renal tubuleCancer, undiagnosed cancer, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer.

[0166] In some embodiments of the present invention, any of the vectors carrying the nucleic acid encoding any of the CARs is any of the lentiviral vectors or retroviral vectors.

[0167] In a second aspect, the present invention provides use of any one of the vectors provided in the first aspect of the present invention in preparing a drug for preventing and / or treating a disease;

[0168] Preferably, the disease is selected from at least one of blood cancer and solid cancer;

[0169] More preferably, the blood cancer is at least one selected from acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL);

[0170] More preferably, the solid cancer is at least one selected from colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

[0171] In some embodiments of the present invention, the blood cancer includes relapsed or refractory blood cancer.

[0172] In some embodiments of the present invention, the blood cancer is selected from at least one of mantle cell lymphoma (MCL) and follicular lymphoma (FL).

[0173] In a third aspect, the present invention provides a composition comprising any one of the carriers provided in the first aspect of the present invention and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives and other auxiliary components.

[0174] In a fourth aspect, the present invention provides a method for transfecting non-activated T cells in vitro, the method comprising contacting non-activated T cells with any of the vectors carrying any of the exogenous nucleic acids provided in the first aspect of the present invention;

[0175] Preferably, the contacting occurs outside the body of a subject, and the subject is an individual to whom the inactivated T cells transfected by the method for transfecting inactivated T cells in vitro are administered;

[0176] More preferably, the inactivated T cells are selected from at least one of the following sources:

[0177] (a) The subject's own non-activated T cells;

[0178] (b) non-activated T cells from the subject's allogeneic individual;

[0179] (c) non-activated T cells derived from iPSCs;

[0180] Still more preferably, the non-activated T cells are contained in the peripheral blood or umbilical cord blood of the subject and / or an individual allogeneic to the subject;

[0181] Still further preferably, the non-activated T cells are contained in PBMCs of the subject and / or an individual allogeneic to the subject;

[0182] Even more preferably, the non-activated T cells are isolated from PBMCs of the subject and / or an individual allogeneic to the subject.

[0183] In some embodiments of the present invention, the non-activated T cells are CD3 T cells isolated from PBMCs of the subject and / or an allogeneic individual of the subject. + T cells.

[0184] In some embodiments of the present invention, the non-activated T cells are not cultured in vitro.

[0185] In some embodiments of the present invention, the non-activated T cells are not serum starved.

[0186] In some embodiments of the present invention, the non-activated T cells are not activated and / or expanded by T cell activation molecules or co-stimulatory molecules;

[0187] Preferably, the T cell activation molecule or co-stimulatory molecule is selected from one or more of an anti-CD3 antibody or an antigen-binding fragment thereof, an antibody or an antigen-binding fragment thereof that specifically binds to CD28, a ligand or a receptor-binding fragment thereof that specifically binds to CD28, and a ligand or a receptor-binding fragment thereof that specifically binds to 4-1BB.

[0188] In some embodiments of the present invention, the vector is in contact with the inactivated T cells for no more than 3 days, no more than 2 days, no more than 47 hours, no more than 46 hours, no more than 45 hours, no more than 44 hours, no more than 43 hours, no more than 42 hours, no more than 41 hours, no more than 40 hours, no more than 39 hours, no more than 38 hours, no more than 37 hours, no more than 36 hours, no more than 35 hours, no more than 34 hours, no more than 33 hours, no more than 32 hours, no more than 31 hours, no more than 30 hours, no more than 29 hours, no more than 28 hours, no more than 27 hours, no more than 26 hours, no more than 25 hours, no more than 24 hours. hours, no more than 23 hours, no more than 22 hours, no more than 21 hours, no more than 20 hours, no more than 19 hours, no more than 18 hours, no more than 17 hours, no more than 16 hours, no more than 15 hours, no more than 14 hours, no more than 13 hours, no more than 12 hours, no more than 11 hours, no more than 10 hours, no more than 9 hours, no more than 8 hours, no more than 7 hours, no more than 6 hours, no more than 5 hours, no more than 4 hours, no more than 3 hours, no more than 2 hours, no more than 1 hour, no more than 59 minutes, no more than 58 minutes, no more than 57 minutes, no more than 56 minutes, no more than 55 minutes, no more than 54 minutes , no more than 53 minutes, no more than 52 minutes, no more than 51 minutes, no more than 50 minutes, no more than 49 minutes, no more than 48 minutes, no more than 47 minutes, no more than 46 minutes, no more than 45 minutes, no more than 44 minutes, no more than 43 minutes, no more than 42 minutes, no more than 41 minutes, no more than 40 minutes, no more than 39 minutes, no more than 38 minutes, no more than 37 minutes, no more than 36 minutes, no more than 35 minutes, no more than 34 minutes, no more than 33 minutes, no more than 32 minutes, no more than 31 minutes, no more than 30 minutes, no more than 29 minutes, no more than 28 minutes, no more than 27 minutes, no more than 26 minutes, No more than 25 minutes, no more than 24 minutes, no more than 23 minutes, no more than 22 minutes, no more than 21 minutes, no more than 20 minutes, no more than 19 minutes, no more than 18 minutes, no more than 17 minutes, no more than 16 minutes, no more than 15 minutes, no more than 14 minutes, no more than 13 minutes, no more than 12 minutes, no more than 11 minutes, no more than 10 minutes, no more than 9 minutes, no more than 8 minutes, no more than 7 minutes, no more than 6 minutes, no more than 5 minutes, no more than 4 minutes, no more than 3 minutes, no more than 2 minutes, no more than 1 minute, no more than 50 seconds, no more than 40 seconds, no more than 30 seconds, no more than 20 seconds or no more than 10 seconds.

[0189] In some embodiments of the present invention, the vector is any one of the lentiviral vectors or retroviral vectors provided in the first aspect of the present invention.

[0190] In some embodiments of the present invention, any of the vectors carrying a nucleic acid encoding any of the CARs provided in the first aspect of the present invention is contacted with inactivated T cells to transfect the inactivated T cells to prepare CAR-T cells; preferably, the vector is any of the lentiviral vectors or retroviral vectors.

[0191] In some embodiments of the present invention, the time required to prepare the CAR-T cells is no more than 14 days, no more than 13 days, no more than 12 days, no more than 11 days, no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, no more than 47 hours, no more than 46 hours, no more than 45 hours, no more than 44 hours, no more than 43 hours, no more than 42 hours, no more than 41 hours, no more than 40 hours, no more than 39 hours, no more than 38 hours, no more than 37 hours, no more than 36 hours, no more than 35 hours, no more than 34 hours, no more than 33 hours, no more than 32 hours, no more than 31 hours, no more than 30 hours, no more than 29 hours, no more than 28 hours , no more than 27 hours, no more than 26 hours, no more than 25 hours, no more than 24 hours, no more than 23 hours, no more than 22 hours, no more than 21 hours, no more than 20 hours, no more than 19 hours, no more than 18 hours, no more than 17 hours, no more than 16 hours, no more than 15 hours, no more than 14 hours, no more than 13 hours, no more than 12 hours, no more than 11 hours, no more than 10 hours, no more than 9 hours, no more than 8 hours, no more than 7 hours, no more than 6 hours, no more than 5 hours, no more than 4 hours, no more than 3 hours, no more than 2 hours or no more than 1 hour.

[0192] In some embodiments of the present invention, the time required to prepare the CAR-T cells is no more than 30 minutes, no more than 20 minutes, or no more than 10 minutes.

[0193] In some embodiments of the present invention, "the time required to prepare the CAR-T cells" refers to the time required from the contact of any of the vectors carrying the nucleic acid encoding any of the CARs with inactivated T cells to the administration of the CAR-T cells to the subject.

[0194] In some embodiments of the present invention, the administration comprises intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection or infusion technique.

[0195] In a fifth aspect, the present invention provides a CAR-T cell, which is prepared by any one of the methods for preparing CAR-T cells in the method for in vitro transfection of non-activated T cells provided in the fourth aspect of the present invention.

[0196] In a sixth aspect, the present invention provides a composition comprising the CAR-T cells provided in the fifth aspect of the present invention and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation fluids and other auxiliary components.

[0197] In a seventh aspect, the present invention provides use of the CAR-T cell provided in the fifth aspect of the present invention or the composition provided in the sixth aspect of the present invention in the preparation of a medicament for treating cancer in a subject or killing cancer cells in a subject;

[0198] Preferably, the cancer is selected from at least one of blood cancer and solid cancer;

[0199] More preferably, the blood cancer is at least one selected from acute lymphocytic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma;

[0200] More preferably, the solid cancer is at least one selected from colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

[0201] In some embodiments of the present invention, the blood cancer includes relapsed or refractory blood cancer.

[0202] In an eighth aspect, the present invention provides a method of administering the CAR-T cells provided in the fifth aspect of the present invention or the composition provided in the sixth aspect of the present invention to a subject, wherein the administration occurs within 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 47 hours, 46 hours, 45 hours, 44 hours, 43 hours, 42 hours, 41 hours, 40 hours, 39 hours, 47 hours, 46 hours, 45 hours, 44 hours, 43 hours, 42 hours, 41 hours, 40 hours, 39 hours, 47 hours, 46 hours, 45 hours, 44 hours, 43 hours, 44 hours, 45 hours, 4 ... Within 38 hours, within 37 hours, within 36 hours, within 35 hours, within 34 hours, within 33 hours, within 32 hours, within 31 hours, within 30 hours, within 29 hours, within 28 hours, within 27 hours, within 26 hours, within 25 hours, within 24 hours, within 23 hours, within 22 hours, within 21 hours, within 20 hours, within 19 hours, within 18 hours, within 17 hours, within 16 hours, within 15 hours, within 14 hours, within 13 hours, within 12 hours, within 11 hours, within 10 hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours or within 1 hour.

[0203] In some embodiments of the present invention, the administration occurs within 30 minutes, 20 minutes, or 10 minutes from the date of production of the CAR-T cells.

[0204] In some embodiments of the present invention, "the CAR-T cells are produced" means that after any of the vectors carrying the nucleic acid encoding any of the CARs is contacted with inactivated T cells, the inactivated T cells contain the nucleic acid encoding the CAR molecule and / or express the CAR molecule.

[0205] In some embodiments of the present invention, the administration comprises intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection or infusion technique.

[0206] In a ninth aspect, the present invention provides a method for treating cancer in a subject or killing cancer cells in a subject, the method comprising administering to the subject the CAR-T cell provided by the fifth aspect of the present invention or the composition provided by the sixth aspect of the present invention;

[0207] Preferably, the cancer is selected from at least one of blood cancer and solid cancer;

[0208] More preferably, the blood cancer is at least one selected from acute lymphocytic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma;

[0209] More preferably, the solid cancer is at least one selected from colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

[0210] In some embodiments of the present invention, the blood cancer includes relapsed or refractory blood cancer.

[0211] In some embodiments of the present invention, the administration occurs within 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 47 hours, 46 hours, 45 hours, 44 hours, 43 hours, 42 hours, 41 hours, 40 hours, 39 hours, 38 hours, 37 hours, 36 hours, 35 hours, 34 hours, 33 hours, 32 hours, 31 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour from the date of production of the CAR-T cells.

[0212] In some embodiments of the present invention, the administration occurs within 30 minutes, 20 minutes, or 10 minutes from the date of production of the CAR-T cells.

[0213] In some embodiments of the present invention, the administration comprises intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection or infusion technique.

[0214] The beneficial effects of the present invention include:

[0215] The vector comprising an antibody or antigen-binding fragment thereof and / or a ligand or receptor-binding fragment thereof targeting an endocytic receptor of non-activated T cells and the method for in vitro transfection of non-activated T cells disclosed in the present invention can directly infect non-activated T cells without the need to pre-activate non-activated T cells using T cell activation stimulating products such as anti-CD3 antibodies and anti-CD28 antibodies, without the need to culture the non-activated T cells such as serum starvation, and without the need to expand and culture the prepared CAR-T cells, which can significantly shorten the time required for patients to receive in vitro CAR-T cell therapy.

