Method of preparing car-t cells and use thereof

Abstract

Disclosed are a method of preparing CAR-T cells, and a composition used in said method. The method comprises: S1, administering, as a pretreatment, lymphocyte depletion therapy to a subject having cancer; and S2, administering to the subject (a) a cell population comprising T cells collected from a haploidentical donor thereof and (b) a viral vector. The method allows for effective preparation of CAR-T cells in a subject.

Description

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

[0001] The present application relates to the field of cell therapy, in particular to a method for preparing CAR-T cells and application 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] At present, CAR-T cell therapy includes in vitro CAR-T cell therapy and in vivo CAR-T cell therapy.

[0004] Generally, in vitro CAR-T cell therapy includes collecting and sorting T cells of a patient, stimulating and activating T cells, transducing T cells to express CAR in vitro, expanding CAR-T cells, and reinfusing (see relevant patent documents, such as Chinese Invention Patent No. CN109652378A and Chinese Invention Patent No. CN112292147A).

[0005] In vitro CAR-T cell therapy has a long preparation period, a complex preparation process, and a high preparation cost, such as general CAR-T cells that need to be knocked out of TCR and HLA genes, and the in vitro CAR-T cell therapy has poor persistency in the patient's body after reinfusion, and the curative effect is limited.

[0006] In vivo CAR-T cell therapy includes using a virus vector (such as an in vivo lentivirus vector carrying a CAR gene) that can transduce T cells in the patient's body to administer to a patient with or without lymphocyte clearance (pre-treatment for lymphocyte clearance); however, after the patient is pre-treated with lymphocyte clearance, the number of T cells available for in vivo lentivirus vector transduction is extremely small due to the large number of T cells in the patient's body, so it is difficult to effectively prepare CAR-T cells in the patient's body; however, if the patient is not pre-treated with lymphocyte clearance, although the patient still has T cells available for in vivo lentivirus vector transduction, the endogenous T cells in the patient's body will release inhibitory signals to inhibit the preparation of CAR-T cells, and it is also difficult to achieve the therapeutic purpose of effectively killing cancer cells in the patient's body.

[0007] Therefore, in vivo CAR-T cell therapy can also administer an in vivo lentivirus vector and a cell population containing T cells to a patient with lymphocyte clearance at the same time, such as administering the in vivo lentivirus vector and the cell population at the same time through a double-channel pipe, mixing the in vivo lentivirus vector and the cell population, and administering the mixture; or administering the in vivo lentivirus vector and the cell population in a short period of time.

[0008] However, when the number and / or function of T cells of a patient is abnormal (e.g., the number of T cells in PBMCs is insufficient or the killing ability is insufficient), even if the patient receives an autologous cell population comprising T cells collected before the patient receives the clear broth therapy, it is difficult for the lentiviral vector in vivo to effectively transduce the abnormal T cells of the patient autologously and effectively prepare CAR-T cells. SUMMARY

[0009] In view of the above, to solve at least one or more of the above technical problems, including the difficulty of lentiviral vectors in vivo to transduce T cells of a patient autologously with insufficient number and / or function, the present disclosure provides a composition comprising a viral vector and a cell population comprising T cells; the cell population comprising T cells is collected from a semi-identical donor of a subject; the subject is an individual to be administered the composition;

[0010] wherein the viral vector:

[0011] (a) comprises a heterologous polynucleotide encoding a chimeric antigen receptor (CAR) that can specifically bind to a cancer-associated antigen; and

[0012] (b) comprises one or more targeting molecules on the surface, which can specifically bind to an endocytosis receptor on the surface of a T cell.

[0013] In some embodiments of the present disclosure, the viral vector is a retroviral vector.

[0014] In some embodiments of the present disclosure, the retroviral vector is a pseudotyped lentiviral vector.

[0015] In some embodiments of the present disclosure, the viral glycoprotein of the viral vector is selected from the group consisting of vesicular stomatitis virus (VSV) strain glycoprotein, NiV glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowlpox virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and T cell-specific choroid plexus meningoencephalitis virus (LCMV) glycoprotein.

[0016] 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.

[0017] In some embodiments of the present application, 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 (Low Density Lipoprotein Receptor, "LDL-R").

[0018] 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.

[0019] In some embodiments of the present application, the VSV-G (wild type) comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0020] In some embodiments of the present application, the full-length protein of the wild type VSV-G (including the signal peptide) comprises an amino acid sequence as shown in SEQ ID NO: 20;

[0021] wherein the amino acid sequence as shown in SEQ ID NO: 20 at position 1-16 is:

[0022] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild type VSV-G.

[0023] In some embodiments of the present application, the Cocal-G (wild type) comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0024] In some embodiments of the present application, the full-length protein of the wild type Cocal-G (including signal peptide) comprises an amino acid sequence as set forth in SEQ ID NO: 27;

[0025] wherein the sequence as set forth in positions 1-17 of SEQ ID NO: 27:

[0026] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild type Cocal-G.

