Method for preparing car-expressing t cell and use

Abstract

The present invention relates to the field of cell therapy, and specifically relates to a method for preparing a CAR-expressing T cell and a use. Disclosed in the present invention is a method for preparing a CAR-T cell, comprising the following steps: S1, collecting a T cell-containing cell population of an autologous or allogeneic donor of a subject; S2, administering lymphodepletion therapy to the subject for pretreatment; and S3, administering the T cell-containing cell population and a viral vector to the subject.

Description

A method for preparing CAR-expressing T cells and use thereof TECHNICAL FIELD

[0001] The present disclosure relates to the field of cell therapy, in particular to a method for preparing CAR-expressing T cells and use thereof. BACKGROUND

[0002] Chimeric antigen receptor-expressing T cells (CAR-T cells) are widely used in the treatment of patients with relapsed / refractory hematological malignancies and other tumor patients.

[0003] Currently, CAR-T cell therapy includes ex vivo CAR-T cell therapy and in vivo CAR-T cell therapy. Generally, ex vivo CAR-T cell therapy includes collecting and sorting T cells from a patient, stimulating the activation of T cells, transducing T cells to express CAR in vitro, expanding CAR-T cells, and reinfusing (see relevant patent documents, such as Chinese Patent No. CN109652378A and Chinese Patent No. CN112292147A); while in vivo CAR-T cell therapy includes using a viral vector (such as an in vivo lentiviral vector) that can transduce T cells in the patient's body to be administered to a patient with or without lymphocyte clearance (lymphocyte clearance pretreatment); however, after the patient is pretreated with lymphocyte clearance, the number of T cells available for in vivo lentiviral vector transduction is extremely small due to the large number of T cells in the patient's body, making it difficult to effectively prepare CAR-T cells in the patient's body; however, if the patient is not pretreated with lymphocyte clearance, although there are still T cells in the patient's body that can be transduced by the in vivo lentiviral vector, the endogenous T cells in the patient's body will release inhibitory signals, inhibiting the preparation of CAR-T cells, and also making it difficult to achieve the therapeutic purpose of effectively killing cancer cells in the patient's body. SUMMARY

[0004] In view of the above, the present disclosure provides a composition comprising a viral vector and a cell population comprising T cells; the viral vector:

[0005] (a) comprises a heterologous polynucleotide encoding a chimeric antigen receptor (CAR) that can specifically bind to an antigen associated with a disease; and

[0006] (b) comprises one or more targeting molecules on the surface, which can specifically bind to an endocytic receptor on the surface of T cells.

[0007] In some embodiments of the present disclosure, the viral vector is a retroviral vector.

[0008] In some embodiments of the present disclosure, the retroviral vector is a pseudotyped lentiviral vector.

[0009] In some embodiments of the present application, the viral glycoprotein of the viral vector is selected from the group consisting of a Vesiculovirus strain glycoprotein, a NiV glycoprotein G, a Measles virus glycoprotein H, a Lentivirus glycoprotein, a Rabies virus glycoprotein (RVG), a GaLV glycoprotein, a MLV-A glycoprotein, a RD114 glycoprotein, a FPV glycoprotein, an EboV glycoprotein, and a LCMV glycoprotein.

[0010] The Vesiculovirus strain glycoprotein is selected from the group consisting of a Vesiculovirus Indiana strain glycoprotein, a Vesiculovirus Cocal strain glycoprotein, a Vesiculovirus Maraba strain glycoprotein, a Vesiculovirus Morreton strain glycoprotein, a Vesiculovirus Alagoas strain glycoprotein, a Vesiculovirus New Jersey strain glycoprotein, a Vesiculovirus Carajas strain glycoprotein, a Vesiculovirus Chandipura strain glycoprotein, a Vesiculovirus Eptesicus strain glycoprotein, a Vesiculovirus Isfahan strain glycoprotein, a Vesiculovirus Jurona strain glycoprotein, a Vesiculovirus Malpais strain glycoprotein, a Vesiculovirus Perinet strain glycoprotein, a Vesiculovirus Piry strain glycoprotein, a Vesiculovirus Radi strain glycoprotein, a Vesiculovirus Rhinolopus strain glycoprotein, and a Vesiculovirus Yug Bogdanovac strain glycoprotein.

[0011] In some embodiments of the present application, the viral glycoprotein is a Vesiculovirus Indiana strain glycoprotein (VSV-G) or a Vesiculovirus Cocal strain glycoprotein (Cocal-G), and the viral glycoprotein receptor is a Low Density Lipoprotein Receptor (“LDL-R”).

[0012] The LDL-R is widely expressed on the surface of various cells, and thus a pseudotyped lentiviral vector comprising VSV-G or Cocal-G has a wide range of infectivity. By inhibiting the ability of VSV-G or Cocal-G to bind to its receptor, the targeting of the pseudotyped lentiviral vector can be effectively improved.

[0013] In some embodiments of the present application, the VSV-G (wild type) comprises an amino acid sequence as set forth in SEQ ID NO: 1.

[0014] In some embodiments of the application, the full-length protein of the wild-type VSV-G (including the signal peptide) comprises an amino acid sequence as set forth in SEQ ID NO: 20;

[0015] wherein the amino acid sequence as set forth in positions 1-16 of SEQ ID NO: 20:

[0016] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.

[0017] In some embodiments of the application, the Cocal-G (wild-type) comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0018] In some embodiments of the application, the full-length protein of the wild-type Cocal-G (including the signal peptide) comprises an amino acid sequence as set forth in SEQ ID NO: 27;

[0019] wherein the sequence as set forth in positions 1-17 of SEQ ID NO: 27:

[0020] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild-type Cocal-G.

[0021] In some embodiments of the application, the ability of the viral glycoprotein to bind to its receptor is inhibited;

[0022] Preferably, the viral glycoprotein is VSV-G or Cocal-G, and the viral glycoprotein receptor is LDL-R.

[0023] More preferably, the viral glycoprotein comprises a first mutation, such that the ability of the viral glycoprotein to bind to its receptor is inhibited.

[0024] Still more preferably, the first mutation comprises one or more of the following mutations:

[0025] (a) a substitution or deletion at amino acid position 8, a substitution or deletion at amino acid position 9, a substitution or deletion at amino acid position 10, a substitution or deletion at amino acid position 47, a substitution or deletion at amino acid position 50, a substitution or deletion at amino acid position 51, a substitution or deletion at amino acid position 183, a substitution or deletion at amino acid position 179, a substitution or deletion at amino acid position 180, a substitution or deletion at amino acid position 182, a substitution or deletion at amino acid position 184, a substitution or deletion at amino acid position 209, a substitution or deletion at amino acid position 347, a substitution or deletion at amino acid position 350, a substitution or deletion at amino acid position 352, a substitution or deletion at amino acid position 353, a substitution at amino acid position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0026] (b) a substitution or deletion at amino acid position 8, a substitution or deletion at amino acid position 9, a substitution or deletion at amino acid position 10, a substitution or deletion at amino acid position 47, a substitution or deletion at amino acid position 50, a substitution or deletion at amino acid position 51, a substitution or deletion at amino acid position 183, a substitution or deletion at amino acid position 179, a substitution or deletion at amino acid position 180, a substitution or deletion at amino acid position 182, a substitution or deletion at amino acid position 184, a substitution or deletion at amino acid position 209, a substitution or deletion at amino acid position 347, a substitution or deletion at amino acid position 350, a substitution or deletion at amino acid position 352, a substitution or deletion at amino acid position 353, a substitution at amino acid position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1 or SEQ ID NO: 2 following the best global alignment;

[0027] Still more preferably, the first mutation comprises one or more of the following mutations:

[0028] (a) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of 1182 of SEQ ID NO: 1;

[0029] (b) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of I182, of SEQ ID NO: 1 following a global optimal alignment of (a) with SEQ ID NO: 1 ;

[0030] (c) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of V182, of SEQ ID NO: 2; and

[0031] (d) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of V182, of SEQ ID NO: 2 following a global optimal alignment of (a) with SEQ ID NO: 2;

[0032] Most preferably, the first mutation comprises a mutation of:

[0033] (a) a deletion of K47 of SEQ ID NO: 1 or SEQ ID NO: 2; or

[0034] (b) a deletion of K47 of SEQ ID NO: 1 or SEQ ID NO: 2 following a global optimal alignment of (a) with SEQ ID NO: 1 or SEQ ID NO: 2.

[0035] The complement system is composed of a series of proteins, which is part of the innate immune system. Complement (C) exists in the serum, tissue fluid and cell membrane surface of normal people and animals, and has enzyme activity after activation, which can occur complex cascade reaction. The complement system is activated by a series of enzymes, and finally forms a membrane attack complex similar to a hole on the target microorganism, which makes the microorganism rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, and can clear immune complexes by lysis, opsonization, phagocytosis and mediation of inflammatory reactions, showing corresponding biological functions. Complement is widely involved in the defense reaction of the body against microbial infection and immune regulation, and also mediates immunopathological damage reaction, which is an important biological effect system and effect method system in the body. Viral glycoproteins such as VSV-G can be recognized and inactivated by complement after entering the serum; therefore, improving the ability of viral glycoprotein to antagonize complement inactivation can effectively improve the ability of pseudotyped lentiviral vectors or retroviral vectors to transduce cells in the body or blood of the subject.

[0036] In some embodiments of the application, the viral glycoprotein of the pseudotyped lentiviral vector or retroviral vector comprises a second mutation that enhances or abolishes the ability of the viral glycoprotein to be inactivated by complement;

[0037] Preferably, the viral glycoprotein is VSV-G or Cocal-G and the viral glycoprotein receptor is LDL-R;

[0038] More preferably, the second mutation comprises one or more mutations at one or more of the following positions:

[0039] (a) at amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0040] (b) at a position corresponding to amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2 after optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0041] (c) at amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0042] (d) at a position corresponding to amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2 after optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0043] (e) at amino acid 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0044] (f) at a position corresponding to amino acid 50 of SEQ ID NO: 1 or SEQ ID NO: 2 after optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0045] (g) at amino acid 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0046] (h) at a position corresponding to amino acid 146 of SEQ ID NO: 1 or SEQ ID NO: 2 after optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0047] Still more preferably, the mutation at the position is selected from the group consisting of substitution, deletion and insertion of an amino acid;

[0048] Yet still more preferably, the mutation at the position is a substitution of an amino acid.