[0216] When the vector is a lentiviral vector or a retroviral vector, the envelope glycoprotein of the lentiviral vector or retroviral vector may be VSV-G or Cocal-G; the envelope glycoprotein may undergo the first mutation, so that the ability of the envelope glycoprotein to specifically bind to LDL-R is reduced or lost compared to before the first mutation, thereby further improving the targeting of the lentiviral vector or retroviral vector to infect non-activated T cells;

[0217] The envelope glycoprotein may also undergo the second mutation, so that the ability of the envelope glycoprotein to antagonize inactivation by complement is enhanced compared to before the second mutation, or the envelope glycoprotein is not inactivated by complement.

[0218] In this article:

[0219] "T cells": T cells are one of the important white blood cells in the human immune system and play an important role in acquired immune responses. One of the main functions of T cells is immune-mediated cell death, which is mainly performed by two T cell subtypes: CD8 + T cells (Cytotoxic T Cell, cytotoxic T cells) and CD4 + T cells (Helper T Cell, helper T cells).

[0220] In some embodiments of the present invention, the T cells are CD4 + / CD8 - 、CD4 - / CD8 + 、CD4 + / CD8+ 、CD4 - / CD8 - T cells or a combination thereof. In some embodiments of the present invention, CD4 + T cells express CAR and bind to target cells such as CD19 + In some embodiments of the present invention, CD8 + T cells lyse antigen-specific target cells after expressing CAR and binding to target cells.

[0221] In some embodiments of the present invention, in the absence of the step of activating and stimulating non-activated T cells, for example, before contacting with non-activated T cells, the non-activated T cells are not exposed to any activation stimulus that can activate non-activated T cells, such as T cell activation molecules or co-stimulatory molecules, and the non-activated T cells are transfected or transduced with any of the vectors carrying any of the CAR molecule genes provided by the present invention, so that the non-activated T cells express the CAR molecules.

[0222] In conventional technical methods for preparing CAR-T cells in vitro using VSV-G lentiviral vectors or retroviral vectors, exogenous activators containing T cell activation or co-stimulatory molecules (such as DYNABEADS, human T-activating factor CD3 / CD28 (Thermo Fisher Scientific) etc.) are usually required to activate and stimulate inactivated T cells (such as inactivated T cells included in human inactivated PBMCs).

[0223] "T cell activation molecule or co-stimulatory molecule": "T cell activation molecules" (also known as "T cell activation primary signaling molecules") bind to T cell surface proteins and participate in T cell receptor (TCR)-mediated T cell activation (TCR-mediated T cell activation). In some embodiments of the present invention, the T cell activation molecule participates in converting the TCR into an active protein tyrosine kinase (PTK), which can phosphorylate a series of substrates, thereby generating a large number of downstream signals. When these signals are appropriately integrated (together with signals from other co-receptors), they lead to T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27:591-619).

[0224] "T cell co-stimulatory molecules": also known as "T cell activation secondary signaling molecules", bind to other T cell surface receptors and provide additional signals necessary for avoiding anergy and effective T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0225] In some embodiments of the present invention, the T cell activation secondary signaling molecule binds to CD28.

[0226] Although other cell surface receptors (co-stimulatory receptors) can also enhance activation signals through TCR, CD28-mediated co-activation stimulation is more potent than other co-stimulatory receptors (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0227] In some embodiments of the present invention, the T cell activation molecule or co-stimulatory molecule includes one or more of an anti-CD3 antibody or an antigen-binding fragment thereof (such as OKT3, CRIS-7 or I2C), a CD28 ligand (CD28L, such as CD80 and CD86) or a receptor-binding fragment thereof, an anti-CD28 antibody or an antigen-binding fragment thereof, and a 4-1BB ligand (4-1BBL or CD137L) or a receptor-binding fragment thereof.

[0228] “Chimeric Antigen Receptor”: i.e. Chimeric Antigen Receptor (CAR), refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells such as lymphocytes and specifically binds to an antigen, which at least includes (1) an extracellular antigen binding region, such as antibodies such as scFv or their antigen binding fragments; (2) a transmembrane region that anchors the CAR molecule to the cell membrane of the immune effector cell, and (3) an intracellular signal transduction domain; the extracellular structure of CAR may further include a hinge region, and the intracellular structure may further include one or more costimulatory molecules to form a costimulatory signal transduction domain. CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding region. In some embodiments of the present invention, the CAR may be monospecific, bispecific (bi-specific) or multispecific (multi-specific).

[0229] “CAR-T cell”: As used herein, “CAR-T cell” includes T cells expressing any of the CAR molecules and T cells comprising a nucleic acid encoding any of the CAR molecules.

[0230] "Antibody" refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to specifically bind to an antigen. "Antibody" herein encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0231] The "antibody" herein includes a typical "four-chain antibody", which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain from its N-terminus to its C-terminus; and, when the full-length antibody is of the IgE isotype, it optionally further includes a heavy chain constant region CH4 domain; the light chain refers to a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) from its N-terminus to its C-terminus; the heavy chains and the light chains are linked by disulfide bonds to form a "Y"-shaped structure.

[0232] "Antibodies" herein also include antibodies that do not contain light chains, such as heavy-chain antibodies, nanobodies and single-chain antibodies, including heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus Dromedarius), Bactrian camels (Camelus Bactrianus), llamas (Lama Glama), guanacos (Lama Guanicoe) and alpacas (Vicugna Pacos), as well as immunoglobulin new antigen receptors (Ig New Antigen Receptor, IgNAR) found in cartilaginous fish such as sharks.

[0233] The terms "VHH domain" and "Nanoantibody" have the same meaning and are used interchangeably herein. They refer to cloning the variable region of a heavy chain antibody to construct a single-domain antibody consisting of only one heavy chain variable region. This is the smallest antigen-binding fragment with complete function. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0234] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0235] Herein, "antigen-binding fragment" refers to a fragment that does not have the entire structure of an intact antibody and only contains a portion or a partial variant of the intact antibody, wherein the portion or partial variant has the ability to bind to an antigen.

[0236] Illustratively, herein, "antibody or antigen-binding fragment thereof" includes but is not limited to at least one of a full-length antibody, a half antibody, Fab, Fab', F(ab')2, Fv, a VHH domain and a scFv; the Fab' fragment and the F(ab')2 fragment may include a partial Fc fragment; the heavy chain variable region (VH) of the scFv is connected to the light chain variable region (VL) via a linker peptide (Linker); in some embodiments of the present invention, there is no particular limitation on the order in which scFv comprises VH or VL from the N-terminus to the C-terminus, such as VH-Linker-VL or VL-Linker-VH from the N-terminus to the C-terminus; the linker peptide may be selected from a flexible linker peptide.

[0237] "Ligand": In receptor-ligand binding, a ligand is generally a molecule that binds to a site on a receptor to generate a signal, such binding typically resulting in a conformational change in the complex structure, thereby inducing the relevant physiological activity.

[0238] "Receptor binding fragment" refers to a fragment that does not possess the entire structure of a complete ligand but only contains a portion or a partial variant of the complete ligand, wherein the portion or partial variant has the ability to bind to the receptor. For example, "receptor binding fragment" herein includes, but is not limited to, the extracellular domain and variable region of the ligand.

[0239] "Nucleic acid" refers to any compound and / or substance including a polymer containing nucleotides, such as a polynucleotide. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed from 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. "Nucleic acid" can be linear or circular. In addition, "nucleic acid" includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Moreover, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues.

[0240] "Exogenous" refers to any molecule that originates from outside an organism, such as exogenous nucleic acids, etc. In contrast, the term "endogenous" refers to any molecule that originates from within an organism (ie, that is produced naturally by the organism).

[0241] "Envelope glycoprotein" refers to the glycoprotein coated on the outer layer of the virus, which plays an important role in the adsorption and penetration of the virus into host cells, pathogenicity, downregulation of host surface protein expression, and increase in virus packaging and budding.

[0242] "Retrovirus" and "retroviral vector" refer to viruses and retroviral vectors. A retrovirus is a virus that can integrate copies of genes contained in its RNA genome into the genome of the host cell it infects, thereby altering the host cell genome. Retroviruses, including lentiviruses and gamma-retroviruses, are used as vectors to deliver nucleic acid molecules into target cells using methods known in the art.

[0243] "Lentivirus": Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain additional genes with regulatory or structural functions. This increased complexity allows the virus to regulate its life cycle, as it does during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV).

[0244] "Lentiviral vector": also known as Lentiviral Vector, which is produced by multiple attenuation of HIV virulence genes through gene editing, genetic engineering and other technical means. For example, the genes env, vif, vpr, vpu and nef are deleted, making the lentiviral vector biosafe.

[0245] Lentiviral vectors can stably integrate target genes, such as shuttle genes, into the chromosomes of target cells, allowing them to express the delivered shuttle genes long-term, offering significant advantages for gene therapy. Furthermore, they do not transfer viral genes, thus avoiding the problem of generating transduced cells that can be destroyed by cytotoxic T cells. Furthermore, they have a relatively large cloning capacity, sufficient for most anticipated clinical applications.

[0246] Lentiviral vectors and lentiviral vector backbone genomes are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey, et al., (1998) J. Virol. 72:9873-9880; Dull, et al., (1998) J. Virol. 72:8463-8471, U.S. Pat. No. 6,013,516, and U.S. Pat. No. 5,994,136, each of which is herein incorporated by reference in its entirety.

[0247] Lentiviral vectors are generally packaged and prepared by a lentiviral vector system in a packaging cell line. For example, the process and method for packaging and preparing lentiviral vectors are described in Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016, which is incorporated herein by reference in its entirety.

[0248] Commonly used lentiviral vector systems include so-called third-generation lentiviral vector systems. The third-generation lentiviral vector system includes four plasmids. "Transfer plasmid" (Transfer Vector, TV; in some embodiments of the present invention, also referred to as "master plasmid") contains target genes such as the lentiviral vector backbone genome and shuttle genes. The transfer plasmid typically has one or more target gene (shuttle gene) sequences flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is typically designed to render the resulting vector replication ineffective. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious lentiviral particles in the host cell. In addition, the transfer plasmid can be designed to lack 3'LTR, thereby rendering the virus "self-inactivated" (Self-Inactivating, SIN). See Dull, et al., J. Virol. 72: 8463-71 (1998); Miyoshi, et al., J. Virol. 72: 8150-57 (1998).

[0249] Third-generation lentiviral vector systems typically also include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically carries the gene encoding VSV-G, which is operably linked to a promoter, typically the CMV promoter. Third-generation lentiviral vector systems use two packaging plasmids, one encoding the genes gag and pol, and the other encoding the gene rev as a further safety feature, an improvement over the single packaging plasmid of so-called second-generation systems. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0250] The use of lentiviral vector systems relies on a "packaging cell line". Generally speaking, a packaging cell line is a cell line whose cells are capable of producing lentiviral vectors that do not have the ability to self-replicate and can infect target cells when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cells. Exemplarily, a transfection method including a chemically mediated transfection method, a physically mediated transfection method, or a biologically mediated transfection method can be used to introduce the plasmid into the packaging cell line. For example, chemically mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran, or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physically mediated transfection methods include transfection methods such as electroporation.

[0251] "Packaging cell line": Generally speaking, a packaging cell line is a cell line that, when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cell line, produces a lentiviral vector capable of infecting host cells. Various methods for introducing plasmids into cells can be used, including but not limited to transfection using chemical reagents such as calcium phosphate, DEAE-dextran, and PEI, or electroporation.