[0027] In some embodiments of the present application, the ability of the viral glycoprotein of the pseudotyped lentiviral vector or retroviral vector to bind to its receptor is inhibited;

[0028] Preferably, the viral glycoprotein is VSV-G or Cocal-G, and the viral glycoprotein receptor is LDL-R.

[0029] More preferably, the viral glycoprotein comprises a first mutation, such that the ability of the viral glycoprotein to bind to its receptor is inhibited.

[0030] Further more preferably, the first mutation comprises one or more of the following mutations:

[0031] (a) substitution or deletion of the amino acid at position 8, substitution or deletion of the amino acid at position 9, substitution or deletion of the amino acid at position 10, substitution or deletion of the amino acid at position 47, substitution or deletion of the amino acid at position 50, substitution or deletion of the amino acid at position 51, substitution or deletion of the amino acid at position 183, substitution or deletion of the amino acid at position 179, substitution or deletion of the amino acid at position 180, substitution or deletion of the amino acid at position 182, substitution or deletion of the amino acid at position 184, substitution or deletion of the amino acid at position 209, substitution or deletion of the amino acid at position 347, substitution or deletion of the amino acid at position 350, substitution or deletion of the amino acid at position 352, substitution or deletion of the amino acid at position 353, substitution of the amino acid at position 354, deletion of the amino acids at positions 1-18, deletion of the amino acids at positions 19-36, deletion of the amino acids at positions 37-51, deletion of the amino acids at positions 314-384, deletion of the amino acids at positions 321-374, deletion of the amino acids at positions 331-364, deletion of the amino acids at positions 344-354, deletion of the amino acids at positions 345-353; and

[0032] (b) a substitution or deletion at an amino acid corresponding to position 8, a substitution or deletion at an amino acid corresponding to position 9, a substitution or deletion at an amino acid corresponding to position 10, a substitution or deletion at an amino acid corresponding to position 47, a substitution or deletion at an amino acid corresponding to position 50, a substitution or deletion at an amino acid corresponding to position 51, a substitution or deletion at an amino acid corresponding to position 183, a substitution or deletion at an amino acid corresponding to position 179, a substitution or deletion at an amino acid corresponding to position 180, a substitution or deletion at an amino acid corresponding to position 182, a substitution or deletion at an amino acid corresponding to position 184, a substitution or deletion at an amino acid corresponding to position 209, a substitution or deletion at an amino acid corresponding to position 347, a substitution or deletion at an amino acid corresponding to position 350, a substitution or deletion at an amino acid corresponding to position 352, a substitution or deletion at an amino acid corresponding to position 353, a substitution at an amino acid corresponding to 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 to SEQ ID NO: 1 or SEQ ID NO: 2;

[0033] Still more preferably, the first mutation comprises one or more of the following mutations:

[0034] (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 1181, of SEQ ID NO: 1 ;

[0035] (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 1181, of SEQ ID NO: 1 following optimal global alignment to SEQ ID NO: 1 ;

[0036] (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

[0037] (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 optimal global alignment to SEQ ID NO: 2;

[0038] Most preferably, the first mutation comprises the following mutation:

[0039] (a) a deletion at K47 of SEQ ID NO: 1 or SEQ ID NO: 2; or

[0040] (b) a deletion at a position corresponding to K47 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] The complement system is composed of a series of proteins, which is part of the innate immune system. Complement (C) exists in normal human and animal serum, tissue fluid and cell membrane surface, 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 and the like may 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 a subject.

[0042] In some embodiments of the application, the viral glycoprotein of the pseudotyped lentiviral vector or retroviral vector comprises a second mutation, which enhances or does not inactivate the ability of the viral glycoprotein to antagonize complement inactivation;

[0043] Preferably, the viral glycoprotein is VSV-G or Cocal-G;

[0044] More preferably, the second mutation comprises one or more mutations at the following positions:

[0045] (a) the 214th amino acid of SEQ ID NO: 1 or SEQ ID NO: 2;

[0046] (b) the 214th amino acid 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] (c) the 352nd amino acid of SEQ ID NO: 1 or SEQ ID NO: 2;

[0048] (d) at the amino acid corresponding to position 352 of SEQ ID NO: 1 or SEQ ID NO: 2 after the best global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0049] (e) at the amino acid corresponding to position 50 of SEQ ID NO: 1 or SEQ ID NO: 2 after the best global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0050] (f) at the amino acid corresponding to position 50 of SEQ ID NO: 1 or SEQ ID NO: 2 after the best global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0051] (g) at the amino acid corresponding to position 146 of SEQ ID NO: 1 or SEQ ID NO: 2 after the best global alignment of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0052] (h) at the amino acid corresponding to position 146 of SEQ ID NO: 1 or SEQ ID NO: 2 after the best global alignment of SEQ ID NO: 1 or SEQ ID NO: 2;

[0053] Still further preferably, the mutation at the site is selected from the group consisting of a substitution, a deletion, and an insertion of an amino acid;

[0054] Yet further preferably, the mutation at the site is a substitution of an amino acid.