[0049] In some embodiments of the application, the second mutation comprises any combination of the following site mutations:

[0050] (a) substitution at (1) T214 and T352 of SEQ ID NO: 1; or (2) T214, T352, K50 and S146 of SEQ ID NO: 1;

[0051] (b) substitution at (1) T214 and T352 of SEQ ID NO: 1; or (2) T214, T352, K50 and S146 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO: 1;

[0052] (c) substitution at (1) K214 and T352 of SEQ ID NO: 2; or (2) K214, T352, K50 and S146 of SEQ ID NO: 2; and

[0053] (d) substitution at (1) K214 and T352 of SEQ ID NO: 2; or (2) K214, T352, K50 and S146 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO: 2;

[0054] Preferably, the mutation comprises any combination of the following site mutations:

[0055] (a) (1) T214N and T352A of SEQ ID NO: 1; or (2) T214N, T352A, K50T and S146T of SEQ ID NO: 1;

[0056] (b) (1) T214N and T352A of SEQ ID NO: 1; or (2) T214N, T352A, K50T and S146T of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO: 1;

[0057] (c) (1) K214N and T352A of SEQ ID NO: 2; or (2) K214N, T352A, K50T and S146T of SEQ ID NO: 2; and

[0058] (d) (1) K214N and T352A of SEQ ID NO: 2; or (2) K214N, T352A, K50T and S146T of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO: 2.

[0059] In some embodiments of the application, the viral glycoprotein is VSV-G and the second mutation comprises any combination of the following site mutations:

[0060] (a) substitutions at (1) T214 and T352 of SEQ ID NO: 1; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; or

[0061] (b) substitutions at (1) T214 and T352 of SEQ ID NO: 1; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; or

[0062] In some embodiments of the application, the viral glycoprotein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-7, 19, 21-26.

[0063] In some embodiments of the application, the viral glycoprotein in which any one of the first mutation and / or the second mutation occurs retains the ability to mediate membrane fusion and lysosomal escape.

[0064] In some embodiments of the application, the endocytic receptor on the surface of the T cell is selected from the group consisting of:

[0065] CD2, CD3, CD3y, CD35, CD3s, TCRy, TCR5, TCRa, TCRp, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD47, CD57, CD62L, CD71, AhR, CD69, CD94, CD95, 4-1BB, CD103, CD122, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), PD-1, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, IL6ST, P2RX7, TIGIT, TIM-3, and LAG-3.

[0066] Endocytosis refers to a process by which substances enter a cell. In endocytosis, a region of the plasma membrane surrounds the substance to be taken in, and the plasma membrane then buds off to form a vesicle containing the 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).

[0067] Endocytic receptor refers to a receptor that can mediate endocytosis. Lymphocytes, such as T cells, NK cells, and B cells, express various endocytic receptors on their surface.

[0068] By constructing a targeting molecule that can specifically bind to an endocytic receptor on the surface of a T cell on a viral vector, the targeting of a pseudotyped lentiviral vector or a retroviral vector can be further improved.

[0069] In some embodiments of the present application, the endocytic receptor on the surface of the T cell is selected from one or more of CD5, CD7, CD3, and CD28.

[0070] In some embodiments of the present application, the endocytic receptor on the surface of the T cell comprises CD3.

[0071] Preferably, the targeting molecule that can specifically bind to CD3 comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

[0072] In some embodiments of the present application, the endocytic receptor on the surface of the T cell comprises CD28.

[0073] Preferably, the targeting molecule that can specifically bind to CD28 comprises one or more of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof.

[0074] More preferably, the CD28 ligand or the receptor-binding fragment thereof is selected from one or more of CD80 or a receptor-binding fragment thereof and CD86 or a receptor-binding fragment thereof.

[0075] In some embodiments of the present application, the targeting molecule comprises a targeting binding region comprising one or more of an antibody or an antigen-binding fragment thereof and a ligand or a receptor-binding fragment thereof that can bind to the endocytic receptor on the surface of the T cell.

[0076] Preferably, when the ligand is a transmembrane protein, the targeting binding region comprises only the receptor binding fragment of the ligand.

[0077] In some embodiments of the application, the receptor binding fragment of the ligand comprises the extracellular region, variable region or binding region of the ligand.

[0078] In some embodiments of the application, the targeting molecule further comprises a transmembrane region, the targeting binding region being directly or indirectly linked to the transmembrane region;

[0079] Preferably, the transmembrane region comprises the transmembrane region of any one of the following proteins:

[0080] CD2, CD3, CD4, CD5, CD7, CD8, CD8a, CD8b, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIg, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1;

[0081] More preferably, the transmembrane region comprises the transmembrane region of CD8a.

[0082] In some embodiments of the application, the targeting molecule further comprises a linker domain, the targeting binding region being indirectly linked to the transmembrane region via the linker domain;

[0083] Preferably, the linker domain is selected from the group consisting of:

[0084] (i) an immunoglobulin hinge region selected from the group consisting of wild type or modified IgGl, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0085] (ii) a hinge region selected from the group consisting of wild type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0086] (iii) all or a portion of an Fc domain selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and

[0087] (iv) a stalk domain of a type II C-lectin selected from a stalk domain of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0088] More preferably, the linker domain is a hinge region of CD8a.

[0089] In some embodiments of the application, any of the aforementioned targeting molecules further comprises a leader signal peptide.

[0090] Preferably, the leader signal peptide is selected from a CD8a signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide.

[0091] More preferably, the leader signal peptide is a CD8a signal peptide.

[0092] In some embodiments of the application, the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0093] In some embodiments of the application, the extracellular antigen binding domain of the CAR can specifically bind to the cancer-associated antigen.

[0094] Preferably, the cancer-associated antigen is selected from:

[0095] 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, GD.2, GD.3, 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-beta, SSEA-4, CD20, AFP, Folate receptor alpha, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin 18.2. one or more of 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 beta, 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, legumain, 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 mutants, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutants, 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 carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFp, APRIL, NKG2D, and GCC (guanylyl cyclase).

[0096] More preferably, the cancer-associated antigen is selected from one or more of CD19, CD20, CD33, CD79A, CD79B, HER2, CEA, and BCMA.

[0097] Still further preferably, the cancer-associated antigen is CD19, the disease comprises indolent non-Hodgkin's lymphoma (iNHL), including follicular lymphoma (FL) and marginal zone lymphoma (MZL); aggressive B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), transformed follicular lymphoma (TFL), and T-cell rich B-cell lymphoma (TCRBCL); and acute B-lymphoblastic leukemia (B-ALL).

[0098] In some embodiments of the application,

[0099] (a) the transmembrane region of the CAR comprises the transmembrane region of any one of the following proteins:

[0100] CD2, CD3, CD4, CD5, CD7, CD8, CD8a, CD8b, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIy, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1;

[0101] Preferably, the transmembrane region of the CAR comprises the transmembrane region of CD8a.

[0102] (b) the intracellular signaling domain of the CAR is selected from the ITAM intracellular signaling domain of CD3e, CD3y, CD35, CD3z, CD79a, CD79b, FceRly, FceRb, FcyRlla, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other proteins comprising at least one ITAM intracellular signaling domain;

[0103] Preferably, the intracellular signaling domain of the CAR is the intracellular signaling domain of CD3z.

[0104] In some embodiments of the application, the CAR further comprises a linker domain and a costimulatory signaling domain; the linker domain links the extracellular antigen-binding region and the transmembrane region of the CAR;

[0105] Preferably, the linking domain of the CAR is selected from:

[0106] (i) an immunoglobulin hinge region selected from wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0107] (ii) a hinge region selected from wild-type or modified hinge regions of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0108] (iii) all or a portion of an Fc domain selected from one or more of a CHI domain, a CH2 domain, and a CH3 domain; and

[0109] (iv) a stalk region of a type II C-lectin selected from stalk regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0110] More preferably, the linking domain of the CAR is a hinge region of CD8a.

[0111] Preferably, the costimulatory signaling domain of the CAR comprises one or more costimulatory signaling domains of CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8a, CD8b, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcaRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-l, LFA-1, LIGHT, JAmL, CD244, CD100, ICOS, CD40, and MyD88.

[0112] More preferably, the costimulatory signaling domain of the CAR comprises a costimulatory signaling domain of 4-1BB.

[0113] In some embodiments of the application, the extracellular antigen-binding region of the CAR comprises one or more of an antibody or antigen-binding fragment thereof that can bind to at least one of the foregoing antigens and a ligand or receptor-binding fragment thereof.

[0114] In some embodiments of the application, the extracellular antigen-binding region of the CAR is monospecific, bi-specific or multi-specific.

[0115] In some embodiments of the application, the extracellular antigen-binding region of the CAR comprises an antibody or antigen-binding fragment thereof that can bind CD19, including but not limited to a scFv derived from FMC63 (FMC63-scFv) and variants thereof, and the like, such as high-affinity variants thereof.

[0116] In some embodiments of the application, the heavy chain variable region (VH region) of the FMC63-scFv comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 24; and the light chain variable region (VL region) of the FMC63-scFv comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 15.

[0117] In some embodiments of the application, the transmembrane region of the CD8a comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 11.

[0118] In some embodiments of the application, the intracellular signaling domain of the CD3 zeta comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 18.

[0119] In some embodiments of the application, the hinge region of the CD8a comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.

[0120] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR includes a 4-1BB costimulatory signal transduction domain, wherein the 4-1BB costimulatory signal transduction domain contains an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:17.

[0121] In some embodiments of the present invention, the polynucleotide encoding the CAR is operatively linked to a polynucleotide encoding a leader signal peptide.

[0122] In some embodiments of the present invention, the CAR comprises a leader signal peptide, an extracellular antigen-binding region, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain, and an intracellular signal transduction domain from the N-terminus to the C-terminus.

[0123] Preferably, the leader signal peptide is selected from CD8α signal peptide, CD28 signal peptide, IgG signal peptide and HLA-A signal peptide;

[0124] More preferably, the leader signal peptide is a CD8α signal peptide.

[0125] In some embodiments, the cell population containing T cells is selected from leukocytes, PBMCs, and CD3. + One or more of the following are found in T cells.

[0126] Preferably, the cell population containing T cells is collected from the subject's own body or an allogeneic donor; the subject is the individual to whom the composition is administered.

[0127] In some embodiments of the present invention, the subject or the subject allogeneic donor has not received any pretreatment with lymphocyte clearance therapy for at least 4 weeks prior to collection.