[0252] The packaging cell line is genetically engineered to improve the immune properties of the lentiviral vector or retroviral vector disclosed herein and / or to facilitate infection of target cells by the lentiviral vector or retroviral vector in other ways; such other ways include, but are not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0253] In some embodiments of the present invention, the packaging cell line includes but is not limited to at least one of the following cell lines: NS0 cell line, Vero cell line, HeLa cell line, COS cell line, CHO cell line, HEK cell line, BHK cell line and MDCKⅡ cell line.

[0254] "Stable integration": also known as "stable transfection" or "stable expression" (Stable Gene Expression), refers to the integration of exogenous nucleic acids into the host cell genome after introduction into the host cell, and their long-term stable expression in the host cell.

[0255] "Subject": As used herein, "subject," "patient," or "individual" includes any animal that exhibits pain, disease, or symptoms that can be treated and / or prevented using the vectors, viruses, compositions, and methods contemplated herein. Subjects include any healthy animal and any animal that suffers from a disease, experiences pain, and / or exhibits any symptoms. Suitable subjects (e.g., patients) include experimental animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs); including non-human primates, and optionally human patients.

[0256] In some embodiments of the present invention, the inactivated T cells include inactivated T cells from one or more of the following sources:

[0257] (a) autologous non-activated T cells of the subject;

[0258] (b) non-activated T cells allogeneic to the subject;

[0259] (c) non-activated T cells allogeneic to the subject;

[0260] (d) non-activated T cells derived from one or more of the following cells: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), and immortalized cells;

[0261] (e) Non-activated T cells isolated from at least one of peripheral blood, umbilical cord blood, lymph nodes, ascites, pleural effusion, thymus, tumor, and bone marrow.

[0262] "Administering": The vectors disclosed herein can be administered by any route, including oral, nasal, intravenous, intraarterial, intramuscular, or intraperitoneal routes. In some embodiments of the present invention, the administration includes intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, orthotopic injection, or sternal injection or infusion techniques.

[0263] “And / or”: should be understood to mean one or two alternatives.

[0264] "About": In this specification, when "about" is used to describe a numerical value "X", "about X" refers to the numerical value "X" itself or a variation within the range of 1%-15% above or below the numerical value "X"; for example, "about 50%" refers to 50% itself or a variation within the range of 1%-15% above or below 50%.

[0265] "Comprising": As used herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, elements, or groups of steps or elements, but not the exclusion of any other steps, elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

[0266] "Embodiments": Reference throughout this specification to "some embodiments," "some embodiments," or combinations thereof means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0267] "Treatment": As used herein, "treatment" includes any beneficial or desired effect associated with treatment. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms.

[0268] "Prevention": As used herein, "prevention" and similar words, such as "preventing," refer to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a condition. As used herein, "prevention" and similar words also include lessening the intensity, effects, symptoms, and / or burden of a disease or condition prior to onset or recurrence.

[0269] "Specific binding": As used herein, the term "specific binding" refers to the binding that occurs between paired molecular species (e.g., a receptor and a ligand). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times greater than nonspecific binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

[0270] "Sequence identity": Generally speaking, "sequence identity" or "sequence homology" refers to the exact correspondence between nucleotides and nucleotides or amino acids and amino acids of two polynucleotides or polypeptide sequences, respectively. Generally, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". Whether it is a nucleic acid or amino acid sequence, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. For example, the advanced BLAST computer program (including version BLAST+2.15.0) purchased from the National Institutes of Health can also be used to compare sequence information to determine the percent identity. In short, the BLAST program defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of shorter symbols in the two sequences. The program can be used to determine the percent identity over the entire length of the protein being compared.

[0271] "Signal peptide": A signal peptide, sometimes also called a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), one of the functions of a signal peptide is to cause cells to transfer proteins, usually to the cell membrane.

[0272] "MOI": Multiplicity of Infection (MOI) refers to the number of virus particles added to each cell during infection. When one million virus particles are added to one million cells, MOI = 1.

[0273] "Operably": A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA of the secreted polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. Generally speaking, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0274] "Autologous": As used herein, the term "autologous" means any material derived from the same individual that is subsequently reintroduced into that individual.

[0275] "Allogeneic": As used herein, "allogeneic" refers to a transplant derived from a different individual of the same species.

[0276] "Transfection": As used herein, the term "transfection," "transformation," or "transduction" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell, packaging cell, or the like. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. This includes the primary subject cell and its progeny.

[0277] "2A peptide": The term "2A peptide" refers to a self-cleaving peptide configured to generate two or more proteins from a single open reading frame, including FT2A peptide, F2A peptide, E2A peptide, T2A peptide and P2A peptide, etc. 2A peptides are 18 to 22 residues long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" can refer to peptides with different amino acid sequences. In the present disclosure, it should be understood that when a lentiviral vector comprises two or more 2A peptides, the 2A peptides may be the same or different from each other. Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012: 199-204.

[0278] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety, to the same extent as if each individual publication, document, or patent not specifically and individually indicated as being incorporated by reference in its entirety was incorporated by reference in its entirety. In the event of a conflict, the present application, including any definitions herein, will control.

[0279] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF THE DRAWINGS

[0280] FIG1 is a schematic diagram of the structure of the lentiviral vector VSVG-A7-19;

[0281] Figure 2: Comparison of the expression abundance of CD5 and CD7 in non-activated PBMCs of Donor-1 and Donor-2;

[0282] FIG3 is a graph showing a comparison of flow cytometry results of the expression efficiency of CAR-19 molecules in the infected PBMCs of each group after the lentiviral vector VSVG-A7-19 and the wild-type VSV-G lentiviral vector were respectively infected into non-activated human PBMCs;

[0283] FIG4 is a comparison of the flow cytometry results of detecting the expression efficiency of CAR-19 molecules in infected PBMCs of each group after the mutant lentiviral vector mVSVG1-A7-19 and the mutant lentiviral vector mVSVG1-A3-19 were respectively infected with human non-activated T cells;

[0284] FIG5 is a flow cytometry result showing the expression efficiency of CAR-19 molecules in non-activated human PBMCs after the lentiviral vector VSVG-A7-19 was infected in vitro for 1 minute, 5 minutes, 10 minutes, and 2 hours, respectively;

[0285] Figure 6: CD19-CAR-T cells prepared after the lentiviral vector VSVG-A7-19 infected non-activated human PBMCs in vitro killed CD19 in mice + The effect of Raji tumor cells;

[0286] Figure 7: To detect the killing of CD33 by CD33-CAR-T cells + Figure 2 shows the detection results of the efficiency of MOLM13 cells;

[0287] Figure 8: To detect the killing of HER2 by the HER2-CAR-T cells + Figure 2 shows the detection results of the efficiency of OVCAR-3 cells;

[0288] Figure 9: To detect the killing of CEA by CEA-CAR-T cells + The test results of the efficiency of T84 cells;

[0289] Figure 10 is a map of the VSVG-A7 envelope plasmid;

[0290] Figure 11: is the plasmid map of the master plasmid-19;

[0291] FIG12 shows the expression efficiency of CAR-19 molecules in non-activated human PBMCs after the lentiviral vector mVSVG2-A7-19 was used to infect non-activated human PBMCs in vitro for 1 minute, 5 minutes, and 15 minutes, respectively.

[0292] Figure 13: The lentiviral vector mVSVG2-A7-19 was used to infect non-activated human PBMCs in vitro for 1 minute, 5 minutes, and 15 minutes, respectively. The CD19-CAR-T cells prepared in each group killed CD19 in vitro. + Figure 2 shows the test results of the killing efficiency of Raij cells;

[0293] Figure 14: The lentiviral vector VSVG-3 / 28 infects human non-activated PBMCs and sorted CD3 + After 1 minute, 5 minutes and 15 minutes, the infected PBMCs and sorted CD3 + The detection results of the expression efficiency of CAR-19 molecules in T cells;

[0294] Figure 15: The lentiviral vector mVSVG2-3 / 28 infects human non-activated PBMCs and sorted CD3 + After 1 minute, 5 minutes and 15 minutes, the infected PBMCs and sorted CD3 +The detection results of the expression efficiency of CAR-19 molecules in T cells;

[0295] Figure 16 shows the results of detecting the expression efficiency of CAR-19 molecules in each group of infected PBMCs after the lentiviral vector CocalG-A7 and the lentiviral vector mCocalG1-A7 infected human non-activated PBMCs in vitro for 1 minute, 5 minutes, and 15 minutes, respectively. DETAILED DESCRIPTION

[0296] The following is a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0297] In the following examples, the experimental methods without specific conditions are based on conventional methods and conditions known in the art, or are selected according to the product specifications. Reagents and raw materials not specified in the present invention are all commercially available.

[0298] Example 1

[0299] Comparison of the expression abundance of CD5 and CD7 on the surface of non-activated T cells.

[0300] Resuscitated 2×10 6 Flow cytometry was performed on frozen non-activated PBMCs from Donor 1 (healthy person) and Donor 2 (healthy person) to detect the expression of CD5 and CD7 in the two groups of PBMCs. The results are shown in Figure 2.

[0301] As shown in Figure 2, the expression abundance of CD7 is higher than that of CD5 in human non-activated PBMCs. Therefore, when vectors containing anti-CD5 or anti-CD7 antibodies on their surface infect human non-activated PBMCs, the endocytosis caused by the specific binding of anti-CD7 antibodies to the endocytic receptor CD7, which is expressed in relatively high abundance on the surface of non-activated T cells, should be stronger than the endocytosis caused by the specific binding of anti-CD5 antibodies to the endocytic receptor CD5, which is expressed in relatively low abundance on the surface of non-activated T cells. Therefore, vectors containing membrane-expressed anti-CD7 antibodies on their surface, such as lentiviral vectors or retroviral vectors, should be more effective in delivering target genes to non-activated T cells than vectors containing membrane-expressed anti-CD5 antibodies on their surface.

[0302] Antibodies used in flow cytometry:

[0303] Anti-CD7 antibody: Brand: BIOLEGEND, Catalog Number: #561604;

[0304] Anti-CD5 antibody: Brand: BIOLEGEND; Product number: #555352.

[0305] Example 2

[0306] Construct a lentiviral vector VSVG-A7-19 targeting non-activated T cells.

[0307] 1. Design of membrane-based expression of anti-CD7 antibodies

[0308] In this embodiment, the membrane-expressing anti-CD7 antibody comprises an anti-CD7 antibody, a CD8α hinge region, and a CD8α transmembrane region. The anti-CD7 antibody is indirectly connected to the CD8α transmembrane region through the CD8α hinge region and is contained in the viral envelope of the lentiviral vector, thereby improving the lentiviral vector's ability to infect CD7. + Cell targeting;

[0309] The structure of the membrane-expressed anti-CD7 antibody from N-terminus to C-terminus is: CD8α signal peptide, heavy chain variable region (VH) of TH-69 (anti-CD7 antibody), connecting peptide (G4SLinker), light chain variable region (VL) of TH-69, CD8α hinge region, and CD8α transmembrane region.

[0310] The nucleic acid encoding the membrane-expressed anti-CD7 antibody is operably linked to the nucleic acid encoding the CD8α signal peptide, and the CD8α signal peptide is located at the N-terminus of the membrane-expressed anti-CD7 antibody;

[0311] (1) The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 8;

[0312] (2) The amino acid sequence of the VH region of TH-69 is shown in SEQ ID NO: 16;

[0313] (3) The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 10;

[0314] (4) The amino acid sequence of the VL region of TH-69 is shown in SEQ ID NO: 17;

[0315] (5) the amino acid sequence of the hinge region of CD8α is shown in SEQ ID NO: 12;

[0316] (6) The amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO: 13.