[0055] In some embodiments of the application, the second mutation comprises a combination of mutations at any one of the following sites:

[0056] (a) substitution of (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1;

[0057] (b) substitution of (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1 after the best global alignment of SEQ ID NO: 1;

[0058] (c) substitution of (1) K214 and T352; or (2) K214, T352, K50, and S146 of SEQ ID NO: 2; and

[0059] (d) substitution of (1) K214 and T352; or (2) K214, T352, K50, and S146 of SEQ ID NO: 2 after the best global alignment of SEQ ID NO: 2;

[0060] Preferably, the mutation comprises a combination of mutations at any one of the following sites:

[0061] (a) at (1) T214N and T352A of SEQ ID NO: 1; or (2) T214N, T352A, K50T, and S146T of SEQ ID NO: 1;

[0062] (b) at (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;

[0063] (c) at (1) K214N and T352A of SEQ ID NO: 2; or (2) K214N, T352A, K50T, and S146T of SEQ ID NO: 2; and

[0064] (d) at (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.

[0065] In some embodiments of the application, the viral glycoprotein is VSV-G, and the second mutation comprises any combination of the following substitutions:

[0066] (a) at (1) T214 and T352 of SEQ ID NO: 1; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; or

[0067] (b) 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;

[0068] 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.

[0069] 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.

[0070] In some embodiments of the application, the endocytic receptor on the surface of the T cell is selected from the group consisting of:

[0071] one or more of CD2, CD3, CD3y, CD3d, CD3s, TCRy, TCRd, TCRa, TCRb, 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.

[0072] "Endocytosis" refers to a process by which substances enter a cell, in which the substance to be taken in is surrounded by a region of the plasma membrane, which then buds off within the cell to form a vesicle containing the substance taken in. 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).

[0073] "Endocytic receptor" refers to a receptor that can mediate endocytosis. The surface of lymphocytes, such as T cells, NK cells, and B cells, expresses a variety of endocytic receptors.

[0074] By constructing a targeting molecule that can specifically bind to an endocytic receptor on the surface of T cells on a viral vector, the targeting of the pseudotyped lentiviral vector or retroviral vector can be further improved.

[0075] In some embodiments of the present application, the endocytic receptor on the surface of T cells is selected from one or more of CD5, CD7, CD3, and CD28.

[0076] In some embodiments of the present application, the endocytic receptor on the surface of T cells comprises CD3.

[0077] Preferably, the targeting molecule that can specifically bind to CD3 comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

[0078] In some embodiments of the application, the endocytic receptor on the surface of the T cell comprises CD28;

[0079] Preferably, 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.

[0080] More preferably, 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.

[0081] In some embodiments of the application, 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 specifically bind the endocytic receptor on the surface of the T cell;

[0082] Preferably, when the ligand is a transmembrane protein, the targeting binding region comprises only a receptor-binding fragment of the ligand.

[0083] In some embodiments of the application, the receptor-binding fragment of the ligand comprises an extracellular region, a variable region or a binding region of the ligand.

[0084] 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;

[0085] Preferably, the transmembrane region comprises a transmembrane region of any one of:

[0086] 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;

[0087] More preferably, the transmembrane region comprises a transmembrane region of CD8a.

[0088] In some embodiments of the application, the targeting molecule further comprises a linker domain, the targeting binding region is indirectly linked to the transmembrane region via the linker domain;

[0089] Preferably, the linker domain is selected from the group consisting of:

[0090] (i) an immunoglobulin hinge region selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0091] (ii) a hinge region selected from the group consisting of 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;

[0092] (iii) all or a portion of an Fc domain selected from the group consisting of one or more of a CHI domain, a CH2 domain and a CH3 domain; and

[0093] (iv) a stalk domain of a type II C-lectin selected from the group consisting of stalk domains of CD23, CD69, CD72, CD94, NKG2A and NKG2D;

[0094] More preferably, the linker domain is a hinge region of CD8a.

[0095] In some embodiments of the application, any of the foregoing targeting molecules further comprises a leader signal peptide.

[0096] Preferably, the leader signal peptide is selected from the group consisting of a CD8a signal peptide, a CD28 signal peptide, an IgG signal peptide and an HLA-A signal peptide.

[0097] More preferably, the leader signal peptide is a CD8a signal peptide.

[0098] In some embodiments of the application, the CAR comprises an extracellular antigen binding region, a transmembrane region and an intracellular signaling domain.

[0099] In some embodiments of the application, the extracellular antigen binding region of the CAR can specifically bind to the cancer-associated antigen;

[0100] Preferably, the cancer-associated antigen is selected from the group consisting of:

[0101] 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).

[0102] More preferably, the cancer-associated antigen is selected from one or more of CD19, CD20, CD33, CD79A, CD79B, HER2, CEA, and BCMA.

[0103] 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).