[0128] In some embodiments of the present invention, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0129] In some embodiments of the present invention, the composition comprises 5 × 10 6 5×10 9 CD3 + The aforementioned cell populations comprising T cells, including terminal values; and 1 × 10 3 -1×10 9 TU / KG is any of the aforementioned viral vectors, including end-values.

[0130] The present application also discloses a method for preparing a chimeric antigen receptor-expressing T cell (CAR-T cell), comprising the following steps:

[0131] S1, collecting a cell population comprising T cells from a subject autologous or allogeneic donor;

[0132] S2, pretreating the subject with lymphocyte depletion therapy; and

[0133] S3, administering the composition to the subject.

[0134] In some embodiments of the present application, in S3, the composition is mixed and administered to the subject;

[0135] Preferably, the composition is mixed for 1 minute to 2 hours, more preferably, the mixing time is 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, or 2 hours.

[0136] In some embodiments of the present application, in S3, after the administration of the cell population comprising T cells or the viral vector in the composition to the subject (first administration), the viral vector or the cell population comprising T cells is administered to the subject (second administration);

[0137] When the first administration is the administration of the cell population comprising T cells to the subject, the second administration is the administration of the viral vector to the subject;

[0138] When the first administration is the administration of the viral vector to the subject, the second administration is the administration of the cell population comprising T cells to the subject.

[0139] In some embodiments of the present application, in S3, the second administration is performed within 1 minute to 7 days of the first administration; preferably, the second administration is performed within 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days of the first administration;

[0140] Preferably, the second administration is performed within 24 hours of the first administration; more preferably, the second administration is performed within 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours of the first administration.

[0141] More preferably, the second administration is performed within 60 minutes of the first administration, more preferably, the second administration is performed within 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes of the first administration.

[0142] In some embodiments of the application, in S3, the cell population comprising T cells and the viral vector are simultaneously administered to the subject using a dual-lumen catheter (dual-lumen administration).

[0143] In some embodiments of the application, in S3, after the administration of the composition to the subject, completion of the second administration, or completion of the dual-lumen administration, the subject can be subjected to one or more of: the dual-lumen administration, the administration of the composition, the administration of the cell population comprising T cells, and the administration of the viral vector.

[0144] In another aspect, the present application also discloses use of any of the aforementioned compositions in the preparation of CAR-T cells.

[0145] In another aspect, the present application also discloses use of any of the aforementioned compositions in the preparation of a medicament for treating a disease.

[0146] Preferably, the disease is cancer, and the cancer is selected from the group consisting of a hematological cancer and a solid cancer.

[0147] More preferably, the hematological cancer is a CD19-expressing hematological cancer.

[0148] Still further preferably, the blood cancer expressing CD19 comprises indolent lymphomas, including follicular lymphoma and marginal zone lymphoma; aggressive B-cell lymphomas, including diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, transformed follicular lymphoma, and T-lymphocyte-rich large B-cell lymphoma; and acute B-lymphoblastic leukemia.

[0149] In another aspect, the present application also discloses a method of treating cancer, the method comprising:

[0150] S1, collecting a cell population comprising T cells from a subject having cancer, which is autologous or allogeneic donor;

[0151] S2, pretreating the subject with lymphodepleting therapy; and

[0152] S3, administering the cell population comprising T cells and a viral vector to the subject; the viral vector:

[0153] (a) comprises a heterologous polynucleotide encoding a chimeric antigen receptor (CAR) that can specifically bind to a cancer-associated antigen; and

[0154] (b) comprises one or more targeting molecules on the surface that can specifically bind to an endocytic receptor on the surface of a T cell.

[0155] In some embodiments of the present application, in S3, the cell population comprising T cells and the viral vector (composition) are mixed, and the composition is administered to the subject;

[0156] Preferably, the cell population comprising T cells and the viral vector are mixed for 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, or 2 hours, and the composition is administered to the subject.

[0157] In some embodiments of the present application, in S3, after the administration of the cell population comprising T cells or the viral vector to the subject (first administration), the viral vector or the cell population comprising T cells is administered to the subject (second administration);

[0158] When the first administration is the administration of the cell population comprising T cells to the subject, the second administration is the administration of the viral vector to the subject;

[0159] When the first administration is the administration of the viral vector to the subject, the second administration is the administration of the cell population comprising T cells to the subject.

[0160] In some embodiments of the application, in S3, the second administration is performed within 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days of the first administration.

[0161] Preferably, the second administration is performed within 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours of the first administration.

[0162] More preferably, the second administration is performed within 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes of the first administration.

[0163] In some embodiments of the application, in S3, the cell population comprising T cells and the viral vector are simultaneously administered to the subject using a double-barreled tube (double-barreled administration).

[0164] In some embodiments of the application, after the administration of the composition to the subject, the completion of the second administration or the completion of the double-barreled administration, the subject can be subjected to one or more of the following: double-barreled administration, administration of the composition, administration of the cell population comprising T cells and administration of the viral vector.

[0165] In some embodiments of the application, the viral vector is any one of the previously described pseudotyped lentiviral vectors or retroviral vectors.

[0166] In some embodiments of the application, the cell population comprising T cells is any one of the previously described cell populations comprising T cells.

[0167] In some embodiments of the present invention, in S1, the subject or the subject allogeneic donor has not received any pretreatment with lymphocyte clearance therapy for at least 4 weeks (inclusive) prior to collection; preferably, the subject allogeneic donor is a healthy donor; more preferably, the subject is a human being.

[0168] In some embodiments of the present invention, in S2, the lymphocyte clearance therapy includes administering an immunosuppressant to the subject;

[0169] Preferably, the subject is given an immunosuppressant daily for 2-4 days, preferably for 3 days.

[0170] In some embodiments of the present invention, the immunosuppressant is selected from one or more of cyclophosphamide and fludarabine;

[0171] Preferably, the immunosuppressant is fludarabine and cyclophosphamide;

[0172] More preferably, the immunosuppressant is 20-40 mg / m² fludarabine and 200-400 mg / m² cyclophosphamide;

[0173] More preferably, the immunosuppressant is 30 mg / m² fludarabine and 300 mg / m² cyclophosphamide.

[0174] In some embodiments of the present invention, in S2, the administration of the lymphocyte clearance therapy is completed 2 to 7 days prior to the administration of the cell population containing T cells and the viral vector to the subject;

[0175] Preferably, the administration of the lymphocyte clearance therapy is completed 2 to 7 days prior to the administration of the composition to the subject or the first administration.

[0176] In some embodiments of the present invention, the cell population containing T cells is selected from leukocytes, PBMCs, and CD3+. + One or more of the following are found in T cells.

[0177] In some embodiments of the present invention, the subject is administered an injection containing 5 × 10 6 5×10 9 CD3 + Any of the aforementioned cell populations containing T cells, including terminal groups; administer 1 × 10 to the subject. 3 -1×10 9 TU / KG is any of the aforementioned viral vectors, including end-values.

[0178] The beneficial effects of this invention include:

[0179] After the patient is subjected to the leukapheresis pretreatment, the number of T cells available for in vivo lentiviral vector transduction is extremely small, and thus it is difficult to effectively produce CAR-T cells in the patient; however, if the patient is not subjected to the leukapheresis pretreatment, although there are T cells available for in vivo lentiviral vector transduction in the patient, the endogenous T cells in the patient release inhibitory signals, which inhibit the production of CAR-T cells, and it is also difficult to achieve the therapeutic goal of effectively killing cancer cells in the patient; and the present application discloses for the first time that the in vivo lentiviral vector and the cell population containing T cells, such as leukocytes, PBMCs and CD3 + T cells are administered to the leukapheresis patient, including simultaneous administration, sequential administration within a short period of time and / or administration using a double-barreled tube, which can effectively kill cancer cells in the patient; relative to conventional ex vivo CAR-T cell therapy, the time required for the patient to receive CAR-T cell treatment is greatly shortened and the treatment cost is reduced; relative to other in vivo CAR-T cell therapies in which only in vivo lentiviral vectors are administered to leukapheresis or non-leukapheresis patients, the efficiency of in vivo production of CAR-T cells and the efficiency of killing cancer cells by CAR-T cells are significantly improved, and the curative effect is better.

[0180] Herein:

[0181] "Inhibit": when referring to the ability of the viral glycoprotein of the pseudotyped lentiviral vector or retroviral vector to transduce a cell expressing the viral glycoprotein receptor by binding to the viral glycoprotein receptor, the term "inhibit" includes completely eliminating the ability of the pseudotyped lentiviral vector or retroviral vector to transduce the cell, and significantly weakening the transduction ability. In specific embodiments, "significantly weaken" means a weakening of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2%, and at least 1%, relative to the viral glycoprotein of the pseudotyped lentiviral vector or retroviral vector containing the first mutation.

[0182] "Lymphocyte": the term "lymphocyte" refers to an immune cell of lymphoid origin, which is a cell that exhibits at least one phenotypic characteristic of a lymphocyte or a precursor or progenitor thereof, which distinguishes the cell from cells of the erythroid lineage or myeloid lineage. The term "lymphocyte" includes T cells, B cells and natural killer (NK) cells.

[0183] "T cell": T cells are one of several white blood cells important in the human immune system and play a major role in the adaptive immune response. One of the main functions of T cells is immune-mediated cell death, which is accomplished primarily by two T cell subtypes: CD8 + T cells (Cytotoxic T Cells) and CD4 + T cells (Helper T Cells).

[0184] In some embodiments of the present application, the T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + , CD4 - / CD8 - T cells or a combination thereof. In some embodiments of the present application, the CD4 + T cells produce IL-2, TFN, TNF or a combination thereof upon expression of a CAR and binding to a target cell, such as a tumor cell. In some embodiments of the present application, the CD8 + T cells lyse antigen-specific target cells upon expression of a CAR and binding to a target cell.

[0185] Non-activated T cells refer to T cells that are not expanded, not differentiated, in a resting state, not recognized antigen, not activated by T cell activation signal molecules such as T cell activation primary and secondary signal molecules (anti-CD3 antibody or CD28-binding ligand, etc.), for example, T cells in the G0 phase of the cell cycle, resting T cells or naïve T cells. Resting T cells are also known as quiescent T cells or natural T cells, which refer to T cells that do not have mitotic activity or have not been exposed to homologous antigens presented on antigen-presenting cells (such as macrophages or dendritic cells).