[0317] 2. Design of chimeric antigen receptor CAR-19 targeting CD19

[0318] A CAR molecule targeting CD19 (CAR-19 molecule) is designed, and the structure of the CAR-19 molecule from N-terminus to C-terminus is: an antigen binding region targeting CD19, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular signaling domain; the antigen binding region is a scFv, FMC63, that specifically binds to CD19, and the heavy chain variable region (VH) of the FMC63 is connected to the light chain variable region (VL) of the FMC63 via the connecting peptide.

[0319] The nucleic acid encoding the CAR-19 molecule is operably linked to the nucleic acid encoding the CD8α signal peptide, and the CD8α signal peptide is located at the N-terminus of the CAR-19 molecule;

[0320] (1) The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 8;

[0321] (2) The amino acid sequence of the VH region of FMC63 is shown in SEQ ID NO: 9, and the amino acid sequence of the VL region of FMC63 is shown in SEQ ID NO: 11; the VH region and the VL region are connected by the connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 10;

[0322] (3) the amino acid sequence of the hinge region of CD8α is shown in SEQ ID NO: 12;

[0323] (4) The amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO: 13;

[0324] (5) the amino acid sequence of the 4-1BB costimulatory signaling domain is shown in SEQ ID NO: 14;

[0325] (6) The amino acid sequence of the intracellular signaling domain of CD3ζ is shown in SEQ ID NO:15.

[0326] 3. Construction of CD7-targeted lentiviral vector VSVG-A7-19

[0327] A lentiviral vector VSVG-A7-19 was constructed. A schematic structural diagram of the lentiviral vector VSVG-A7-19 is shown in Figure 1 . The viral envelope of the lentiviral vector VSVG-A7-19 comprises a membrane-expressed anti-CD7 antibody. Exemplarily, the structure of the membrane-expressed anti-CD7 antibody is as described above. The envelope glycoprotein of the lentiviral vector VSVG-A7-19 is the envelope glycoprotein of the wild-type vesicular stomatitis virus Indiana strain (VSV-G), and the extracellular domain of the wild-type VSV-G comprises the amino acid sequence shown in SEQ ID NO: 1.

[0328] Wild-type VSV-G has difficulty in effectively infecting non-activated T cells by specifically binding to LDL-R; however, non-activated T cells express the endocytosis receptor CD7, so the lentiviral vector VSVG-A7-19, whose viral envelope contains anti-CD7 antibodies, can enter and infect non-activated T cells by specifically binding to the endocytosis receptor CD7, and then through endocytosis, through the membrane fusion ability of its viral envelope glycoprotein VSV-G, it fuses with the endosomal / lysosomal membrane, mediating lysosomal escape, so that the exogenous genes carried by the lentiviral vector VSVG-A7-19, such as the nucleic acid encoding the CAR molecule, are successfully integrated into the genome of non-activated T cells.

[0329] A. Packaging and preparation of lentiviral vector VSVG-A7-19:

[0330] (1) preparing the following four plasmids: an envelope plasmid (VSVG-A7 envelope plasmid) carrying nucleic acid encoding the wild-type VSV-G and nucleic acid encoding the membrane-expressing anti-CD7 antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a main plasmid (main plasmid-19) carrying nucleic acid encoding the CAR-19 molecule, wherein the structure of the CAR-19 molecule is as described above; the VSVG-A7 envelope plasmid and the main plasmid-19 are synthesized by conventional molecular cloning methods;

[0331] The plasmid maps of the VSVG-A7 envelope plasmid and the main plasmid-19 are shown in Figures 10 and 11, respectively.

[0332] The full-length protein of wild-type VSV-G (including the signal peptide) comprises the amino acid sequence shown in SEQ ID NO: 25:

[0333] Wherein, the amino acid sequence shown at positions 1 to 16 of SEQ ID NO: 25:

[0334] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of wild-type VSV-G.

[0335] (2) Packaging and preparing lentiviral vectors: mixing the four plasmids and transfecting the four plasmids into the packaging cell line HEK-293T cell line using PEI reagent. The specific steps are as follows:

[0336] On Day 0, add 199ug of the main plasmid, 4ug of the pMDLg / pRRE packaging plasmid, 2ug of the pRSV-REV packaging plasmid, and 2ug of the VSVG-A7 envelope plasmid to 1mL of Opti-MEM medium. Shake well and add 64uL of PEI reagent. After pipetting evenly, let it stand for 10 minutes and then add it to the culture medium of HEK-293T cells. After 6 hours, the culture medium was renewed. 48 hours after transfection, the culture supernatant was collected and filtered using a 0.45um filter membrane. Centrifuge at 50,000g for 2.5h, discard the supernatant, resuspend in 200uL of F12 medium, and freeze at -80℃.

[0337] Opti-MEM alpha Reduced Serum Medium: Brand: GIBCO, Catalog Number: #SP0272;

[0338] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

[0339] F12 culture medium: Brand: GIBCO, catalog number: #C11330500BT;

[0340] Syringe filter: Brand: SORFA, item number: #622120.

[0341] B. Infected human non-activated PBMCs culture system (MOI=2):

[0342] Day 0, take 4×10 6100 μL of resuscitated human inactivated PBMCs (healthy subjects) were resuspended in 500 μL of normal saline. Methods for resuscitating frozen PBMCs are well known to those skilled in the art. At an MOI of 2, the lentiviral vector VSVG-A7-19 was added to the PBMCs and mixed evenly. After infection at room temperature for 10 minutes, 10 mL of DPBS buffer was added and mixed evenly. The mixture was then centrifuged at 500 g for 3 minutes, the supernatant was discarded, and the PBMCs were resuspended in 1 mL of XVT medium containing 20 ng / mL IL-7 and 20 ng / mL IL-15. In vitro cell culture was performed in an incubator at 37° C. and 5% CO2 (for quality control purposes, not necessary for the rapid preparation of CAR-T cells by infecting inactivated PBMCs with the lentiviral vector VSVG-A7-19). On Day 5, flow cytometry was used to detect the expression of the CAR-19 molecule in the infected PBMCs. The results are shown in the right figure of FIG3 .

[0343] DPBS buffer: Brand: Seville, Product No.: #G4200-500mL;

[0344] XVT culture medium: Brand: IRVINE, Trade name: PRIME-XV T cell CDM, Catalog number: #91154;

[0345] IL-7: Brand: Sino Biological, Trade Name: IL-7 Protein, Human, Recombinant, Catalog Number: #11821-HNAE;

[0346] IL-15: Brand: Sino Biological, Trade Name: IL-15 Protein, Human, Recombinant (His Tag), Catalog Number: #10360-H07E).

[0347] C. Packaging of wild-type VSV-G lentiviral vector for control group:

[0348] (1) Prepare the following four plasmids: pMD2.G (wild-type VSV-G) envelope plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid-19;

[0349] (2) Packaging a control group lentiviral vector: Referring to the packaging method of the above-mentioned lentiviral vector VSVG-A7-19, a control group, i.e., a wild-type VSV-G lentiviral vector, was packaged; the viral envelope of the wild-type VSV-G lentiviral vector did not contain an anti-CD7 antibody or an antigen-binding fragment thereof;

[0350] (3) Infection of PBMCs: Referring to the above-mentioned method of infecting non-activated human PBMCs with the lentiviral vector VSVG-A7-19, the wild-type VSV-G lentiviral vector was used to infect 4×10 6 On Day 5, human non-activated PBMCs were recovered and the expression of the CAR-19 molecule in the infected PBMCs was detected by flow cytometry. The results are shown in the left figure of Figure 3.

[0351] As can be seen from Figure 3, the wild-type VSV-G lentiviral vector whose viral envelope does not contain anti-CD7 antibodies is difficult to effectively infect non-activated T cells, so it is difficult to deliver the nucleic acid encoding the CAR-19 molecule to the genome of non-activated T cells; while the lentiviral vector VSVG-A7-19 whose viral envelope contains anti-CD7 antibodies specifically binds to the endocytic receptor CD7 on the surface of non-activated T cells, and then successfully infects non-activated T cells in human non-activated PBMCs through endocytosis, and integrates the nucleic acid encoding the CAR molecule it carries into the genome of non-activated T cells.

[0352] Flow cytometry antibodies used in the assay:

[0353] Flow cytometry antibody for detecting the CAR-19 molecule: Trade name: PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (0.03% Proclin) DMF Filed, Brand: ACRO, Product Number: #FM3-PY54A2-200 tests.

[0354] Example 3

[0355] Comparison of infection efficiency between lentiviral vectors targeting CD3 and CD7.

[0356] 1. Construction of a CD3-targeted mutant lentiviral vector mVSVG1-A3-19

[0357] A. Construction of membrane-bound anti-CD3 antibody expression:

[0358] A membrane-expressing anti-CD3 antibody was constructed. The structure of the membrane-expressing anti-CD3 antibody from N-terminus to C-terminus was: UCHT1-scFv, CD8α hinge region, and CD8α transmembrane region.

[0359] The amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO: 18; the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO: 12; and the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO: 13.

[0360] The nucleic acid encoding the membrane-expressed anti-CD3 antibody is operably linked to the nucleic acid encoding the CD8α signal peptide; the CD8α signal peptide is located at the N-terminus of the membrane-expressed anti-CD3 antibody.

[0361] B. Prepare the following four plasmids: an envelope plasmid carrying nucleic acid encoding mutant VSV-G1 and nucleic acid encoding the membrane-expressing anti-CD3 antibody (mutant VSVG1-A3 envelope plasmid), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the master plasmid-19; the mutant VSVG1-A3 envelope plasmid is synthesized by conventional molecular cloning methods;

[0362] The extracellular domain of the mutant VSV-G1 comprises the amino acid sequence shown in SEQ ID NO: 7; relative to SEQ ID NO: 1, SEQ ID NO: 7 comprises R354Q, T214N and T352A.

[0363] C. Packaging and preparation of lentiviral vector mVSVG1-A3-19: Referring to the method for packaging lentiviral vector VSVG-A7-19 described in Example 2, the mutant lentiviral vector mVSVG1-A3-19 was packaged.

[0364] 2. Construction of a mutant lentiviral vector mVSVG1-A7-19 targeting CD7

[0365] The following four plasmids were prepared: an envelope plasmid carrying a nucleic acid encoding the mutant VSV-G1 and a nucleic acid encoding a membrane-expressing anti-CD7 antibody as described in Example 2 (mutant VSVG1-A7 envelope plasmid), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the master plasmid-19; the mutant VSVG1-A7 envelope plasmid was synthesized by conventional molecular cloning methods;

[0366] Referring to the packaging method of the lentiviral vector VSVG-A7-19 described in Example 2, the mutant lentiviral vector mVSVG1-A7-19 was packaged.

[0367] 3. Infection of human non-activated PBMCs with lentiviral vectors mVSVG1-A3-19 and mVSVG1-A7-19

[0368] The PBMCs cryopreservation solutions of Donor 1 and Donor 2 (healthy individuals) were taken for resuscitation.

[0369] After recovery, PBMCs from Donor 1 and Donor 2 were divided into three groups (six groups in total), each containing 1×10 6 PBMCs, one of which was a blank control group.

[0370] On Day 0, at an MOI of 5, the mutant lentiviral vector mVSVG1-A7-19 and the mutant lentiviral vector mVSVG1-A3-19 were added to the remaining two groups of PBMCs of Donor 1 and Donor 2, respectively, except the blank control group; 10 minutes after infection at room temperature, 10 mL of DPBS buffer was added to the three groups of PBMCs of Donor 1 and Donor 2, respectively, and mixed, followed by centrifugation at 500 g for 3 minutes, the supernatant was discarded, and the PBMCs were resuspended in 1 mL of XVT medium containing 20 ng / mL IL-7 and 20 ng / mL IL-15, and in vitro cell culture was performed in an incubator at 37°C and a CO2 concentration of 5% (for quality control purposes); on Day 2, the expression of CAR-19 molecules in each group of PBMCs was detected by flow cytometry, and the results are shown in Figure 4.