[0104] In some embodiments of the application,

[0105] (a) the transmembrane region of the CAR comprises the transmembrane region of any one of the following proteins:

[0106] 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;

[0107] Preferably, the transmembrane region of the CAR comprises the transmembrane region of CD8a.

[0108] (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, 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;

[0109] Preferably, the intracellular signaling domain of the CAR is the intracellular signaling domain of CD3z.

[0110] 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;

[0111] Preferably, the linking domain of the CAR is selected from:

[0112] (i) an immunoglobulin hinge region selected from wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0113] (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;

[0114] (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

[0115] (iv) a stalk region of a type II C-lectin selected from stalk regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0116] More preferably, the linking domain of the CAR is a hinge region of CD8a.

[0117] 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.

[0118] More preferably, the costimulatory signaling domain of the CAR comprises a costimulatory signaling domain of 4-1BB.

[0119] 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 cancer-associated antigens and a ligand or receptor-binding fragment thereof.

[0120] In some embodiments of the application, the extracellular antigen-binding region of the CAR is monospecific, bi-specific, or multi-specific.

[0121] 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.

[0122] 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: 14; 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] In some embodiments of the application, the co-stimulatory signaling domain of the CAR is a co-stimulatory signaling domain of 4-1BB comprising 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: 17.

[0127] In some embodiments of the application, the polynucleotide encoding the CAR is operably linked to a polynucleotide encoding a leader signal peptide.

[0128] In some embodiments of the application, the CAR comprises, from N-terminus to C-terminus, a leader signal peptide, an extracellular antigen binding region, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signaling domain.

[0129] Preferably, the leader signal peptide is selected from the group consisting of a CD8a signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide.

[0130] More preferably, the leader signal peptide is a CD8a signal peptide.

[0131] In some embodiments of the application, the population of cells is selected from the group consisting of white blood cells, peripheral blood mononuclear cells (PBMCs), and CD3 + T cells.

[0132] In some embodiments of the application, the white blood cells, PBMCs, or CD3 + T cells are collected by apheresis.

[0133] In some embodiments of the application, the PBMCs are isolated from peripheral blood of a semi-allogeneic donor of the subject by venipuncture.

[0134] In some embodiments of the application, the composition comprises any of the foregoing viral vectors (including endpoints) comprising 5 x 10 6 - 5 x 10 9 CD3 + T cells per TU / KG (including endpoints). 3 - 1 x 10 9 TU / KG (including endpoints).

[0135] In some embodiments of the application, the subject has an abnormal number and / or function of T cells.

[0136] In some embodiments of the application, the subject has an abnormal number of T cells.

[0137] In some embodiments of the application, the subject has an abnormal killing ability of T cells.

[0138] In some embodiments of the application, the subject is an end-line patient.

[0139] In some embodiments of the application, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0140] In another aspect, a method of preparing a chimeric antigen receptor-expressing T cell (CAR-T cell) is disclosed, comprising the steps of:

[0141] S1, administering a lymphodepleting therapy to a subject having cancer for pretreatment; and

[0142] S2, administering the composition to the subject.

[0143] In some embodiments of the application, in S2, the cell population and the viral vector in the composition are mixed, and the composition is administered to the subject.

[0144] Preferably, the cell population 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.

[0145] In some embodiments of the application, in S2, after the administration of the cell population or the viral vector to the subject (first administration), the viral vector or the cell population is administered to the subject (second administration).

[0146] When the first administration is the administration of the cell population to the subject, the second administration is the administration of the viral vector to the subject.

[0147] When the first administration is the administration of the viral vector to the subject, the second administration is the administration of the cell population to the subject.

[0148] In some embodiments of the application, in S2, 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.

[0149] 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.

[0150] 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.

[0151] In some embodiments of the application, in S2, the cell population and the viral vector are simultaneously administered to the subject using a dual-lumen catheter (dual-lumen catheter administration); wherein the cell population is administered using one lumen of the dual-lumen catheter and the viral vector is administered using the other lumen of the dual-lumen catheter.

[0152] In some embodiments of the application, in S2, 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 subjected to one or more of: the dual-lumen catheter administration, the administration of the composition, the administration of the cell population, and the administration of the viral vector.

[0153] In some embodiments of the application, in S1, the lymphodepleting therapy comprises administering to the subject an immunosuppressive agent.

[0154] Preferably, the immunosuppressive agent is administered to the subject daily for 2-4 days; more preferably, for 3 days.

[0155] In some embodiments of the application, the immunosuppressive agent comprises one or more of cyclophosphamide and fludarabine;

[0156] Preferably, the immunosuppressive agent comprises fludarabine and cyclophosphamide;

[0157] More preferably, the immunosuppressive agent comprises 20-40 mg / m2 fludarabine and 200-400 mg / m2 cyclophosphamide;

[0158] Further more preferably, the immunosuppressive agent is 30 mg / m2 fludarabine and 300 mg / m2 cyclophosphamide.

[0159] In some embodiments of the application, in S1, the administration of the lymphodepleting therapy is completed within 2 to 7 days prior to the performance of S2.