[0186] “Chimeric antigen receptor”: i.e., Chimeric Antigen Receptor (CAR), refers to an artificial cell surface receptor engineered to be expressed on immune effector cells including T cells, which can specifically bind to an antigen, comprising at least: (1) an extracellular antigen-binding region, such as scFv or VHH, (2) a transmembrane region anchoring the CAR to the cell membrane, and (3) an intracellular signaling domain; the extracellular structure of CAR can further comprise a hinge region; the intracellular structure of CAR can further comprise a costimulatory signaling domain. CAR can redirect T cells and other immune effector cells to a selected target, such as a cancer cell, in a non-MHC restricted manner using the extracellular antigen-binding region. In some embodiments of the application, the CAR further comprises a leader signal peptide.

[0187] “Chimeric”: The term “chimeric” refers to any nucleic acid molecule or protein that is non-endogenous and comprises a combination of sequences that are not naturally joined or linked together in nature. For example, a chimeric nucleic acid molecule can comprise nucleic acids encoding various domains from multiple different genes. As another example, a chimeric nucleic acid molecule can comprise regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a manner different from that found in nature.

[0188] “Antigen”: The terms “antigen” and “Ag” refer to a molecule capable of inducing an immune response. The induced immune response can include antibody production and / or activation of specific immunocompetent cells. Macromolecules including proteins, glycoproteins, and glycolipids can serve as antigens. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be (i) encoded solely by a full-length nucleotide sequence of a gene or (ii) encoded entirely by a gene. An antigen can be generated or synthesized, or an antigen can be derived from a biological sample. Such biological samples can include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0189] “Flexible Linker”: i.e. Flexible Linkers, flexible linkers are typically used when the domains to be linked require some degree of movement or interaction (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10): 1357-69.). Flexible linkers are typically composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990;211:943-958.). These small size amino acids provide flexibility while also allowing movement of the functional domains to be linked. Commonly used flexible linkers are described in Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10): 1357-69., which is incorporated herein by reference in its entirety.

[0190] “Antibody”: refers to a polypeptide or combination of polypeptides that comprises a sufficient sequence from an immunoglobulin heavy chain variable region and / or a sufficient sequence from an immunoglobulin light chain variable region to specifically bind to an antigen. “Antibody” herein encompasses various formats and various structures as long as they exhibit the desired antigen binding activity.

[0191] “Antibody” herein includes a typical “four-chain antibody” which belongs to 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, a heavy chain constant region CH3 domain in the direction from the N-terminus to the C-terminus thereof; and, when the full-length antibody is of an IgE isotype, 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) in the direction from the N-terminus to the C-terminus thereof; the heavy chain and the heavy chain are connected by a disulfide bond, and the heavy chain and the light chain are connected by a disulfide bond, forming a “Y”-shaped structure.

[0192] In the context of antibodies, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains (VH and VL regions, respectively) of natural antibodies generally have similar structures, each domain including four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th Ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL region can be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds a particular antigen can be isolated using a VH or VL region from an antibody that binds the particular antigen to screen a library of complementary VL or VH regions, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0193] The terms "complementarity determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions that impart antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0194] The term "antibody" herein also includes antibodies that do not comprise light chains, such as heavy-chain antibodies (Heavy-Chain Antibodies, HC Abs) produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and Vicugna pacos, and the like, and immunoglobulin new antigen receptors (Ig New Antigen Receptors, IgNARs) found in cartilaginous fishes, such as sharks.

[0195] The terms "VHH domain," "nanoantibody," and "single domain antibody" (sdAb) have the same meaning and are used interchangeably herein to refer to the variable region of a heavy chain antibody that is cloned to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full functionality. Generally, a heavy chain antibody naturally missing a light chain and a heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0196] The term "antibody" herein also includes a monoclonal antibody or an antigen binding portion thereof. The monoclonal antibody or antigen binding portion thereof can be non-human, chimeric, humanized or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0197] The term "antibody" herein can be derived from any animal, including but not limited to human and non-human animals, which can be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, monkeys (e.g., cynomolgus and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or chondrichthyan (e.g., sharks).

[0198] The term "antigen binding fragment" herein refers to a fragment that does not possess the entire structure of a complete antibody, but only contains a part or a part of variant of a complete antibody, which has the ability to bind to an antigen.

[0199] Exemplarily, the term "antibody or antigen binding fragment thereof" herein includes but is not limited to: immunoglobulin (full-length antibody), half antibody, Fab, Fab', F(ab')2, Fv fragment, single-chain variable region fragment (scFv), disulfide-stabilized antibody (dsFv), heavy chain variable region (VH) or light chain variable region (VL) of an antibody, Fd fragment consisting of VH and CH1 domains, linear antibody, heavy chain antibody and nanobody (VHH).

[0200] In some embodiments of the present application, the order of the scFv containing VH region or VL region from N-terminus to C-terminus is not particularly limited, such as VH-Linker-VL or VL-Linker-VH from N-terminus to C-terminus; the connecting peptide can be selected from a flexible connecting peptide.

[0201] "Ligand": In receptor-ligand binding, the ligand is usually a molecule that binds to a site on the receptor to produce a signal, which usually leads to a conformational change of the complex structure, thereby inducing the relevant physiological activity.

[0202] "Receptor binding fragment": refers to a fragment that does not possess the entire structure of a complete ligand, but only contains a part or a part of variant of a complete ligand, which has the ability to bind to a receptor. Exemplarily, the term "receptor binding fragment" herein includes but is not limited to the extracellular domain, binding region and variable region of the ligand.

[0203] “Variant”: A variant refers to a mutant that is at least 50% identical to the amino acid sequence of a non-mutant (wild type), “at least 50% identical” means that the variant is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of a non-mutant (wild type); or, a variant refers to a mutant in which the nucleic acid sequence encoding the variant is at least 50% identical to the nucleic acid sequence encoding a non-mutant (wild type), “at least 50% identical” means that the nucleic acid sequence encoding the variant is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence encoding a non-mutant (wild type). In some embodiments of the application, a variant includes a mutant that comprises conservative substitutions relative to a non-mutant. “Conservative substitutions” are recognized in the art (see, e.g., WO 97 / 09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY (1975), pages 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8).

[0204] “Transgene”: As used herein, the term “transgene” refers to a gene or polynucleotide that encodes a protein of interest (e.g., a CAR) that is desired to be expressed in a host cell and has been transferred into the cell by genetic engineering techniques. Transgenes can encode proteins of therapeutic interest as well as proteins that are reporters, tags, markers, suicide proteins, etc. Transgenes can be from natural sources, modifications of natural genes, or recombinant or synthetic molecules. In certain embodiments, a transgene is a component of a vector, such as a pseudotyped lentiviral vector or a retroviral vector.

[0205] “Expression Cassette”: As used herein, the term “expression cassette” refers to a unique set of components of a vector nucleic acid comprising at least one transgene and regulatory sequences (e.g., a promoter, a 3’ UTR) that control its expression in a host cell. A tandem expression cassette refers to a set of components of a vector nucleic acid comprising at least two transgenes under a set of the same regulatory sequences for expressing the at least two transgenes in tandem. In certain embodiments, a tandem expression cassette comprises at least two transgenes under the control of the same promoter. In certain embodiments, a first transgene and a second transgene are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to allow co-expression of two proteins from a single mRNA.

[0206] “Nucleic Acid”: Refers to any chemical and / or substance comprising a polymer of nucleotides, such as a polynucleotide. As used herein, “nucleic acid,” “polynucleotide,” and “gene” are used synonymously. 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 the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5’ to 3’. As used herein, the term “nucleic acid” encompasses deoxyribonucleic acids (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acids (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. A “nucleic acid” can be linear or circular. Furthermore, a “nucleic acid” includes both the sense (coding) strand and the antisense (template) strand, as well as single- and double-stranded forms. Also, a “nucleic acid” as described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases modified with derivatized sugars, phosphate backbone linkages, or chemically modified residues.

[0207] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds.

[0208] “Encoding” refers to the inherent property of specific polynucleotide sequences, such as DNA, cDNA and mRNA sequences, to serve as templates for synthesis of other polymers and macromolecules in biological processes having the appropriate cellular machinery. Thus, if an mRNA corresponding to a particular polynucleotide is translated by a cell or other biological system, then the polynucleotide encodes the protein specified by the mRNA. Either or both of the sense and anti-sense strands of a polynucleotide can be referred to as encoding the protein or other product specified by the polynucleotide, unless otherwise indicated. “Nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences which, as a result of the reading frame used by molecular biology techniques, results in the synthesis of a polypeptide having the amino acid sequence specified by that nucleotide sequence.

[0209] “Exogenous”: refers to any molecule, including nucleic acids, proteins, polypeptides, or small molecule compounds, etc. that originates from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0210] “Promoter”: The term “promoter” as used herein is defined as a DNA sequence recognized by the cellular or introduced synthetic machinery for starting the specific transcription of a polynucleotide sequence. The term “promoter / regulatory sequence” as used herein means a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence can be a core promoter sequence and in other cases the sequence can include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence can be, for example, a sequence that expresses the gene product in a tissue-specific manner.

[0211] A “constitutive” promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0212] An “inducible” promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0213] A “tissue-specific” promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only when the cell is of a tissue type corresponding to the promoter.

[0214] “Viral envelope”: refers to the outermost layer of various viruses (HURLBERT, RONALD E., Fundamentals of Microbiology, 102. Chapter #11: Viruses. Archived from the original on 2008-11-10.). The viral envelope protects the genetic material during its life cycle when the virus shuttles in host cells. Not all viruses have a viral envelope. Various human pathogenic viruses are wrapped in a lipid bilayer, which they use to infect target cells by fusing the viral envelope with the cell membrane. Viruses with viral envelopes include retroviruses, etc.

[0215] “Lentivirus”: Lentiviruses are complex retroviruses that contain additional genes with regulatory or structural functions in addition to the common retroviral genes Gag, Pol, and env. The higher complexity enables the virus to regulate its life cycle, as it does during latent infection. Lentiviruses belong to the retrovirus genus that can infect dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type I and HIV type II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0216] “Lentiviral vector”: Lentiviral vector is a vector derived from lentivirus, and contains one or more lentivirus packaging proteins and / or lentivirus proteins necessary for expressing one or more genes carried by the vector. It is produced by multiple attenuations of virulence genes of HIV and other lentiviruses through gene editing, genetic engineering, etc. For example, deleting genes env, vif, vpr, vpu, and nef makes the lentiviral vector biologically safe.