[0371] As shown in Figure 4, the mutant lentiviral vector mVSVG1-A7-19 has a better infection efficiency in non-activated T cells than the mutant lentiviral vector mVSVG1-A3-19, and the lentiviral vector constructed using membrane-expressing anti-CD7 antibodies has a better infection efficiency in non-activated T cells than the lentiviral vector constructed using membrane-expressing anti-CD3 antibodies.

[0372] Antibody used in flow cytometry: Anti-FMC63, brand: ACROBIOSYSTEM; product number: #FM3-PY54A2-25tests.

[0373] Example 4

[0374] The expression of the CAR-19 molecule is detected when rapidly preparing CAR-T cells.

[0375] On Day 0, resuscitated non-activated PBMCs (healthy subjects) were divided into 4 groups, each containing 4 × 10 6Individual non-activated PBMCs were infected with the lentiviral vector VSVG-A7-19 described in Example 2 at an MOI of 5 for 1 minute, 5 minutes, 10 minutes, and two hours, respectively. 10 mL of DPBS buffer was then added to each group of PBMCs, and the mixture was centrifuged at 500 g for 3 minutes. The supernatant was removed, and 1 mL of XVT medium containing 20 ng / mL IL-7 and 20 ng / mL IL-15 was then added to each group of PBMCs to resuspend the PBMCs. In vitro cell culture was performed in an incubator at 37°C and 5% CO2 (for quality control purposes). On Day 5, flow cytometry was used to detect the expression of the CAR-19 molecule and CD3 in the four groups of non-activated PBMCs to confirm the preparation of CAR-T cells. The test results are shown in Figure 5.

[0376] As shown in Figure 5, the expression of CAR-19 molecules was detected on Day 5 in the four groups of PBMCs infected with the lentiviral vector VSVG-A7-19 for 1 minute, 5 minutes, 10 minutes and two hours, respectively. When the lentiviral vector VSVG-A7-19 infected the non-activated T cells for 1 minute, 5 minutes, 10 minutes or two hours, especially when the infection time was extremely short at 1 minute, 5 minutes or 10 minutes, it specifically bound to the endocytic receptor CD7 on the surface of the non-activated T cells, and then entered and infected the non-activated T cells through endocytosis, integrating the nucleic acid encoding the CAR-19 molecule into the genome of the non-activated T cells, thereby successfully preparing CAR-T cells targeting CD19.

[0377] Antibodies used in flow cytometry:

[0378] Anti-CD3 antibody: Trade name: FITC Mouse Anti-Human CD3; Brand: BIOLEGEND, Catalog Number: #555339.

[0379] Example 5

[0380] Detect the efficiency of CAR-T cells in killing tumor cells Raji cells in mice.

[0381] On Day 0, three groups of mice (4 mice in each group) were injected with 1×10 6 Raji (CD19 + Human B cell lymphoma cells)-Luci tumor cells were then injected into the mouse's peritoneal cavity with 200 μL of 15 mg / mL D-luciferin sodium salt working solution (brand: Yisheng Biological, trade name: D-Luciferin, Sodium Salt, product number: #40901ES10). In vivo imaging showed that the model was successfully established.

[0382] Day 0, take 4×10 7 Human inactivated PBMCs (healthy subjects) after resuscitation were resuspended in 1 mL of normal saline. The lentiviral vector VSVG-A7-19 was added to the PBMCs at an MOI of 1 and mixed evenly. After infection at room temperature for 10 minutes, 10 mL of DPBS buffer was added and mixed evenly. The mixture was then centrifuged at 500 g for 3 minutes, the supernatant was discarded, and 800 μL of DPBS buffer was added to resuspend the mixture and divided equally into 4 portions to obtain infected PBMCs. 1×10 7 The infected PBMCs (Raji+CAR-T cell group) (the time from infection of PBMCs to injection of CAR-T cells into mice was about 30 minutes); control group 1 consisted of mice bearing tumors alone (Raji group), and control group 2 consisted of mice injected with 1×10 7 mice containing resuscitated human non-activated PBMCs (Raji+PBMC group); in vivo imaging detection was performed on each group of mice on Day 0, Day 7 and Day 14, and the results are shown in Figure 6.

[0383] As shown in Figure 6, the CD19-CAR-T cells targeting CD19, which were rapidly prepared by inactivated T cells in inactivated human PBMCs infected with the lentiviral vector VSVG-A7-19, can sustainably and effectively kill tumor cells Raji cells in mice, and the killing effect on Day 7 and Day 14 is significantly better than that of inactivated human PBMCs that are not infected with the lentiviral vector VSVG-A7-19.

[0384] In the above process of preparing CD19-CAR-T cells, the lentiviral vector VSVG-A7-19 specifically binds to the endocytic receptor CD7 on the surface of non-activated T cells, and then infects non-activated T cells through endocytosis to prepare CD19-CAR-T cells.

[0385] Compared to conventional technical solutions for preparing CAR-T cells in vitro, when using the lentiviral vector VSVG-A7-19 whose viral envelope contains anti-CD7 antibodies or antigen-binding fragments thereof to infect inactive T cells in inactive human PBMCs, there is no need to pre-activate and stimulate inactive T cells with activating stimulating antibody products such as anti-CD3 antibodies and anti-CD8 antibodies (such as magnetic beads), nor is there a need to perform cell culture such as serum starvation on the inactive cells. The lentiviral vector VSVG-A7-19 infects inactive PBMCs for only 10 minutes, and the infected PBMCs are then infused into mice and can sustainably kill Raji-Luci tumor cells in the mice.

[0386] It only takes about 30 minutes from the time the lentiviral vector VSVG-A7-19 is used to infect inactive PBMCs to the time the prepared CAR-T cells are infused into mice. Conventional in vitro CAR-T cell preparation processes, starting with the activation and stimulation of inactive T cells, followed by a series of CAR-T cell amplification and culture procedures, take much longer (often taking dozens of days) to infuse the prepared CAR-T cells into subjects.

[0387] The method of using the lentiviral vector VSVG-A7-19 whose viral envelope contains anti-CD7 antibodies or antigen-binding fragments thereof to transfect non-activated T cells in vitro and then prepare CAR-T cells can significantly shorten the time required for subjects to receive CAR-T cell therapy.

[0388] Example 6

[0389] A lentiviral vector VSVG-A7-33 containing a nucleic acid encoding a CAR molecule targeting CD33 (CAR-33 molecule) was constructed.

[0390] 1. Design of CAR-33 molecules

[0391] Referring to the structure of the CAR-19 molecule described in Example 2, a CAR-33 molecule targeting CD33 was designed; the structure of the CAR-33 molecule from N-terminus to C-terminus is: an antigen binding region targeting CD33, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular signaling domain; the antigen binding region comprises an scFv (scFv-33) targeting CD33, and the scFv-33 comprises a heavy chain variable region and a light chain variable region derived from the antibody Gemtuzumab, and the heavy chain variable region (VH) of the scFv-33 is connected to the light chain variable region (VL) of the scFv-33 via the connecting peptide.

[0392] The nucleic acid encoding the CAR-33 molecule is operably linked to the nucleic acid encoding the CD8α signal peptide, and the CD8α signal peptide is located at the N-terminus of the CAR-33 molecule;

[0393] The VH region of the scFv-33 comprises the amino acid sequence shown in SEQ ID NO: 19;

[0394] The VL region of the scFv-33 comprises the amino acid sequence shown in SEQ ID NO: 20.

[0395] 2. Packaging of Lentiviral Vector VSVG-A7-33

[0396] The lentiviral vector VSVG-A7-33 containing a nucleic acid encoding a CAR-33 molecule targeting CD33 was packaged. The specific method was as follows: referring to the method for packaging the lentiviral vector VSVG-A7-19 in Example 2, the VSVG-A7 envelope plasmid, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, and the master plasmid (master plasmid-33) carrying the nucleic acid encoding the CAR-33 molecule were transfected into the packaging cell line HEK-293T cell line using a PEI reagent, and the lentiviral vector VSVG-A7-33 was packaged and collected; the master plasmid-33 was synthesized by conventional molecular cloning methods.

[0397] 3. Infecting non-activated human PBMCs to prepare CD33-targeted CAR-T cells

[0398] Day 0, at MOI=5, the lentiviral vector VSVG-A7-33 was added to 4×10 6 The virus was mixed with non-activated PBMCs (healthy people) at room temperature for 10 minutes. 10 mL of DPBS buffer was added to the infected PBMCs, centrifuged at 500 g for 3 minutes, the supernatant was removed, and 800 μL of DPBS buffer was added to resuspend the virus-infected PBMCs. 3 × 10 5 PBMCs infected with the virus were mixed with 1×10 5 CD33 + MOLM13 cells (human acute myeloid leukemia cells, brand: Punosai, catalog number: #CL-0681) were mixed; the control group was 3×10 5 Non-activated PBMCs from uninfected humans were mixed with 1×10 5 A mixture of MOLM13 cells (effector-target ratio E:T = 3:1);

[0399] On Day 5, flow cytometry was performed to record the expression of CD33 in the mixed cells and to detect the killing efficiency of the rapidly prepared CAR-T cells against MOLM13 cells. The results are shown in Figure 7.

[0400] As shown in Figure 7, the CD33-CAR-T cells prepared by infecting non-activated human PBMCs in vitro with the lentiviral vector VSVG-A7-33 can effectively kill CD33 + MOLM13 cells.

[0401] Example 7

[0402] A lentiviral vector VSVG-A7-HER2 containing a nucleic acid encoding a CAR molecule targeting HER2 (CAR-HER2 molecule) was constructed.

[0403] 1. Designing CAR-HER2 molecules

[0404] Referring to the structure of the CAR-19 molecule described in Example 2, a CAR-HER2 molecule targeting HER2 was designed; the structure of the CAR-HER2 molecule from N-terminus to C-terminus is: an antigen binding region targeting HER2, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular signaling domain; the antigen binding region comprises a scFv targeting HER2 (scFv-HER2), the scFv-HER2 comprises a heavy chain variable region and a light chain variable region derived from the antibody Pertuzumab, and the heavy chain variable region (VH) of the scFv-HER2 is connected to the light chain variable region (VL) of the scFv-HER2 via the connecting peptide.

[0405] The nucleic acid encoding the CAR-HER2 molecule is operably linked to the nucleic acid encoding the CD8α signal peptide, and the CD8α signal peptide is located at the N-terminus of the CAR-HER2 molecule;

[0406] The VH region of the scFv-HER2 comprises the amino acid sequence shown in SEQ ID NO: 21;

[0407] The VL region of the scFv-HER2 comprises the amino acid sequence shown in SEQ ID NO: 22.

[0408] 2. Packaging of Lentiviral Vector VSVG-A7-HER2

[0409] The lentiviral vector VSVG-A7-HER2 containing a nucleic acid encoding a CAR-HER2 molecule targeting HER2 was packaged. The specific method was as follows: referring to the method for packaging the lentiviral vector VSVG-A7-19 in Example 2, the VSVG-A7 envelope plasmid, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, and the main plasmid (main plasmid-HER2) carrying the nucleic acid encoding the CAR-HER2 molecule were transfected into the packaging cell line HEK-293T cell line using a PEI reagent, and the lentiviral vector VSVG-A7-HER2 was packaged and collected; the main plasmid-HER2 was synthesized by conventional molecular cloning methods.