[0160] In some embodiments of the application, in S2, the haploidentical donor of the subject is a healthy donor; more preferably, the subject is a human.

[0161] In some embodiments of the application, the preparation of the CAR-T cells occurs in vivo and / or ex vivo in the subject;

[0162] Preferably, the subject is a human.

[0163] In some embodiments of the application, the preparation of the CAR-T cells occurs in vivo in the subject;

[0164] Preferably, the subject is a human.

[0165] In some embodiments of the application, the subject is administered a combination of any of the foregoing cell populations and any of the foregoing viral vectors, and the preparation of the CAR-T cells occurs in vivo and / or ex vivo in the subject.

[0166] In some embodiments of the application, the subject is administered any of the foregoing first administration and second administration, and the preparation of the CAR-T cells occurs in vivo in the subject.

[0167] In some embodiments of the application, the subject is administered the dual access administration, and the preparation of the CAR-T cells occurs in vivo in the subject.

[0168] In some embodiments of the application, the hematological cancer is a relapsed / refractory hematological cancer.

[0169] In some embodiments of the application, the administering is selected from one or more of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection, nodal injection, infusion techniques, oral, nasal, intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, transportal, intralesional, sustained release system, and implanted device.

[0170] In some embodiments of the application, the subject is administered 5 x 10 6 In some embodiments of the application, the subject is administered 5 x 10 9 In some embodiments of the application, the subject is administered 5 x 10 + In some embodiments of the application, the subject is administered 1 x 10 3 In some embodiments of the application, the subject is administered 1 x 10 9 In some embodiments of the application, the subject is administered 1 x 10

[0171] In some embodiments of the application, the subject has abnormal number and / or function of T cells.

[0172] In some embodiments of the application, the subject has abnormal number of T cells.

[0173] In some embodiments of the application, the subject has abnormal killing ability of T cells.

[0174] In some embodiments of the application, the subject is an end-line patient.

[0175] In another aspect, the present application also discloses use of any one of the aforementioned compositions in the preparation of CAR-T cells.

[0176] In another aspect, the present application also discloses use of any one of the aforementioned compositions in the preparation of a medicament for treating cancer, the cancer being selected from one or more of a hematological cancer and a solid cancer.

[0177] Preferably, the hematological cancer is a CD19-expressing hematological cancer.

[0178] More preferably, the CD19-expressing hematological cancer includes 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-cell rich large B-cell lymphoma; and acute B-lymphoblastic leukemia.

[0179] In another aspect, the present application also discloses a method for treating cancer, the method comprising:

[0180] S1, administering a lymphodepleting therapy to a subject having cancer for preconditioning; and

[0181] S2, administering to the subject (a) a cell population comprising T cells (cell population) collected from a half-matched donor thereof and (b) a viral vector; the viral vector:

[0182] (a) comprises a heterologous polynucleotide encoding a chimeric antigen receptor (CAR) that can specifically bind to a cancer-associated antigen; and

[0183] (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.

[0184] The functional normal T cells from the half-matched donor can help to reconstitute or partially reconstitute the T cells of the patient; and, while the CAR-T cells prepared in vivo can kill cancer cells, the GvL (Graft-Versus-Leukemia) that can be induced by the T cells from the half-matched donor can also help to kill cancer cells.

[0185] In some embodiments of the present application, in S2, the cell population and the viral vector are mixed (composition), and the composition is administered to the subject;

[0186] Preferably, the cell population 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.

[0187] In some embodiments of the present application, in S2, after the administration of the cell population or the viral vector to the subject (first administration), the viral vector or the cell population is administered to the subject (second administration);

[0188] When the first administration is the administration of the cell population to the subject, the second administration is the administration of the viral vector to the subject;

[0189] When the first administration is the administration of the viral vector to the subject, the second administration is the administration of the cell population to the subject.

[0190] In some embodiments of the application, in S2, 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 completing the first administration.

[0191] 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 completing the first administration.

[0192] 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 completing the first administration.

[0193] In some embodiments of the application, in S2, the cell population and the viral vector are simultaneously administered to the subject using a double-barreled tube (double-barreled administration); wherein the cell population is administered using one barrel of the double-barreled tube and the viral vector is administered using the other barrel of the double-barreled tube.

[0194] 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: the double-barreled administration, the administration of the composition, the administration of the cell population, and the administration of the viral vector.

[0195] In some embodiments of the application, the viral vector is any one of the previously described pseudotyped lentiviral vectors or retroviral vectors.

[0196] In some embodiments of the application, the cell population is any one of the foregoing cell populations comprising T cells.

[0197] In some embodiments of the application, in S1, the lymphodepleting therapy comprises administering to the subject an immunosuppressive agent;

[0198] Preferably, the immunosuppressive agent is administered to the subject daily for 2-4 days; more preferably, for 3 days.