[0217] Lentiviral vectors or retroviral vectors are generally packaged in packaging cells by lentiviral vector packaging systems or retroviral vector packaging systems. For example, see Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. (2016), 3, 16017, which is incorporated by reference in its entirety.

[0218] Commonly used pseudotyped lentiviral vectors include so-called third generation lentiviral vector packaging systems. Third generation lentiviral vector packaging systems include four plasmids, typically including a transfer plasmid and three packaging plasmids: a transfer plasmid comprising a gene of interest (GOI), such as a transgene, a GagPol plasmid, a Rev plasmid, and an envelope plasmid (comprising a viral glycoprotein gene such as VSV-G or a variant thereof or Cocal-G or a variant thereof).

[0219] A “transfer plasmid” comprises a lentiviral vector backbone genome and a transgene. The transfer plasmid typically has one or more transgenes flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transgene contained by the transfer plasmid into the host genome. The LTRs are responsible for the reverse transcription and integration processes of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell. For safety reasons, the transfer plasmid is typically designed such that the resulting viral vector is unable to self-replicate. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious lentiviral particles in a host cell. In addition, the transfer plasmid can be designed to have a 3’ LTR deleted, rendering the virus “self-inactivating.” Compared to traditional second generation pseudotyped lentiviral vector packaging systems (typically a single packaging plasmid comprising nucleic acids encoding Gag, Pol, Rev, and Tat and a separate envelope plasmid), the TAT gene is eliminated from third generation pseudotyped lentiviral vector packaging systems by adding a chimeric 5’ LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) on the transfer plasmid. The transfer plasmid typically includes a Ψ sequence (Psi sequence, also known as Ψ packaging signal) downstream of the 5’ LTR, which is responsible for packaging the transgene RNA into viral particles. The Ψ sequence ensures that only RNA containing the transgene is packaged into viral particles. The transfer plasmid can also optionally include an Internal Ribosome Entry Site (“IRES”) to allow for the translation of two or more open reading frames (ORFs) on one mRNA, enabling multi-gene expression. Some transfer plasmids, such as the lentivirus-GFP plasmid used in some embodiments of the present application, can also include a selection marker gene, such as an antibiotic resistance gene (e.g., PuroR, encoding puromycin resistance) or a fluorescent protein gene (e.g., GFP), for screening or tracking transduced cells.

[0220] For transfer plasmids in lentiviral vector packaging systems, see, e.g., DμLl, et al., J. Virol. 72:8463-71 (1998); Miyoshi, et al., J. Virol. 72:8150-57 (1998).

[0221] Third generation lentiviral vector systems also typically include three packaging plasmids: a GagPol plasmid, a Rev plasmid, and an envelope plasmid. The envelope plasmid typically carries a viral glycoprotein gene, with wild-type VSV-G or Cocal-G being one of the commonly used viral glycoproteins; the viral glycoprotein gene is operably linked to a promoter, typically a CMV promoter, which initiates transcription of the viral glycoprotein gene. Third generation lentiviral vector systems also include two packaging plasmids, one containing genes encoding Gag and Pol proteins (GagPol packaging plasmid), and the other containing a gene encoding Rev protein (Rev plasmid) as a further safety feature, which is an improvement over the single packaging plasmid of the so-called second generation packaging system. The Gag gene encodes a Gag polyprotein precursor that contains matrix, capsid, and nucleocapsid structural proteins of the lentivirus; the Pol gene encodes a Pol polyprotein precursor that contains protease, reverse transcriptase, and integrase enzymes necessary for replication; the Rev gene encodes a Rev protein that binds to a Rev response element (RRE) on the viral RNA to allow nuclear export of unspliced and singly spliced HIV RNAs during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral particle production. The Rev protein binds to the Rev response element (RRE) sequence on the viral RNA, facilitating transport of unspliced viral RNA from the nucleus to the cytoplasm by interacting with the host cell’s nuclear export machinery. These unspliced RNAs can be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral particles.

[0222] Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0223] 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; DμLl, et al., (1998) J. Virol. 72:8463-8471, U.S. Patent No. 6,013,516, and U.S. Patent No. 5,994,136, each of which is incorporated by reference herein in its entirety.

[0224] In comparison to pseudotyped lentiviral vector packaging systems, pseudotyped retroviral vector packaging systems generally do not include a Rev plasmid, as genomic RNA derived from retroviruses such as Moloney Murine Leukemia Virus ("MMLV") can naturally be transported from the nucleus to the cytoplasm for translation and assembly, thus not requiring reliance on a specific nuclear export mechanism such as a Rev protein. Pseudotyped retroviral vector packaging systems generally include one transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transfer plasmid includes a transgene sequence flanked by long terminal repeat sequences ("LTRs"), which facilitate integration of the transfer plasmid sequence into the host genome. Generally, the sequence between and including the LTRs will be integrated into the host genome during viral transduction. The backbone genome of MMLV or Murine Stem Cell Virus ("MSCV"), which includes its respective LTRs, is often utilized in the construction of the transfer plasmid in a pseudotyped retroviral vector packaging system. The GagPol packaging plasmid includes Gag and Pol genes; the envelope plasmid generally includes a polynucleotide encoding a viral glycoprotein such as VSV-G or Cocal-G.

[0225] In some embodiments, the production cells are transfected with a defined ratio of the transfer plasmid, the GagPol plasmid, the envelope plasmid, and the Rev plasmid. In some embodiments, the ratio of each plasmid is determined by mass, which is not particularly limited as long as a biologically active pseudotyped lentiviral vector is packaged. In some embodiments, the mass of each of the transfer plasmid and the GagPol plasmid is higher than the mass of each of the envelope plasmid and the Rev plasmid. In some embodiments, the defined ratio of the transfer plasmid, the GagPol plasmid, the envelope plasmid, and the Rev plasmid is 1 : 1 : 1 : 1 to 9:4:2:2; in some embodiments of the present application, the envelope plasmid can include a nucleic acid encoding a targeting molecule.

[0226] In some embodiments, the present disclosure provides a pseudotyped lentiviral vector packaging system, wherein the envelope plasmid comprises a tandem expression cassette encoding VSV-G or a variant thereof or Cocal-G or a variant thereof and a targeting molecule as disclosed herein. In specific embodiments, the tandem expression cassette comprised in the envelope plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding a targeting molecule, a polynucleotide encoding one of an internal ribosome entry site (IRES), a furin cleavage site, or a viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding VSV-G or a variant thereof or Cocal-G or a variant thereof. In certain embodiments, the polynucleotide encoding VSV-G or a variant thereof or Cocal-G or a variant thereof is located 5' to the polynucleotide encoding the targeting molecule. In other embodiments, the polynucleotide encoding VSV-G or a variant thereof or Cocal-G or a variant thereof is located 3' to the polynucleotide encoding the targeting molecule. The polynucleotide encoding the targeting molecule and the polynucleotide encoding VSV-G or a variant thereof or Cocal-G or a variant thereof are separated in the tandem cassette by a polynucleotide encoding an IRES, a furin cleavage site, or a viral 2A peptide, which allows co-expression of both proteins from a single mRNA. In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof.

[0227] The use of lentiviral vector systems relies on a "packaging cell line". Generally, a packaging cell line is a cell line whose cells are capable of producing lentiviral vectors that are not self-replicating, and that can infect / transduce target cells upon introduction of a transfer plasmid, one or more packaging plasmids into the cells. An overview of available packaging lines is provided in JM Coffin, SM Hughes, et al. Cold Spring Harbour Laboratory Press, 1997, p. 447, which is incorporated herein by reference in its entirety.

[0228] Exemplarily, various plasmids can be introduced into the packaging cell line using transfection methods including chemical-mediated transfection methods, physical-mediated transfection methods, or biological-mediated transfection methods, etc. For example, chemical-mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran, or PEI (Polyethylenimine, a transfection reagent), etc. Physical-mediated transfection methods include transfection using electroporation, etc.

[0229] The packaging cells can be genetically engineered to otherwise improve the immunological properties of the lentiviral and retroviral vectors disclosed herein and / or to increase the efficiency of transduction of target cells by the lentiviral and retroviral vectors; such otherwise including but not limited to the addition of genes, deletion of genes, and introduction of point mutations into genes.

[0230] Production cells useful for the production of the pseudotyped lentiviral or retroviral vectors disclosed herein include human embryonic kidney (HEK) 293 cells and derivatives thereof. The production cells can be an adherent cell line such as HEK293T production cells, or a suspension cell line such as HEK293T / 17SF production cells.

[0231] Illustratively, the packaging cells are selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, and 211 cells.

[0232] Preferably, the packaging cells are HEK-293T cells.

[0233] “Retrovirus” and “Retroviral Vector”: i.e., Retrovirus and Retroviral Vector. “Retrovirus” refers to an RNA virus having a single-stranded positive-sense RNA molecule. Retroviruses contain a reverse transcriptase and an integrase. Upon entry into a target cell, a retrovirus utilizes its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the DNA molecule is integrated into the host cell genome using the integrase. Upon integration into the host cell genome, the sequence from the retrovirus is referred to as a provirus (e.g., a sequence of a provirus or a proviral sequence). Retroviral vectors generally refer to pseudotyped retroviral vectors derived from a retrovirus, illustratively, a gamma-retrovirus. Unlike lentiviral vectors, which can transduce both dividing and non-dividing cells, retroviral vectors can only transduce dividing cells, and the exogenous transgenes they can carry are generally relatively small. For a comparison and discussion of lentiviral and retroviral vectors, see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, K.K., Vorburger, S.A., Swisher, S.G. (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0234] “Viral glycoprotein”: refers to a glycoprotein that coats the outer layer of a virus and plays an important role in adsorption and penetration of the virus into host cells, pathogenicity, downregulation of expression of host surface proteins, and increase in the process of viral packaging and budding.

[0235] “Pharmaceutically acceptable excipient or carrier”: Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives, and / or adjuvants. The excipient is preferably non-toxic or substantially non-toxic to the recipient at the dosages and concentrations employed.

[0236] “Subject”: As used herein, “subject,” “patient,” and “individual” are used synonymously and include, but are not limited to, a mammal, a human or non-human mammal, such as a domestic animal, an agricultural animal, or a wild animal, as well as birds and aquatic animals. A “patient” is a subject afflicted with, at risk of developing, or otherwise in need of any one of the pseudotyped lentiviral vectors or retroviral vectors, engineered immune cells, compositions, or methods of treatment provided herein.