[0410] 3. Infecting non-activated human PBMCs to prepare HER2-targeted CAR-T cells

[0411] Day 0, at MOI=5, the lentiviral vector VSVG-A7-HER2 was added to 4×10 6The virus was mixed with non-activated PBMCs (healthy people) at room temperature for 10 minutes. 10 mL of DPBS buffer was added to the infected PBMCs, centrifuged at 500 g for 3 minutes, the supernatant was removed, and 800 μL of DPBS buffer was added to resuspend the virus-infected PBMCs. 3 × 10 5 PBMCs infected with the virus were mixed with 1×10 5 HER2 + OVCAR-3 cells (human ovarian cancer cells, brand: Punosai, catalog number: #CL-0178) were mixed; the control group was 3×10 5 Non-activated PBMCs from uninfected humans were mixed with 1×10 5 A mixture of 100 OVCAR-3 cells (effector-target ratio E:T = 3:1);

[0412] On Day 5, flow cytometry was performed to record the expression of HER2 in the mixed cells and to detect the killing efficiency of the rapidly prepared CAR-T cells against OVCAR-3 cells. The results are shown in Figure 8.

[0413] As shown in Figure 8, the HER2-CAR-T cells prepared by infecting non-activated human PBMCs in vitro using the lentiviral vector VSVG-A7-HER2 can effectively kill HER2 + OVCAR-3 cells.

[0414] Example 8

[0415] A lentiviral vector VSVG-A7-CEA containing a nucleic acid encoding a CAR molecule (CAR-CEA molecule) targeting CEA (Carcinoembryonic Antigen, "CEA") was constructed.

[0416] 1. Design of CAR-CEA molecules

[0417] Referring to the structure of the CAR-19 molecule described in Example 2, a CAR-CEA molecule targeting CEA was designed; the structure of the CAR-CEA molecule from N-terminus to C-terminus is: an antigen binding region targeting CEA, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular signaling domain; the antigen binding region targeting CEA comprises a scFv targeting CEA (scFv-CEA), and the heavy chain variable region (VH) of the scFv-CEA is connected to the light chain variable region (VL) of the scFv-CEA through the connecting peptide.

[0418] The nucleic acid encoding the CAR-CEA molecule is operably linked to the nucleic acid encoding the CD8α signal peptide, and the CD8α signal peptide is located at the N-terminus of the CAR-CEA molecule;

[0419] The VH region of the scFv-CEA comprises the amino acid sequence shown in SEQ ID NO: 23;

[0420] The VL region of the scFv-CEA comprises the amino acid sequence shown in SEQ ID NO: 24.

[0421] 2. Packaging of Lentiviral Vector VSVG-A7-CEA

[0422] The lentiviral vector VSVG-A7-CEA containing a nucleic acid encoding a CAR-CEA molecule targeting CEA was packaged. The specific method was as follows: referring to the method for packaging the lentiviral vector VSVG-A7-19 in Example 2, the VSVG-A7 envelope plasmid, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, and the master plasmid (master plasmid-CEA) carrying the nucleic acid encoding the CAR-CEA molecule were transfected into the packaging cell line HEK-293T cell line using a PEI reagent, and the lentiviral vector VSVG-A7-CEA was packaged and collected; the master plasmid-CEA was synthesized by conventional molecular cloning methods.

[0423] 3. Infecting non-activated human PBMCs to prepare CEA-targeted CAR-T cells

[0424] Day 0, at MOI=5, the lentiviral vector VSVG-A7-CEA was added to 4×10 6 The virus was mixed with non-activated PBMCs (healthy people) at room temperature for 10 minutes. 10 mL of DPBS buffer was added to the infected PBMCs, centrifuged at 500 g for 3 minutes, the supernatant was removed, and 800 μL of DPBS buffer was added to resuspend the virus-infected PBMCs. 3 × 10 5 PBMCs infected with the virus were mixed with 1×10 5 CEA + T84 cells (human colon adenocarcinoma lung metastasis cells, brand: Punosai, catalog number: #CL-0229) were mixed; the control group was 3×10 5 Non-activated PBMCs from uninfected humans were mixed with 1×10 5 A mixture of 10 T84 cells (effector-target ratio E:T=3:1);

[0425] On Day 5, flow cytometry was performed to record the expression of CEA in the mixed cells and to detect the killing efficiency of the rapidly prepared CAR-T cells against T84 cells. The results are shown in Figure 9.

[0426] As shown in Figure 9, the CEA-CAR-T cells prepared by infecting non-activated human PBMCs in vitro using the lentiviral vector VSVG-A7-CEA can effectively kill CEA + T84 cells.

[0427] Example 9

[0428] 1. Packaging of Lentiviral Vector mVSVG2-A7-19

[0429] The following four plasmids were prepared: an envelope plasmid carrying a nucleic acid encoding a mutant VSV-G2 and a nucleic acid encoding a membrane-expressing anti-CD7 antibody as described in Example 2 (a mutant VSVG2-A7 envelope plasmid), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the master plasmid-19; the mutant VSVG2-A7 envelope plasmid was synthesized by conventional molecular cloning methods;

[0430] The extracellular domain of the mutant VSV-G2 comprises the amino acid sequence shown in SEQ ID NO: 3; relative to SEQ ID NO: 1, SEQ ID NO: 3 comprises a K47 deletion.

[0431] Referring to the packaging method of the lentiviral vector VSVG-A7-19 described in Example 2, the mutant lentiviral vector mVSVG2-A7-19 was packaged.

[0432] 2. Infection of Non-activated Human PBMCs

[0433] Referring to the method for rapid infection of non-activated human PBMCs in Example 4, on Day 0, at an MOI of 5, the lentiviral vector mVSVG2-A7-19 was added to three groups of 4×10 6 The cells were mixed with non-activated PBMCs (healthy subjects) of individuals / groups and infected at room temperature for 1 minute, 5 minutes, or 15 minutes. On Day 5, flow cytometry was performed to record the expression of the CAR-19 molecule in the virus-infected PBMCs of each group. The test results are shown in FIG12 .

[0434] As shown in Figure 12, the lentiviral vector mVSVG2-A7-19 can still enter and infect human non-activated T cells through the endocytosis triggered by the anti-CD7 antibody contained in its viral envelope binding to the endocytic receptor CD7 on the surface of non-activated T cells after infecting non-activated PBMCs for only 1 minute, 5 minutes or 15 minutes, thereby efficiently delivering the CAR-19 molecule gene.

[0435] 3. Rapid preparation of CAR-T cells to kill target cells in vitro

[0436] Day 0, at MOI=5, the lentiviral vector mVSVG2-A7-19 was added into three groups of 4×10 6 The cells were mixed with non-activated PBMCs (healthy people) of each group and infected at room temperature for 1 minute, 5 minutes or 15 minutes. 10 mL of DPBS buffer was added to each group of PBMCs, and the cells were centrifuged at 500 g for 3 minutes. The supernatant was removed and 800 μL of DPBS buffer was added to resuspend the virus-infected PBMCs. 3 × 10 5 According to the effect-target ratio E:T=3:1, 1×10 5 CD19 + The control group was 3×10 5 Non-activated PBMCs from uninfected humans were mixed with 1×10 5 A mixture of 10 Raji cells (effector-target ratio E:T=3:1);

[0437] On Day 5, flow cytometry was performed to record the expression of CD19 in the mixed cells of each group and to detect the killing efficiency of each group of rapidly prepared CAR-T cells against Raji cells. The results are shown in Figure 13.

[0438] As shown in Figure 13, the lentiviral vector mVSVG2-A7-19 was used to infect human non-activated PBMCs in vitro for only 1 minute, 5 minutes or 15 minutes. The CAR-T cells of each group prepared rapidly could effectively kill CD19. + Raji cells.

[0439] Example 10

[0440] The lentiviral vectors VSVG-3 / 28 and mVSVG2-3 / 28 were packaged with anti-CD3 and anti-CD28 antibodies in the viral envelope;

[0441] The viral envelope of the lentiviral vector VSVG-3 / 28 comprises anti-CD3 antibodies, anti-CD28 antibodies and wild-type VSV-G;

[0442] The viral envelope of the lentiviral vector mVSVG2-3 / 28 contains anti-CD3 antibodies, anti-CD28 antibodies and the mutant VSV-G2.

[0443] Non-activated T cells express endocytic receptors CD3 and CD28. Therefore, lentiviral vectors whose viral envelope contains anti-CD3 antibodies and anti-CD28 antibodies can enter and infect non-activated T cells through endocytosis triggered by binding to CD3 and CD28 on the surface of non-activated T cells, thereby delivering CAR molecule genes.

[0444] 1. Design of membrane-bound anti-CD3 antibody × anti-CD28 antibody structure

[0445] Referring to the membrane-expressed anti-CD7 antibody designed in Example 2, a membrane-expressed anti-CD3 antibody × anti-CD28 antibody structure (membrane-expressed CD3 × CD28 dual antibody) was designed.

[0446] In this embodiment, the nucleic acids encoding the membrane-expressing CD3×CD28 dual antibody are as follows from the 5' end to the 3' end: nucleic acid encoding CD8α signal peptide, nucleic acid encoding anti-CD3 antibody (the UCHT1-scFv), nucleic acid encoding CD8α hinge region, nucleic acid encoding CD8α transmembrane region, nucleic acid encoding FT2A peptide, nucleic acid encoding CD8α signal peptide, nucleic acid encoding anti-CD28 antibody (scFv), nucleic acid encoding CD8α hinge region, nucleic acid encoding CD8α transmembrane region;

[0447] (1) The amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 26;

[0448] (2) The amino acid sequence of the anti-CD28 antibody (scFv) 15E8 is shown in SEQ ID NO: 27.

[0449] 2. Packaging of lentiviral vector VSVG-3 / 28 and lentiviral vector mVSVG2-3 / 28

[0450] Prepare the following four plasmids: mutant VSVG2-3 / 28 envelope plasmid or VSVG-3 / 28 envelope plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid-19;

[0451] The mutant VSVG2-3 / 28 envelope plasmid carries the nucleic acid encoding the mutant VSV-G2 and the nucleic acid encoding the membrane-type CD3×CD28 dual antibody;

[0452] The VSVG-3 / 28 envelope plasmid carries the nucleic acid encoding the wild-type VSV-G and the nucleic acid encoding the membrane-expressing CD3×CD28 dual antibody;

[0453] The mutant VSVG2-3 / 28 envelope plasmid or VSVG-3 / 28 envelope plasmid is synthesized by conventional molecular cloning methods;

[0454] Referring to the packaging method of the lentiviral vector VSVG-A7-19 described in Example 2, the lentiviral vector VSVG-3 / 28 and the lentiviral vector mVSVG2-3 / 28 were packaged.

[0455] 3. Lentiviral vectors infect CD3+ T cells and human non-activated PBMCs

[0456] Isolation of CD3+ T cells: CD3 MicroBeads, human-lyophilized (brand: MILTENYI BIOTEC, catalog number: #130-097-043) were used to isolate CD3+ T cells from non-activated PBMCs of healthy individuals. + T cells (for the sorting method, refer to the user manual of CD3 MicroBeads, human-lyophilized) (https: / / static.miltenyibiotec.com / asset / 150655405641 / document_crlhfrrfc57ij2fda27q9l4e04?content-disposition=inline).

[0457] Referring to the method for rapid infection of human non-activated PBMCs in Example 4, on Day 0, at an MOI of 5, the lentiviral vector VSVG-3 / 28 or lentiviral vector mVSVG2-3 / 28 was added to three groups of human non-activated PBMCs and three groups of selected CD3 + The T cells were mixed and infected at room temperature for 1 minute, 5 minutes or 15 minutes respectively; on Day 5, flow cytometry was performed to record the number of PBMCs and sorted CD3 + The expression of the CAR-19 molecule in the T cells.

[0458] The lentiviral vector VSVG-3 / 28 infects non-activated human PBMCs and sorted CD3 + The results of the detection of the infection efficiency of T cells for 1 minute, 5 minutes or 15 minutes are shown in the left and right figures of Figure 14 respectively;

[0459] As shown in Figure 14, the lentiviral vector VSVG-3 / 28 was effective in infecting non-activated human PBMCs and sorted CD3 + The T cells were able to efficiently infect human non-activated T cells and sorted CD3 T cells in just 1 minute, 5 minutes or 15 minutes. + T cells, successfully delivering the CAR-19 molecule gene.