[0199] In some embodiments of the application, the immunosuppressive agent comprises one or more of cyclophosphamide and fludarabine;

[0200] Preferably, the immunosuppressive agent comprises fludarabine and cyclophosphamide;

[0201] More preferably, the immunosuppressive agent comprises 20-40 mg / m2 fludarabine and 200-400 mg / m2 cyclophosphamide;

[0202] Still more preferably, the immunosuppressive agent comprises 30 mg / m2 fludarabine and 300 mg / m2 cyclophosphamide.

[0203] In some embodiments of the application, in S1, the administration of the lymphodepleting therapy is completed within 2 to 7 days prior to the performance of S2.

[0204] In some embodiments of the application, the cell population is selected from the group consisting of white blood cells, PBMCs and CD3 + T cells.

[0205] In some embodiments of the application, in S2, the haploidentical donor of the subject is a healthy donor; more preferably, the subject is a human.

[0206] In some embodiments of the application, the cell population and the viral vector generate CAR-T cells in vivo in the subject that kill cancer cells.

[0207] In some embodiments of the application, the subject is administered any one of the foregoing cell populations comprising T cells comprising 5 x 10 6 cells; more preferably, 5 x 10 9 CD3 + T cells; more preferably, 1 x 10 3 cells; more preferably, 1 x 10 9 TU / KG of any one of the foregoing viral vectors.

[0208] In some embodiments of the application, the subject is administered a therapeutically effective amount of any one of the foregoing cell populations comprising T cells and any one of the foregoing viral vectors.

[0209] In some embodiments of the application, the subject has abnormal number and / or function of T cells.

[0210] In some embodiments of the application, the subject has abnormal number of T cells.

[0211] In some embodiments of the application, the subject has abnormal killing ability of T cells.

[0212] In some embodiments of the application, the subject is an end-line patient.

[0213] The beneficial effects of the present application include:

[0214] The inventors of the present application first discovered that the technical problem of using autologous T cells of a patient with insufficient number and / or function of T cells to effectively prepare CAR-T cells in vivo using a lentiviral vector, and the technical problem of using a semi-compatible donor of the patient containing a T cell-containing cell population, such as PBMCs or leukocytes, to effectively prepare CAR-T cells in vivo using a lentiviral vector.

[0215] The method for preparing CAR-T cells disclosed in the present application uses a semi-compatible donor of a patient with abnormal number and / or function of autologous T cells containing a T cell-containing cell population, such as PBMCs, to replace the autologous T cell-containing cell population of the patient, and is mixed with a lentiviral vector in vivo, for example, by using a double-channel tube, mixed and administered, and / or administered before and after a short period of time, to effectively prepare CAR-T cells in the patient, thereby effectively solving the technical problem that the prior art is difficult to solve, i.e., it is difficult for a lentiviral vector in vivo to effectively transduce autologous T cells of a patient with abnormal number and / or function of autologous T cells, thereby effectively preparing CAR-T cells;

[0216] Furthermore, the normal T cells from the semi-compatible donor can help to rebuild or partially rebuild the T cells of the patient; while the CAR-T cells prepared in vivo can kill cancer cells, the GvL induced by the T cells from the semi-compatible donor can also help to kill cancer cells.

[0217] Herein:

[0218] “Haploidentical Donor”: As used herein, “Haploidentical Donor” refers to a tissue donor that is partially matched (typically 50%) to the HLA tissue type of a recipient of a stem cell, organ transplant, or cell population comprising T cells. HLA is a group of markers present on the surface of human cells and tissues that play an important role in the body’s immune response to foreign substances. Prior to a transplant, the recipient’s HLA tissue type is matched with the HLA type of potential donors. When a fully or highly matched donor cannot be found, a haploidentical donor, such as a parent, child, or sibling, can be used for a stem cell or organ transplant (https: / / www.cancer.gov / publications / dictionaries / cancer-terms / def / haploidentical-donor).

[0219] “Inhibit”: The term “inhibit” includes complete abrogation of the ability of the pseudotyped lentiviral vector or retroviral vector to transduce a cell expressing a viral glycoprotein receptor by binding to the viral glycoprotein receptor, as well as a significant attenuation of the transduction ability. In particular embodiments, “significantly attenuate” means a reduction selected from 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 pseudotyped lentiviral vector or retroviral vector comprising the first mutation in the viral glycoprotein.

[0220] “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 cell thereof that distinguishes the cell from a cell of the erythroid lineage or myeloid lineage. The term “lymphocyte” includes T cells, B cells, and natural killer (NK) cells.

[0221] “T cell”: T cells are one of several white blood cells important to 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).

[0222] In some embodiments of the application, the T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + , CD4 - / CD8 - T cells or a combination thereof. In some embodiments of the application, the CD4 + T cells produce IL-2, TFN, TNF or a combination thereof upon expression of the CAR and binding to a target cell, such as a tumor cell. In some embodiments of the application, the CD8 + T cells lyse an antigen-specific target cell upon expression of the CAR and binding to the target cell.