[0237] A "disease" is a state of health of a subject in which the subject is unable to maintain homeostasis and in which the health of the subject continues to deteriorate if the disease is not ameliorated. In contrast, a "condition" or "adverse condition" of a subject is a state of health of the subject in which the subject is able to maintain homeostasis, but in which the state of health of the subject is not as advantageous as in the absence of the condition or adverse condition. A condition or adverse condition does not necessarily result in a further decrease in the state of health of the subject if left untreated.

[0238] "Cancer": The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. The aberrant cells can form a solid tumor or constitute a hematological malignancy. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

[0239] "Treat" or "treatment": As used herein, "treatment" or "treat" includes any beneficial or desired effect on the treatment. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms. Herein, "treatment" includes administering to a subject a provided pseudotyped lentiviral vector or retroviral vector, composition of the application or employing the therapeutic / prophylactic methods described herein to achieve at least one positive therapeutic effect (such as, for example, a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell infiltration into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth). The method of treatment effective to treat a patient can vary depending on a variety of factors such as the disease state, age, body weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0240] "Prevent" or "prevention": As used herein, "prevent," and like words, such as "preventing," and the like, indicate an approach for preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a condition. As used herein, "prevent," and like words also include lessening the intensity, effect, symptoms, and / or burden of a disease or condition prior to onset or recurrence.

[0241] "and / or": Is to be taken as specific alternatives, one or both.

[0242] "Comprise": Herein, unless otherwise required by context, the word "comprising" is to be understood to imply the inclusion of any recited steps or elements - or group of steps or elements - but not the exclusion of any other unrecited steps or elements - or group of steps or elements. In some embodiments of the application, the terms "including," "has," "have," and "comprises" are synonymous with each other.

[0243] “Embodiments”: Reference throughout this specification to “some embodiments” and “some examples”, or combination thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. The appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0244] “Stably integrated”: Also referred to as “stably transduced” or “stable gene expression”, refers to the integration of an exogenous nucleic acid into the genome of a host cell, which is stably expressed in the host cell over a long period of time.

[0245] “Specific binding”: As used herein, the term “specific binding” refers to binding that occurs between pairs of molecular species (e.g., a receptor and a ligand, an antibody and an antigen). Binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions when the interaction of the two species results in a non-covalently bound complex. In various embodiments, specific binding between one or more species is direct. In some embodiments of the application, the affinity of specific binding is 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold or more greater than background binding (non-specific binding).

[0246] "Sequence identity": Generally, "sequence identity" or "sequence homology" refers to the exact correspondence of nucleotides and nucleotides or amino acids and amino acids of two nucleic acid sequences or protein / polypeptide sequences. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a nucleic acid and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a control nucleotide or amino acid sequence. Two or more sequences (nucleic acid or amino acid) can be compared by determining their "percent identity." Whether a nucleic acid or amino acid sequence, the percent identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100. For example, sequence information can also be compared using the advanced BLAST computer program, available from the National Institutes of Health, to determine percent identity. The BLAST program is based on the following alignment method: Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (typically nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine percent identity over the entire protein / polypeptide length of the sequences being compared.

[0247] “Signal Peptide”: Signal Peptide, sometimes also referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).) that includes the leader signal peptide encoded by the leader sequence; signal peptides are found at the N-terminus of most newly synthesized proteins that are destined for the secretory pathway (occasionally non-classically at the C-terminus or internally) (Owji, et al., A comprehensive Review of signal peptides: Structure, roles, and applications, European Journal of Cell Biology. 97 (6):422-441. (2018)) (Blobel G, Dobberstein B, et al., Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, The Journal of Cell Biology, 67 (3):835-51. (1975)). Signal peptides are short peptides present at the N-terminus of newly synthesized proteins, which are dedicated to the plasma membrane or secretory pathway. Signal sequences usually contain a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a signal sequence cleavage site. In eukaryotes, signal sequences cause the translocation of newly synthesized proteins to the endoplasmic reticulum, where they are cleaved by signal peptidases to produce mature proteins that then go to their proper destination. The diversity in signal sequence length and amino acid composition makes it difficult to accurately predict cleavage sites. For the polypeptide sequences disclosed herein, when reference is made to a signal sequence, polypeptide sequences are also contemplated that lack a signal sequence or have a partial signal sequence.

[0248] “MOI”: i.e., “Multiplicity of Infection (MOI)”, refers to the number of virions added to each cell during a viral infection or transduction. For example, when one million virions are added to one million cells, MOI = 1.

[0249] "operably linked": nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid. For example, nucleic acid sequences that are operably linked can be contiguous with each other; and, for example, when

[0250] "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. A 2A peptide is an 18- to 22-residue long viral oligopeptide that mediates "cleavage" of a polypeptide during translation in eukaryotic cells. "2A peptide" can refer to a peptide having different amino acid sequences. In the present disclosure, it is understood that where a vector such as a lentiviral vector or a retroviral vector comprises two or more 2A peptides, the 2A peptides can be the same as 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.

[0251] "autologous": As used herein, the term "autologous" means any material derived from the same individual to which the material is subsequently reintroduced.

[0252] "allogeneic": As used herein, "allogeneic" refers to a graft derived from a different individual of the same species.

[0253] “Therapeutically effective amount”: As used herein, a “therapeutically effective amount” is the amount of a composition or active agent thereof administered to a subject that is sufficient to provide a beneficial effect or otherwise reduce deleterious, non-beneficial events, such as the amount of viral vector and cell population comprising T cells or the amount of CAR-T cells provided by the present invention. A “therapeutically effective dose” herein means the dose or doses that result in one or more of the desired or intended (e.g., beneficial) effects as a result of its administration, which is carried out one or more times over a specified period of time. The exact dose will depend on the purpose of the treatment, and can be determined by one of skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0254] “Transduction”: As used herein, the terms “transfection,” “transformation,” and “transduction” are used synonymously to refer to the process of transfer or introduction of exogenous nucleic acid into a host cell, packaging cell. A “transfected,” “transformed,” or “transduced” cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the original recipient subject cell and its progeny.

[0255] Methods of introducing a vector, such as a viral particle, or an isolated polynucleotide into a mammalian cell are known in the art. The described vectors can be transferred into the immune effector cells by physical, chemical, or biological methods.

[0256] Physical methods of introducing a vector or an isolated polynucleotide into an immune effector cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, the vector is introduced into the cell by electroporation. In some embodiments of the present invention, the vector is introduced into the cell by PEI transfection reagent.

[0257] All publications, documents, and patent references cited herein are hereby incorporated by reference in their entirety as if each had been individually incorporated by reference herein, to the extent that they provide exemplary, procedural, or other appropriate guidance to the practice of the application. In case of conflict, the present application, including any definitions herein, will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not intended to be, and shall not be given any construal that it is prior art with respect to the present application.

[0258] 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 DRAWINGS

[0259] Figure 1: is the map of the envelope plasmid in Example 1;

[0260] Figure 2: is the map of the main plasmid in Example 1;

[0261] Figure 3: is the flow cytometry result of detecting CD3 + T cells in the PBMCs collected from the patient before the treatment;

[0262] Figure 4: is the flow cytometry result of detecting the expression of CD19 in the peripheral blood of the patient before the treatment, before the reinfusion, D1, D4, D7, D14, D21, D28, and D28;

[0263] Figure 5: is the flow cytometry result of detecting the expression of CAR-19 in the peripheral blood of the patient D7, D14, D21, and D28. DETAILED DESCRIPTION

[0264] The concept and the technical effects of the present application will be described in detail below in combination with the embodiments, so as to fully understand the technical solutions, the solved technical problems and the beneficial effects of the present application. Obviously, the described embodiments are only some embodiments of the present application, but not all the embodiments; based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0265] The experimental methods not specified in the following examples are selected according to the conventional methods and conditions known in the art, or according to the instructions of the commodity. The reagents and raw materials not specified in the present application are commercially available.

[0266] Example 1

[0267] 1. Preparation of lentiviral vector mG1A3 / 28-CAR19

[0268] The virus envelope prepared in advance and frozen contains a membrane type expressing anti-CD3 antibody and anti-CD28 antibody and a mutated VSV-G (mutant VSV-G1), and a lentiviral vector mG1A3 / 28-CAR19 carrying a polynucleotide encoding a CAR molecule targeting CD19 (CAR-19); the mutant VSV-G1 contains an amino acid sequence as shown in SEQ ID NO: 6; relative to SEQ ID NO: 1, SEQ ID NO: 6 contains a K47 deletion, T214N and T352A.

[0269] The method for preparing the lentiviral vector mG1A3 / 28-CAR19 is as follows:

[0270] A. Construction of membrane type expressing anti-CD3 antibody x anti-CD28 antibody

[0271] In this example, a membrane type expressing anti-CD3 antibody x anti-CD28 antibody (double antibody) is constructed, and the structure of the membrane type expressing double antibody from N-terminus to C-terminus is as follows: CD8a signal peptide, anti-CD3 antibody (scFv-UCHT1), CD8a hinge region, CD8a transmembrane region, FT2A peptide, CD8a signal peptide, anti-CD28 antibody (scFv-15E8), CD8a hinge region, CD8a transmembrane region;

[0272] (1) The amino acid sequence of the CD8a signal peptide is as shown in SEQ ID NO: 8;

[0273] (2) The amino acid sequence of the anti-CD3 antibody (scFv) UCHT1 is as shown in SEQ ID NO: 9;

[0274] (3) The amino acid sequence of the CD8a hinge region is as shown in SEQ ID NO: 10;

[0275] (4) The amino acid sequence of the CD8a transmembrane region is as shown in SEQ ID NO: 11;

[0276] (5) The amino acid sequence of the FT2A peptide is as shown in SEQ ID NO: 12;

[0277] (6) The amino acid sequence of the anti-CD28 antibody (scFv-15E8) is as shown in SEQ ID NO: 13.

[0278] B. Construction of CAR-19

[0279] In this embodiment, a chimeric antigen receptor (CAR-19) targeting CD19 is constructed, which has the structure of CD8a signal peptide, extracellular antigen binding region targeting CD19, CD8a hinge region, CD8a transmembrane region, 4-1BB co-stimulatory signaling domain and CD3 zeta intracellular signaling domain from N-terminus to C-terminus; the extracellular antigen binding region targeting CD19 is scFv derived from FMC63 (scFv-FMC63), which comprises VH region and VL region of FMC-63, and the VH region is connected to the VL region through the (G4S)3linker peptide.