[0460] The lentiviral vector mVSVG2-3 / 28 infected human non-activated PBMCs and sorted CD3+ The results of the detection of the infection efficiency of T cells for 1 minute, 5 minutes or 15 minutes are shown in the left and right figures of Figure 15 respectively;

[0461] As shown in Figure 15, the lentiviral vector mVSVG2-3 / 28 was effective in infecting non-activated human PBMCs or sorted CD3 + The T cells were able to efficiently infect human non-activated T cells and sorted CD3 T cells in just 1 minute, 5 minutes or 15 minutes. + T cells, successfully delivering the CAR-19 molecule gene.

[0462] Example 11

[0463] The envelope glycoprotein (Cocal-G) of the wild-type vesicular stomatitis virus Cocal strain or the mutant Cocal-G1 is used to construct the viral envelope of the lentiviral vector, and an anti-CD7 antibody is simultaneously constructed on the viral envelope of the lentiviral vector.

[0464] The extracellular domain of the wild-type Cocal-G comprises the amino acid sequence shown in SEQ ID NO: 2; the extracellular domain of the mutant Cocal-G1 comprises the amino acid sequence shown in SEQ ID NO: 28; relative to SEQ ID NO: 2, SEQ ID NO: 28 comprises a K47 deletion;

[0465] The sequence of the full-length protein of wild-type Cocal-G (including the signal peptide) is shown in SEQ ID NO: 29;

[0466] Wherein, the sequence shown in positions 1 to 17 of SEQ ID NO: 29:

[0467] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of wild-type Cocal-G.

[0468] 1. Packaging of lentiviral vector CocalG-A7 or lentiviral vector mCocalG1-A7

[0469] Prepare the following four plasmids: CocalG-A7 envelope plasmid or mutant CocalG1-A7 envelope plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid-19;

[0470] The CocalG-A7 envelope plasmid carries the nucleic acid encoding the wild-type Cocal-G and the nucleic acid encoding the membrane-expressing anti-CD7 antibody;

[0471] The mutant CocalG1-A7 envelope plasmid carries the nucleic acid encoding the mutant Cocal-G1 and the nucleic acid encoding the membrane-expressing anti-CD7 antibody;

[0472] The CocalG-A7 envelope plasmid or mutant CocalG1-A7 envelope plasmid is synthesized by conventional molecular cloning methods;

[0473] Referring to the packaging method of the lentiviral vector VSVG-A7-19 described in Example 2, the lentiviral vector CocalG-A7 or the lentiviral vector mCocalG1-A7 was packaged.

[0474] 2. Infection of Non-activated Human PBMCs

[0475] Referring to the method for rapid infection of human non-activated PBMCs in Example 4, the lentiviral vector CocalG-A7 or lentiviral vector mCocalG1-A7 was added to three groups of human non-activated PBMCs at an MOI of 5, mixed evenly, and infected at room temperature for 1 minute, 5 minutes, or 15 minutes, respectively;

[0476] On Day 5, flow cytometry was performed to record the expression of the CAR-19 molecule in PBMCs of each group. The results are shown in FIG16 ;

[0477] As shown in Figure 16, when the lentiviral vector CocalG-A7 or the lentiviral vector mCocalG1-A7 infects human non-activated PBMCs for only 1 minute, 5 minutes or 15 minutes respectively, it can still enter and infect human non-activated T cells through the endocytosis caused by the anti-CD7 antibody contained in its viral envelope binding to the endocytic receptor CD7 on the surface of non-activated T cells, thereby efficiently delivering the CAR-19 molecule gene.

Claims

1. A carrier, characterized in that The surface of the carrier comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to endocytic receptors of non-activated T cells; the endocytic receptor is not LDL-R.

2. The carrier according to claim 1, characterized in that The endocytic receptor of the non-activated T cells is CD7.

3. The carrier according to claim 1, characterized in that The endocytic receptor of the non-activated T cells is CD3.

4. The carrier according to claim 1, characterized in that The endocytic receptors of the non-activated T cells are CD3 and CD28.

5. The carrier according to any one of claims 1 to 4, characterized in that The vector is selected from the following vectors: lipid nanoparticles and viruses; the virus is a virus with a viral envelope; preferably, the virus with a viral envelope is a lentiviral vector or a retroviral vector.

6. The carrier according to claim 5, characterized in that The virus with a viral envelope is a lentiviral vector or a retroviral vector, and the envelope glycoprotein of the lentiviral vector or the retroviral vector is selected from the following envelope glycoproteins and variants thereof: envelope glycoproteins of vesicular stomatitis virus strains and variants thereof, envelope glycoproteins of baboon endogenous retrovirus BaEV and variants thereof, envelope glycoproteins of feline endogenous retrovirus RD114 and variants thereof, and envelope glycoproteins of gibbon ape leukemia virus GALV and variants thereof; The envelope glycoprotein of the vesicular stomatitis virus strain and its variants are selected from the following envelope glycoproteins and their variants: the envelope glycoprotein of the vesicular stomatitis virus Indiana strain and its variants, the envelope glycoprotein of the vesicular stomatitis virus Cocal strain and its variants, the envelope glycoprotein of the vesicular stomatitis virus Indiana strain and its variants, The envelope glycoprotein of Maraba strain of vesicular stomatitis virus and its variants, the envelope glycoprotein of Morreton strain of vesicular stomatitis virus and its variants, the envelope glycoprotein of Alagoas strain of vesicular stomatitis virus and its variants, the envelope glycoprotein of New The envelope glycoproteins of Jersey strain and variants thereof, the envelope glycoproteins of Carajas strain and variants thereof, the envelope glycoproteins of Chandipura strain and variants thereof, the envelope glycoproteins of Eptesicus strain and variants thereof, the envelope glycoproteins of Isfahan strain and variants thereof, the envelope glycoproteins of Jurona strain and variants thereof, the envelope glycoproteins of Malpais strain and variants thereof, the envelope glycoproteins of Perinet strain and variants thereof, the envelope glycoproteins of Piry strain and variants thereof, the envelope glycoproteins of Radi strain and variants thereof, the envelope glycoproteins of Rhinolopus strain and variants thereof, and the envelope glycoproteins of Yug Bogdanovac strain and variants thereof.

7. The carrier according to claim 6, characterized in that The envelope glycoprotein undergoes a first mutation, which weakens or loses the ability of the envelope glycoprotein to specifically recognize a receptor relative to before the first mutation.

8. The carrier according to claim 7, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus or a variant thereof, and the receptor is a low-density lipoprotein receptor LDL-R; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 1; (b) After optimal global alignment with SEQ ID NO: 1, the position corresponding to SEQ ID NO:1: substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; More preferably, the first mutation comprises that the amino acid sequence comprises at least one of the following Amino acid mutations: substitution of K47, deletion of K47, substitution of R354; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: the 47th amino acid is replaced by lysine K to glutamine Q, the 354th amino acid is replaced by arginine R to glutamine Q, and the 47th amino acid lysine is deleted; Most preferably, the first mutation comprises a mutation in which the amino acid sequence comprises the following amino acid: a deletion of K47.

9. The carrier according to claim 7, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the receptor is a low-density lipoprotein receptor LDL-R; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:2; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO:2, substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO:2; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: substitution of K47, deletion of K47, substitution of R354; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: the 47th amino acid is replaced by lysine K to glutamine Q, the 354th amino acid is replaced by arginine R to glutamine Q, and the 47th amino acid lysine is deleted; Most preferably, the first mutation comprises a mutation in which the amino acid sequence comprises the following amino acid: a deletion of K47.

10. The carrier according to any one of claims 6 to 9, characterized in that The envelope glycoprotein undergoes a second mutation, which enhances the ability of the envelope glycoprotein to antagonize inactivation by complement relative to that before the second mutation or prevents the envelope glycoprotein from being inactivated by complement.

11. The carrier according to claim 10, characterized in that The envelope glycoprotein is an envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus or a variant thereof; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1; Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 1; (b) after optimal global alignment with SEQ ID NO: 1, is located at the 214th amino acid corresponding to SEQ ID NO: 1; (c) amino acid position 352 of SEQ ID NO: 1; (d) after optimal global alignment with SEQ ID NO: 1, located at the amino acid position equivalent to 352 of SEQ ID NO: 1; (e) amino acid position 50 of SEQ ID NO: 1; (f) after optimal global alignment with SEQ ID NO: 1, is located at the 50th amino acid corresponding to SEQ ID NO: 1; (g) amino acid position 146 of SEQ ID NO: 1; (h) after optimal global alignment with SEQ ID NO: 1, is located at the 146th amino acid corresponding to SEQ ID NO: 1; More preferably, the amino acid mutation includes deletion, insertion or substitution of amino acids; Still more preferably, the amino acid sequence comprises at least one of the following amino acid substitutions: (a) amino acid position 214 of SEQ ID NO: 1; (b) After optimal global alignment with SEQ ID NO: 1, the position corresponding to SEQ ID NO: 1 The 214th amino acid of (c) amino acid position 352 of SEQ ID NO: 1; (d) after optimal global alignment with SEQ ID NO: 1, located at the amino acid position equivalent to 352 of SEQ ID NO: 1; (e) amino acid position 50 of SEQ ID NO: 1; (f) after optimal global alignment with SEQ ID NO: 1, is located at the 50th amino acid corresponding to SEQ ID NO: 1; (g) amino acid position 146 of SEQ ID NO: 1; (h) After optimal global alignment with SEQ ID NO: 1, it is located at the 146th amino acid corresponding to SEQ ID NO:

1.

12. The carrier according to claim 11, characterized in that The second mutation includes that the amino acid sequence contains one or more of the following site mutations: substitution of T214, substitution of T352, substitution of K50, substitution of S146; Preferably, the amino acid sequence comprises one or more of the following site mutations: amino acid 214 is replaced by threonine T to asparagine N, amino acid 352 is replaced by threonine T to alanine A, amino acid 50 is replaced by lysine K to threonine T, and amino acid 146 is replaced by serine S to threonine T; Preferably, the amino acid sequence comprises any combination of the following site mutations: (a) Replacement of T214 and T352; (b) Replacement of T214, T352, K50 and S146; More preferably, the amino acid sequence comprises a combination of any of the following site mutations: (a) The amino acid at position 214 was replaced by threonine T to asparagine N and the amino acid at position 352 The amino acid was replaced by threonine T to alanine A; (b) The amino acid at position 214 is replaced by threonine T to asparagine N, the amino acid at position 352 is replaced by threonine T to alanine A, the amino acid at position 50 is replaced by lysine K to threonine T, and the amino acid at position 146 is replaced by serine S to threonine T.

13. The carrier according to claim 10, characterized in that The envelope glycoprotein is an envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:2; Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 2, is located at the 214th amino acid corresponding to SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 2, is located at the amino acid position corresponding to SEQ ID NO: 2 at position 352; (e) amino acid position 50 of SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 2, is located at the 50th amino acid corresponding to SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 2; (h) after optimal global alignment with SEQ ID NO: 2, is located at the amino acid position corresponding to 146 of SEQ ID NO: 2; More preferably, the amino acid mutation includes deletion, insertion or substitution of amino acids; Still more preferably, the amino acid sequence comprises at least one of the following amino acid substitutions: (a) amino acid position 214 of SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 2, is located at the 214th amino acid corresponding to SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 2, is located at the amino acid position corresponding to SEQ ID NO: 2 at position 352; (e) amino acid position 50 of SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 2, is located at the 50th amino acid corresponding to SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 2; (h) After optimal global alignment with SEQ ID NO: 2, it is located at the 146th amino acid corresponding to SEQ ID NO:

2.