[0223] Non-Activated T cells refer to T cells that are not expanded, not differentiated, resting, not recognized antigen, not activated by T cell activation signaling molecules such as T cell activation primary and secondary signaling molecules (anti-CD3 antibody or CD28 binding ligand, etc.), for example, T cells in the cell cycle G0 phase, 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.

[0224] 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 antigens, and at least contains (1) an extracellular antigen binding region, such as scFv or VHH, outside the cell; (2) a transmembrane region that anchors the CAR to the cell membrane, and (3) an intracellular signaling domain; the extracellular structure of the CAR can further contain a hinge region; the intracellular structure of the CAR can further contain a costimulatory signaling domain. CAR can use the extracellular antigen binding region to redirect T cells and other immune effector cells to the selected target, such as cancer cells, in a non-MHC restricted manner. In some embodiments of the application, the CAR further comprises a leader signal peptide.

[0225] “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.

[0226] “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.

[0227] “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.

[0228] “Antibody”: refers to a polypeptide or combination of polypeptides comprising sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region such that it specifically binds to an antigen. “Antibody” herein encompasses various formats and various structures as long as they exhibit the desired antigen binding activity.

[0229] “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 its N-terminus to C-terminus; and, when the full-length antibody is of 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 its N-terminus to C-terminus; 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” shape.

[0230] 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).

[0231] The terms "complementarity determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to a non-contiguous sequence of amino acids within antibody variable regions which confer 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).

[0232] 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, etc., and immunoglobulin new antigen receptor (Ig New Antigen Receptor, IgNAR) found in cartilaginous fishes such as sharks.

[0233] 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 function. Generally, a heavy chain antibody naturally lacking 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.

[0234] 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).

[0235] 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).

[0236] 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.

[0237] 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).

[0238] 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.

[0239] "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.

[0240] "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.

[0241] "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); alternatively, a variant refers to a mutant that 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. A "conservative substitution" is considered in the art to be the replacement of one amino acid with another that has similar properties. Exemplary conservative substitutions are well known 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).

[0242] "Transgene": As used herein, the term "transgene" refers to a gene or polynucleotide that encodes a protein of interest (e.g., a CAR) whose expression in a host cell is desired 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, and the like. 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.

[0243] “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., promoters, 3’ UTRs) 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.

[0244] “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 acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising 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.

[0245] 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.

[0246] “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.

[0247] “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).

[0248] “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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] “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.

[0253] “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).

[0254] “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.

[0255] 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.

[0256] 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).

[0257] 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.

[0258] For 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).

[0259] 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 a 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 incompletely spliced 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.

[0260] Exemplary, the packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0261] Lentiviral vectors and lentiviral vector backbones 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.

[0262] 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.

[0263] 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.

[0264] 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 the two 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.

[0265] 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.

[0266] 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.

[0267] 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, the deletion of genes, and the introduction of point mutations into genes.

[0268] 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.

[0269] 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.

[0270] Preferably, the packaging cells are HEK-293T cells.

[0271] “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 gammaretrovirus. 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.

[0272] “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.

[0273] “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.

[0274] “Subject”: As used herein, “subject,” “patient,” and “individual” are used synonymously and include, but are not limited to, a mammal, such as a human or a non-human mammal, e.g., 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.

[0275] 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.

[0276] "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.

[0277] "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.

[0278] "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.

[0279] "and / or": It is to be understood that "and / or" is used to mean

[0280] "Comprise": As used herein, unless otherwise required by context, the word "comprise" will be understood to imply the inclusion of stated steps, or elements, or steps or elements, but not the exclusion of any other steps, or elements, or steps or elements.

[0281] “Embodiments”: Reference throughout this specification to “some embodiments” and “some examples”, or combinations 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 the 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.

[0282] “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.

[0283] “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).

[0284] "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.

[0285] “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 destinations. 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.

[0286] “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.

[0287] "Operably linked": Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid. For example, nucleotide sequences that are operably linked to each other can be contiguous; and, for example, when two protein coding regions are to be joined, are in the same reading frame. For example, DNA for a leader or secretory leader sequence is operably linked to the DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked with a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked nucleic acids are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are employed in accordance with conventional practice.

[0288] "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, among others. 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 peptides 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.

[0289] "Autologous": As used herein, the term "autologous" means any material derived from the same individual to which the material is subsequently reintroduced.

[0290] "Allogeneic": As used herein, "allogeneic" refers to a graft derived from a different individual of the same species.

[0291] "Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preserving agents, preservatives, and / or adjuvants. The excipient is preferably nontoxic or substantially nontoxic to recipients at the dosages and concentrations employed.

[0292] “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 application. 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)).

[0293] “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.

[0294] 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 an immune effector cell by physical, chemical, or biological methods.

[0295] 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 application, the vector is introduced into the cell by electroporation. In some embodiments of the present application, the vector is introduced into the cell by PEI transfection reagent.