[0280] (1) the amino acid sequence of the VH region of FMC-63 is shown as SEQ ID NO: 14, and the amino acid sequence of the VL region of FMC-63 is shown as SEQ ID NO: 15;

[0281] (2) the amino acid sequence of the 4-1BB co-stimulatory domain is shown as SEQ ID NO: 17;

[0282] (3) the amino acid sequence of the intracellular signaling domain of CD3 zeta is shown as SEQ ID NO: 18;

[0283] (4) the amino acid sequence of the (G4S)3linker peptide is shown as SEQ ID NO: 16.

[0284] C. Packaging lentiviral vector mG1A3 / 28-CAR19

[0285] Prepare the following four kinds of plasmids: envelope plasmid (envelope plasmid 1, as shown in FIG. 1) containing polynucleotide encoding the mutant VSV-G1 and polynucleotide encoding the membrane type expression double antibody, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and transfer plasmid / master plasmid (master plasmid CAR-19, as shown in FIG. 2) carrying polynucleotide encoding the CAR-19; the envelope plasmid 1 and the master plasmid CAR-19 are synthesized by conventional molecular cloning method.

[0286] Mix the four kinds of plasmids, and transfect the four kinds of plasmids into packaging cells HEK-293T cells by PEI reagent, and the specific steps are as follows:

[0287] Add 9ug of the main plasmid CAR-19, 4ug of pMDLg / pRRE packaging plasmid, 2ug of pRSV-REV packaging plasmid and the 2ug of envelope plasmid 1 into 1mL of Opti-MEM medium, shake well, then add 64uL of PEI reagent, blow evenly, stand for 10 minutes, then add into the culture medium of HEK-293T cells, update the culture medium after 6 hours, collect the supernatant of the culture medium 48 hours after transfection, filter with 0.45um filter membrane, centrifuge at 50000g for 2.5h, discard the supernatant, resuspend the lentiviral vector mG1A3 / 28-CAR19 with 200uL of F12 culture medium and freeze at-80℃.

[0288] Opti-MEM alpha reduced serum medium, brand: GIBCO, item number: #SP0272;

[0289] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: brand: GIBCO, item number: #C12430500BT; FBS: brand: EXCELL, item number: #FSP500;

[0290] F12 culture medium: brand: GIBCO, item number: #C11330500BT;

[0291] Needle filter: brand: SORFA, item number: #622120.

[0292] 2. D-18~D-7: S1, collect the PBMCs of the patient, perform red cell lysis treatment

[0293] D-18~D-7:

[0294] S1, collect the PBMCs of the B-ALL patient using apheresis machine, perform red cell lysis treatment (known in the art); use flow cytometry on the PBMCs to detect the proportion of CD3 + T cells in the PBMCs, and the results are shown in Figure 3;

[0295] As can be seen from Figure 3, the proportion of CD3 + T cells in the PBMCs is 89.94%, and 5.6x10 8 PBMCs are frozen, that is, the 5.6x10 8 PBMCs contain at least 5x10 8 CD3 + T cells.

[0296] 3. D-7~D-2: S2, administer lymphocyte depletion therapy to the patient

[0297] D-7~D-2:

[0298] S2, administering 30 mg / m2 fludarabine and 300 mg / m2 melphalan to the patient for 3 days for lymphocyte-depleting conditioning (lymphodepletion); observing the patient for 2 days.

[0299] 4. D0: S3, mixing PBMCs and lentiviral vector, administering the composition to the patient

[0300] D0:

[0301] Thawing the 5.6 x 10 8 PBMCs and mixing (composition) with 2 x 10 8 TU of the lentiviral vector mG1A3 / 28-CAR19 in vitro, and then administering the composition to the patient without in vitro culture or sorting.

[0302] 5. Detecting the expression of CD19 and the preparation of CAR-T cells in the peripheral blood of the patient

[0303] Collecting the peripheral blood of the patient before lymphodepletion, before reinfusion, on D1, D4, D7, D14, D21, D28, and D28, isolating PBMCs, and detecting the expression of CD19 (B cell marker) and the preparation of CAR-T cells (detecting the expression of FMC-63) in the isolated PBMCs using flow cytometry, and the results are shown in Figures 4 and 5.

[0304] As shown in Figure 4, after the patient is administered the composition, the number of CD19 + target cells in the peripheral blood of the patient is significantly reduced, and on D21 and D28, the peripheral blood of the patient almost does not contain CD19 + target cells;

[0305] As shown in Figure 5, the presence of CAR-T cells is detected on D14, D21, and D28, and on D21, the number of CAR-T cells in the PBMCs of the patient is as high as 22.55%;

[0306] Therefore, the composition can efficiently prepare CAR-T cells in the patient, and then effectively kill CD19 + target cells.

Claims

1. A composition characterized in that, The composition comprises a viral vector and a population of cells comprising T cells; the viral vector: (a) comprises a heterologous polynucleotide encoding a chimeric antigen receptor (CAR) that can specifically bind an antigen associated with a disease; and (b) comprises one or more targeting molecules on the surface that can specifically bind an endocytic receptor on the surface of a T cell.

2. The composition of claim 1, wherein, The viral vector is a retroviral vector.

3. The composition of claim 2, wherein, The retroviral vector is a pseudotyped lentiviral vector.

4. The composition according to claim 2 or 3, characterized in that, The viral glycoprotein of the viral vector is selected from the group consisting of Vesiculovirus strain glycoprotein, NiV glycoprotein G, Measles virus glycoprotein H, Lentivirus glycoprotein, Rabies virus glycoprotein (RVG), GaLV glycoprotein, MLV-A glycoprotein, RD114 glycoprotein, FPV glycoprotein, EboV glycoprotein, and LCMV glycoprotein. The Vesiculovirus strain glycoprotein is selected from the group consisting of Vesiculovirus Indiana strain glycoprotein, Vesiculovirus Cocal strain glycoprotein, Vesiculovirus Maraba strain glycoprotein, Vesiculovirus Morreton strain glycoprotein, Vesiculovirus Alagoas strain glycoprotein, Vesiculovirus New Jersey strain glycoprotein, Vesiculovirus Carajas strain glycoprotein, Vesiculovirus Chandipura strain glycoprotein, Vesiculovirus Eptesicus strain glycoprotein, Vesiculovirus Isfahan strain glycoprotein, Vesiculovirus Jurona strain glycoprotein, Vesiculovirus Malpais strain glycoprotein, Vesiculovirus Perinet strain glycoprotein, Vesiculovirus Piry strain glycoprotein, Vesiculovirus Radi strain glycoprotein, Vesiculovirus Rhinolopus strain glycoprotein, and Vesiculovirus Yug Bogdanovac strain glycoprotein.

5. The composition of claim 4, wherein, The viral glycoprotein is Vesiculovirus Indiana strain glycoprotein (VSV-G) or Vesiculovirus Cocal strain glycoprotein (Cocal-G), and the viral glycoprotein receptor is Low Density Lipoprotein Receptor ("LDL-R").

6. The composition according to any one of claims 2-5, characterized in that, The viral glycoprotein of the viral vector is inhibited in its ability to bind its receptor.

7. The composition of claim 6, wherein, The viral glycoprotein is VSV-G or Cocal-G, and corresponding variants, and the viral glycoprotein is inhibited in its ability to bind its receptor LDL-R.

8. The composition of claim 7, wherein, The viral glycoprotein comprises a first mutation that inhibits the viral glycoprotein's ability to bind its receptor.

9. The composition of claim 8, wherein, The first mutation comprises one or more of the following mutations: (a) a substitution or deletion at amino acid position 8, a substitution or deletion at amino acid position 9, a substitution or deletion at amino acid position 10, a substitution or deletion at amino acid position 47, a substitution or deletion at amino acid position 50, a substitution or deletion at amino acid position 51, a substitution or deletion at amino acid position 183, a substitution or deletion at amino acid position 179, a substitution or deletion at amino acid position 180, a substitution or deletion at amino acid position 182, a substitution or deletion at amino acid position 184, a substitution or deletion at amino acid position 209, a substitution or deletion at amino acid position 347, a substitution or deletion at amino acid position 350, a substitution or deletion at amino acid position 352, a substitution or deletion at amino acid position 353, a substitution at amino acid position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a substitution or deletion at amino acid position 8, a substitution or deletion at amino acid position 9, a substitution or deletion at amino acid position 10, a substitution or deletion at amino acid position 47, a substitution or deletion at amino acid position 50, a substitution or deletion at amino acid position 51, a substitution or deletion at amino acid position 183, a substitution or deletion at amino acid position 179, a substitution or deletion at amino acid position 180, a substitution or deletion at amino acid position 182, a substitution or deletion at amino acid position 184, a substitution or deletion at amino acid position 209, a substitution or deletion at amino acid position 347, a substitution or deletion at amino acid position 350, a substitution or deletion at amino acid position 352, a substitution or deletion at amino acid position 353, a substitution at amino acid position 354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 following optimal global alignment of SEQ ID NO: 1 or SEQ ID NO: 2 to SEQ ID NO: 1 or SEQ ID NO:

2.

10. The composition of claim 9, wherein, The first mutation comprises one or more of the following mutations: (a) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of 1182 of SEQ ID NO:

1. (b) is a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of I182, of SEQ ID NO: l following a global optimal alignment of SEQ ID NO: l; (c) is a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of V182, of SEQ ID NO: 2; and (d) is a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, a substitution of R354, a substitution of V182, of SEQ ID NO: 2 following a global optimal alignment of SEQ ID NO:

2.

11. The composition of claim 10, wherein, the first mutation comprises a mutation at: (a) K47 of SEQ ID NO: l or SEQ ID NO: 2; or (b) K47 of SEQ ID NO: l or SEQ ID NO: 2 following a global optimal alignment of SEQ ID NO: l or SEQ ID NO:

2.

12. The composition according to any one of claims 8-11, characterized in that, the viral glycoprotein of the viral vector comprises a second mutation that enhances or abolishes the ability of the viral glycoprotein to be inactivated by complement.

13. The composition of claim 12, wherein, the viral glycoprotein is VSV-G or Cocal-G and the viral glycoprotein receptor is LDL-R.