14. The carrier according to claim 13, characterized in that The second mutation includes that the amino acid sequence contains one or more of the following site mutations: substitution of K214, substitution of T352, substitution of K50, substitution of S146; Preferably, the amino acid sequence comprises one or more of the following site mutations: the 214th amino acid is replaced by lysine K to asparagine N, the 352nd amino acid is replaced by threonine T The amino acid at position 50 was replaced by alanine A, the amino acid at position 50 was replaced by lysine K to threonine T, and the amino acid at position 146 was replaced by serine S to threonine T; Preferably, the amino acid sequence comprises any combination of the following site mutations: (a) Replacement of K214 and T352; (b) Replacement of K214, T352, K50 and S146; More preferably, the amino acid sequence comprises a combination of any of the following site mutations: (a) amino acid 214 is replaced by lysine K to asparagine N and amino acid 352 is replaced by threonine T to alanine A; (b) The amino acid at position 214 is replaced by lysine K to asparagine N, the amino acid at position 352 is replaced by threonine T to alanine A, the amino acid at position 50 is replaced by lysine K to threonine T, and the amino acid at position 146 is replaced by serine S to threonine T.

15. The carrier according to any one of claims 1 to 14, characterized in that The antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof is directly or indirectly connected to the transmembrane region and is contained on the surface of the carrier; Preferably, the transmembrane region is selected from the transmembrane regions of the following proteins: CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A , CD79B, CD80, CD86, CD95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L) , CD200R, CD223(LAG3), CD270(HVEM), CD272(BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD- 1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70; More preferably, the transmembrane region includes the transmembrane region of CD8α; Preferably, the antibody or antigen-binding fragment thereof and / or the ligand or receptor-binding fragment thereof is indirectly connected to the transmembrane region via a linker domain; More preferably, the linking domain is selected from: (i) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge region; (ii) a hinge region selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (iii) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and (iv) a stem region of a type II C-lectin, wherein the type II C-lectin is selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the connecting domain includes the hinge region of CD8α.

16. The vector according to any one of claims 1 to 15, characterized in that The vector carries one or more exogenous substances, and the exogenous substances are nucleic acids.

17. The carrier according to claim 16, characterized in that The nucleic acid encodes a chimeric antigen receptor; preferably, the chimeric antigen receptor comprises an antigen binding region, a transmembrane region and an intracellular signaling domain; More preferably, the antigen binding region can bind to an antigen selected from the group consisting of: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.

2. Prostase, PAP, ELF2M, Ephrin B2, IGF-Ⅰ receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2 ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3,. PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, and NKG2D; More preferably, the chimeric antigen receptor further comprises a hinge region and a co-stimulatory signaling domain consisting of one or more co-stimulatory molecules.

18. Use of the carrier according to any one of claims 1 to 17 in the preparation of a drug for preventing and / or treating a disease; Preferably, the disease is selected from at least one of blood cancer and solid cancer; More preferably, the blood cancer is selected from at least one of acute lymphoblastic leukemia (Acute Lymphoblastic Leukemia, "ALL"), diffuse large B-cell lymphoma (Diffuse Large B-cell Lymphoma, "DLBCL"), primary mediastinal large B-cell lymphoma (Primary Mediastinal B-cell Lymphoma, "PMBCL"), chronic lymphocytic leukemia (Chronic Lymphocytic Leukemia, "CLL"), multiple myeloma (Multiple Myeloma, "MM"), Hodgkin Lymphoma (Hodgkin Lymphoma, "HL") and non-Hodgkin Lymphoma (Non-Hodgkin Lymphoma, "NHL"); More preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

19. A composition, characterized in that The composition comprises the carrier according to any one of claims 1 to 17 and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation solutions and other auxiliary components.

20. A method for in vitro transfection of non-activated T cells, characterized in that: Contacting non-activated T cells with the vector according to claim 16 or 17; Preferably, the contacting occurs outside the body of a subject, and the subject is an individual to whom the inactivated T cells transfected by the in vitro transfection method of inactivated T cells are administered; More preferably, the inactivated T cells are selected from at least one of the following sources: (a) the subject's own non-activated T cells; (b) non-activated T cells from the subject's allogeneic individual; (c) non-activated T cells derived from iPSCs (induced pluripotent stem cells); Still further preferably, the non-activated T cells are contained in the peripheral blood or umbilical cord blood of the subject and / or an individual allogeneic to the subject; Still further preferably, the non-activated T cells are contained in PBMCs of the subject and / or an individual allogeneic to the subject; Even more preferably, the non-activated T cells are isolated from PBMCs of the subject and / or an individual allogeneic to the subject.

21. The method according to claim 20, characterized in that The non-activated T cells are not subjected to in vitro cell culture.

22. The method according to claim 20 or 21, characterized in that The non-activated T cells were not serum starved.

23. The method according to any one of claims 20 to 22, characterized in that The non-activated T cells are not activated and / or expanded by T cell activation molecules or co-stimulatory molecules; Preferably, the T cell activation molecule or co-stimulatory molecule is selected from one or more of an anti-CD3 antibody or an antigen-binding fragment thereof, an antibody or an antigen-binding fragment thereof that specifically binds to CD28, a ligand or a receptor-binding fragment thereof that specifically binds to CD28, and a ligand or a receptor-binding fragment thereof that specifically binds to 4-1BB.

24. The method according to any one of claims 20 to 23, characterized in that The vector is in contact with the non-activated T cells for no more than 2 days, no more than 47 hours, no more than 46 hours, no more than 45 hours, no more than 44 hours, no more than 43 hours, no more than 42 hours, no more than 41 hours, no more than 40 hours, no more than 39 hours, no more than 38 hours, no more than 37 hours, no more than 36 hours, no more than 35 hours, no more than 34 hours, no more than 33 hours, no more than 32 hours, no more than 31 hours, no more than 30 hours, no more than 29 hours, no more than 28 hours, no more than 27 hours, no more than 26 hours, no more than 25 hours, no more than 24 hours, no more than 23 hours, no more than 22 hours, no more than 21 hours, no more than 20 hours, no more than 19 hours, no more than 18 hours, no more than 17 hours , no more than 16 hours, no more than 15 hours, no more than 14 hours, no more than 13 hours, no more than 12 hours, no more than 11 hours, no more than 10 hours, no more than 9 hours, no more than 8 hours, no more than 7 hours, no more than 6 hours, no more than 5 hours, no more than 4 hours, no more than 3 hours, no more than 2 hours, no more than 1 hour, no more than 59 minutes, no more than 58 minutes, no more than 57 minutes, no more than 56 minutes, no more than 55 minutes, no more than 54 minutes, no more than 53 minutes, no more than 52 minutes, no more than 51 minutes, no more than 50 minutes, no more than 49 minutes, no more than 48 minutes, no more than 47 minutes, no more than 46 minutes, no more than 45 minutes, no more than 44 minutes, no more than 43 minutes, no more than 42 minutes, no more than 41 minutes, no more than 40 minutes, no more than 39 minutes, no more than 38 minutes, no more than 37 minutes, no more than 36 minutes, no more than 35 minutes, no more than 34 minutes, no more than 33 minutes, no more than 32 minutes, no more than 31 minutes, no more than 30 minutes, no more than 29 minutes, no more than 28 minutes, no more than 27 minutes, no more than 26 minutes, no more than 25 minutes, no more than 24 minutes, no more than 23 minutes, no more than 22 minutes, no more than 21 minutes, no more than 20 minutes, no more than 19 minutes, no more than 18 minutes, no more than 17 minutes, no more than 16 minutes, no more than 15 minutes, no more than 14 minutes, no more than 13 minutes, no more than 12 minutes, no more than 11 minutes, no more than 10 minutes, no more than 9 minutes, no more than 8 minutes, no more than 7 minutes, no more than 6 minutes, no more than 5 minutes, no more than 4 minutes, no more than 3 minutes, no more than 2 minutes, no more than 1 minute, no more than 50 seconds, no more than 40 seconds, no more than 30 seconds, no more than 20 seconds or no more than 10 seconds.

25. The method according to any one of claims 20 to 24, characterized in that The vector according to claim 17 is contacted with the non-activated T cells to transfect the non-activated T cells to prepare CAR-T cells.

26. The method according to claim 25, characterized in that The time required to prepare the CAR-T cells is no more than 14 days, no more than 13 days, no more than 12 days, no more than 11 days, no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, no more than 47 hours, no more than 46 hours, no more than 45 hours, no more than 44 hours, no more than 43 hours, no more than 42 hours, no more than 41 hours, no more than 40 hours, no more than 39 hours, no more than 38 hours, no more than 37 hours, no more than 36 hours, no more than 35 hours, no more than 34 hours, no more than 33 hours, no more than 32 hours, no more than 31 hours no more than 30 hours, no more than 29 hours, no more than 28 hours, no more than 27 hours, no more than 26 hours, no more than 25 hours, no more than 24 hours, no more than 23 hours, no more than 22 hours, no more than 21 hours, no more than 20 hours, no more than 19 hours, no more than 18 hours, no more than 17 hours, no more than 16 hours, no more than 15 hours, no more than 14 hours, no more than 13 hours, no more than 12 hours, no more than 11 hours, no more than 10 hours, no more than 9 hours, no more than 8 hours, no more than 7 hours, no more than 6 hours, no more than 5 hours, no more than 4 hours, no more than 3 hours, no more than 2 hours or no more than 1 hour.

27. A CAR-T cell, characterized in that: The CAR-T cells are prepared by the method of claim 25 or 26.

28. A composition, characterized in that The composition comprises the CAR-T cells of claim 27 and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation fluids and other auxiliary components.

29. Use of the CAR-T cell of claim 27 or the composition of claim 28 in the preparation of a drug for treating cancer in a subject or killing cancer cells in a subject; Preferably, the cancer is selected from at least one of a blood cancer and a solid cancer; More preferably, the blood cancer is selected from at least one of acute lymphocytic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma; More preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

30. A method of administering the CAR-T cell of claim 27 or the composition of claim 28 to a subject, characterized in that: The administration occurs within 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, within 47 hours, within 46 hours, within 45 hours, within 44 hours, within 43 hours, within 42 hours, within 41 hours, within 40 hours, within 39 hours, within 38 hours, within 37 hours, within 36 hours, within 35 hours, within 34 hours, within 33 hours, within 32 hours, within 31 hours, within 30 hours, within 29 hours, within 28 hours, within 27 hours, within 26 hours, within 25 hours, within 24 hours, within 23 hours, within 22 hours, within 21 hours, within 20 hours, within 19 hours, within 18 hours, within 17 hours, within 16 hours, within 15 hours, within 14 hours, within 13 hours, within 12 hours, within 11 hours, within 10 hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours or within 1 hour from the date of production of the CAR-T cells.

31. A method for treating cancer in a subject or killing cancer cells in a subject, characterized in that: Administering the CAR-T cell of claim 27 or the composition of claim 28 to the subject; Preferably, the cancer is selected from at least one of a blood cancer and a solid cancer; More preferably, the blood cancer is selected from at least one of acute lymphocytic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma; More preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

32. The method according to claim 31, characterized in that The administration occurs within 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, within 47 hours, within 46 hours, within 45 hours, within 44 hours, within 43 hours, within 42 hours, within 41 hours, within 40 hours, within 39 hours, within 38 hours, within 37 hours, within 36 hours, within 35 hours, within 34 hours, within 33 hours, within 32 hours, within 31 hours, within 30 hours, within 29 hours, within 28 hours, within 27 hours, within 26 hours, within 25 hours, within 24 hours, within 23 hours, within 22 hours, within 21 hours, within 20 hours, within 19 hours, within 18 hours, within 17 hours, within 16 hours, within 15 hours, within 14 hours, within 13 hours, within 12 hours, within 11 hours, within 10 hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours or within 1 hour from the date of production of the CAR-T cells.

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