14. The composition of claim 13, wherein, the second mutation comprises a mutation at one or more of the following positions: (a) amino acid 214 of SEQ ID NO: l or SEQ ID NO: 2; (b) amino acid 214 of SEQ ID NO: l or SEQ ID NO: 2 following a global optimal alignment of SEQ ID NO: l or SEQ ID NO: 2; (c) amino acid 352 of SEQ ID NO: l or SEQ ID NO: 2; (d) amino acid 352 of SEQ ID NO: l or SEQ ID NO: 2 following a global optimal alignment of SEQ ID NO: l or SEQ ID NO: 2; (e) amino acid 50 of SEQ ID NO: l or SEQ ID NO: 2; (f) amino acid 50 of SEQ ID NO: l or SEQ ID NO: 2 following a global optimal alignment of SEQ ID NO: l or SEQ ID NO: 2; (g) amino acid 146 of SEQ ID NO: l or SEQ ID NO: 2; and (h) amino acid 146 of SEQ ID NO: l or SEQ ID NO: 2 following a global optimal alignment of SEQ ID NO: l or SEQ ID NO:

2. the mutation at the position is selected from the group consisting of a substitution, a deletion, and an insertion of an amino acid.

15. The composition of claim 14, wherein, the mutation at the position is a substitution of an amino acid. the mutation at the position is a substitution of an amino acid.

16. The composition of claim 15, wherein, The second mutation comprises any combination of the following site mutations: (a) substitutions at (1) T214 and T352 of SEQ ID NO: 1; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; (b) substitutions at (1) T214 and T352 of SEQ ID NO: 1; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO: 1; (c) substitutions at (1) K214 and T352 of SEQ ID NO: 2; or (2) K214, T352, K50, and S146 of SEQ ID NO: 2; and (d) substitutions at (1) K214 and T352 of SEQ ID NO: 2; or (2) K214, T352, K50, and S146 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:

2.

17. The composition of claim 16, wherein, The second mutation comprises any combination of the following site mutations: (a) T214N and T352A of SEQ ID NO: 1; or (2) T214N, T352A, K50T, and S146T of SEQ ID NO: 1; (b) T214N and T352A of SEQ ID NO: 1; or (2) T214N, T352A, K50T, and S146T of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO: 1; (c) K214N and T352A of SEQ ID NO: 2; or (2) K214N, T352A, K50T, and S146T of SEQ ID NO: 2; and (d) K214N and T352A of SEQ ID NO: 2; or (2) K214N, T352A, K50T, and S146T of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:

2.

18. The composition of any one of claims 1-17, wherein, The endocytic receptor on the surface of the T cell is selected from one or more of: CD2, CD3, CD3y, CD35, CD3s, TCRy, TCR5, TCRa, TCRp, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD47, CD57, CD62L, CD71, AhR, CD69, CD94, CD95, 4-1BB, CD103, CD122, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), PD-1, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, IL6ST, P2RX7, TIGIT, TIM-3, and LAG-3.

19. The composition of any one of claims 1-18, wherein, The endocytic receptor on the surface of the T cell comprises CD3; The targeting molecule that can specifically bind CD3 comprises an anti-CD3 antibody or antigen-binding fragment thereof.

20. The composition of any one of claims 1-19, wherein, the endocytic receptor on the surface of the T cell comprises CD28; the targeting molecule that can specifically bind CD28 comprises one or more of an anti-CD28 antibody or antigen-binding fragment thereof and a CD28 ligand or receptor-binding fragment thereof.

21. The composition of claim 20, wherein, the CD28 ligand or receptor-binding fragment thereof is selected from one or more of CD80 or a receptor-binding fragment thereof and CD86 or a receptor-binding fragment thereof.

22. The composition of any one of claims 1-21, wherein, the targeting molecule comprises a targeting binding region comprising one or more of an antibody or antigen-binding fragment thereof and a ligand or receptor-binding fragment thereof that can bind to an endocytic receptor on the surface of a T cell.

23. The composition of claim 22, wherein, when the ligand is a transmembrane protein, the targeting binding region comprises only a receptor-binding fragment of the ligand.

24. The composition of claim 22 or 23, wherein, the targeting molecule further comprises a transmembrane region, the targeting binding region being directly or indirectly linked to the transmembrane region.

25. The composition of claim 24, wherein, the transmembrane region comprises a transmembrane region of any one of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8a, CD8b, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIg, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

26. The composition of claim 25, wherein, the transmembrane region comprises a transmembrane region of CD8a.

27. The composition of any one of claims 22-26, wherein, the targeting molecule further comprises a linker domain, the targeting binding region being indirectly linked to the transmembrane region via the linker domain.

28. The composition of claim 27, wherein, the linker domain is selected from: (i) an immunoglobulin hinge region selected from wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (ii) a hinge region selected from wild-type or modified hinge regions of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (iii) all or a portion of an Fc domain selected from one or more of a CHI domain, a CH2 domain, and a CH3 domain; and (iv) a stalk region of a type II C-lectin selected from stalk regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D.

29. The composition of claim 27, the linker domain being a hinge region of CD8a.

30. The composition of any one of claims 1-29, wherein, The CAR comprises an extracellular antigen binding region, a transmembrane region, and an intracellular signaling domain.

31. The composition of claim 30, wherein, The extracellular antigen binding region of the CAR can specifically bind to an antigen associated with a disease; (a) the transmembrane region of the CAR comprises a transmembrane region of any one of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8a, CD8b, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIy, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; (b) the intracellular signaling domain of the CAR is selected from the ITAM intracellular signaling domain of CD3e, CD3y, CD35, CD3zeta, CD79a, CD79b, FceRly, FceRb, FcyRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other proteins comprising at least one ITAM intracellular signaling domain.

32. The composition of claim 31, wherein, The transmembrane region of the CAR comprises the transmembrane region of CD8a; the intracellular signaling domain of the CAR is the intracellular signaling domain of CD3zeta.

33. The composition of any one of claims 30-32, wherein, The CAR further comprises a linker domain and a costimulatory signaling domain; the linker domain links the extracellular antigen binding region and the transmembrane region of the CAR; The linker domain of the CAR is selected from: (i) an immunoglobulin hinge region, the immunoglobulin hinge region being selected from the wild type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region; (ii) a hinge region, the hinge region being selected from the wild type or modified hinge region of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (iii) all or a portion of an Fc domain, the Fc domain being selected from one or more of the CH1 domain, the CH2 domain, and the CH3 domain; and (iv) a stalk region of a type II C-lectin, the type II C-lectin being selected from the stalk region of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; The costimulatory signaling domain of the CAR comprises a costimulatory signaling domain of one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8a, CD8b, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcaRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-l, LFA-1, LIGHT, JAmL, CD244, CD100, ICOS, CD40, and MyD88.

34. The composition of any one of claims 1-33, wherein, The cell population comprising T cells is selected from the group consisting of leukocytes, PBMCs, and CD3 + one or more of the T cells.

35. The composition of claim 34, wherein, The cell population comprising T cells is collected from an autologous or allogeneic donor of the subject; the subject is the individual to whom the composition is administered.

36. The composition of claim 35, wherein, The subject or the allogeneic donor of the subject has not received any lymphodepleting therapy for conditioning at least 4 weeks (inclusive) prior to the collection.

37. The composition of any one of claims 1-36, wherein, The composition further comprises a pharmaceutically acceptable carrier or excipient.

38. A method of making a chimeric antigen receptor-expressing T cell (CAR-T cell), characterized in that, The method comprises the following steps: S1, collecting a cell population comprising T cells from an autologous or allogeneic donor of the subject; S2, conditioning the subject with a lymphodepleting therapy; and S3, administering the composition of claims 1-37 to the subject.

39. The method of claim 38, wherein, In S3, the composition is mixed and administered to the subject.

40. The method of claim 39, wherein, The composition is mixed for 1 minute to 2 hours and administered to the subject.

41. The method of claim 38, wherein, In S3, after the administration of the cell population comprising T cells or the viral vector in the composition to the subject (first administration), the viral vector or the cell population comprising T cells is administered to the subject (second administration); When the first administration is the administration of the cell population comprising T cells to the subject, the second administration is the administration of the viral vector to the subject; When the first administration is the administration of the viral vector to the subject, the second administration is the administration of the cell population comprising T cells to the subject.

42. The method of claim 41, wherein, In S3, the second administration is performed within 1 minute to 7 days of the first administration.

43. The method of claim 42, wherein, In S3, the second administration is performed within 24 hours of the first administration.

44. The method of claim 43, wherein, In S3, the second administration is performed within 60 minutes of the first administration.

45. The method of claim 38, wherein, In S3, the cell population comprising T cells and the viral vector in the composition are simultaneously administered to the subject using a dual-lumen catheter (dual-lumen catheter administration).

46. The method of any one of claims 38-45, wherein, In S3, after the administration of the composition to the subject, the completion of the second administration, or the completion of the dual-lumen catheter administration, the subject can be further subjected to one or more of the following: dual-lumen catheter administration, administration of the composition, administration of the cell population comprising T cells, and administration of the viral vector.

47. The method of any one of claims 38-46, wherein, In S1, the subject or the allogeneic donor of the subject has not received any lymphodepleting therapy for conditioning at least 4 weeks (inclusive) prior to the collection.

48. The method of any one of claims 38-47, wherein, In S2, the lymphodepleting therapy comprises administering to the subject an immunosuppressive agent.

49. The method of claim 48, wherein, The subject is administered an immunosuppressive agent daily for 2-4 days.

50. The method of claim 49, wherein, The immunosuppressive agent is selected from one or more of cyclophosphamide and fludarabine.

51. The method of claim 50, wherein, The immunosuppressive agent is 20-40 mg / m2 fludarabine and 200-400 mg / m2 cyclophosphamide.

52. The method of any one of claims 38-51, wherein, In S2, the lymphodepleting therapy is administered within 2 to 7 days prior to administering the composition to the subject.

53. The method of claim 39, wherein, The preparation of the CAR-T cells occurs in vivo and / or ex vivo in the subject.

54. The method of any one of claims 40-46, wherein, The preparation of the CAR-T cells occurs in vivo in the subject.

55. Use of the composition of any one of claims 1-37 in the manufacture of a CAR-T cell.

56. Use of the composition of any one of claims 1-37 in the manufacture of a medicament for the treatment of a disease.

57. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-56. a step comprising a method of making a chimeric antigen receptor-expressing T cell (CAR-T cell) as in any one of claims 38-54.

58. The method of treatment according to claim 57, wherein administering to the subject a cell population comprising 5 x 10 6 -5 x 10 9 CD3 + T cells; administering to the subject 1 x 10 3 -1 x 10 9 TU / KG of the viral vector.

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