[0296] All publications, documents, and patent references mentioned herein are hereby incorporated by reference in their entirety as if each had been individually incorporated. In the case of conflict between the present application, including any definitions herein, and any incorporated reference, the present application, including any definitions herein, controls. However, any reference, article, publication, patent, patent publication, and patent application, cited herein, is not, and should not be, construed as being a recognition of any form of admission.

[0297] 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

[0298] Figure 1: is the map of the envelope plasmid in Example 1;

[0299] Figure 2: is the map of the main plasmid in Example 1;

[0300] Figure 3: is the flow cytometry result of detecting CD3 + T cells in PBMCs of semi-compatible donor 01;

[0301] Figure 4: is the flow cytometry result of detecting CD19 expression in peripheral blood of D4, D7, D14, D21 of patient 01;

[0302] Figure 5: is the flow cytometry result of detecting CAR-19 expression in peripheral blood of D7, D14, D21 of the patient. DETAILED DESCRIPTION

[0303] The concept and the technical effects of the present application are described below in combination with the examples, so as to fully understand the technical solutions, the solved technical problems and the beneficial effects of the present application. Obviously, the described examples are only some of the 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.

[0304] 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. EXAMPLE

[0305] In this embodiment, patient 01 suffers from acute B lymphoblastic leukemia (B-ALL), and the donor (D01) who is the brother of the patient is detected for HLA molecular matching, which meets the standard of semi-compatible donor.

[0306] 1. Preparation of lentiviral vector mG1A3 / 28-CAR19

[0307] The virus envelope prepared in advance and cryopreserved 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.

[0308] The method for preparing the lentiviral vector mG1A3 / 28-CAR19 is as follows:

[0309] A. Construction of membrane type expressing anti-CD3 antibody x anti-CD28 antibody

[0310] 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;

[0311] (1) The amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO: 8;

[0312] (2) The amino acid sequence of the anti-CD3 antibody (scFv) UCHT1 is shown in SEQ ID NO: 9;

[0313] (3) The amino acid sequence of the CD8a hinge region is shown in SEQ ID NO: 10;

[0314] (4) The amino acid sequence of the CD8a transmembrane region is shown in SEQ ID NO: 11;

[0315] (5) The amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 12;

[0316] (6) The amino acid sequence of the anti-CD28 antibody (scFv-15E8) is shown in SEQ ID NO: 13.

[0317] B. Construction of CAR-19

[0318] In this embodiment, a chimeric antigen receptor (CAR-19) targeting CD19 is constructed, which has the structure of CD8α signal peptide, extracellular antigen binding region targeting CD19, CD8α hinge region, CD8α transmembrane region, 4-1BB co-stimulatory signaling domain and CD3ζ 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.

[0319] (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;

[0320] (2) the amino acid sequence of the 4-1BB co-stimulatory domain is shown as SEQ ID NO: 17;

[0321] (3) the amino acid sequence of the intracellular signaling domain of CD3ζ is shown as SEQ ID NO: 18;

[0322] (4) the amino acid sequence of the (G4S)3linker peptide is shown as SEQ ID NO: 16.

[0323] C. Packaging lentiviral vector mG1A3 / 28-CAR19

[0324] Prepare the following four kinds of plasmids: envelope plasmid (envelope plasmid 1, as shown in Figure 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 Figure 2) carrying polynucleotide encoding the CAR-19; the envelope plasmid 1 and the master plasmid CAR-19 are synthesized by conventional molecular cloning method.

[0325] 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:

[0326] 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 to 1mL of Opti-MEM medium, shake well, then add 64uL of PEI reagent, blow evenly, stand for 10 minutes, then add to the culture medium of HEK-293T cells, update the culture medium after 6 hours, collect the culture medium supernatant 48 hours after transfection, filter with 0.45um filter membrane, centrifuge at 50000g for 2.5h, aspirate the supernatant, resuspend the lentiviral vector mG1A3 / 28-CAR19 with 200uL of F12 culture medium and freeze at-80℃.

[0327] Opti-MEM alpha reduced serum medium, brand: GIBCO, item number: #SP0272;

[0328] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: brand: GIBCO, item number: #C12430500BT; FBS: brand: EXCELL, item number: #FSP500;

[0329] F12 medium: brand: GIBCO, item number: #C11330500BT;

[0330] Needle filter: brand: SORFA, item number: #622120.

[0331] 2. D-18~D-7: Collect PBMCs of the patient's semi-compatible donor

[0332] Collect PBMCs of the semi-compatible donor D01, after red blood cell lysis, use flow cytometry to detect the proportion of CD3 - T cells in the lymphocytes (CD11B + ) of the PBMCs of the semi-compatible donor D01, the results are shown in Figure 3.

[0333] As shown in Figure 3, the proportion of CD3 + T cells in the lymphocytes of the PBMCs of the semi-compatible donor D01 is 60.96%, the number of T cells in the PBMCs of the semi-compatible donor D01 is normal, and can be used to prepare CAR-T cells in patient 01.

[0334] Collect PBMCs of the semi-compatible donor D01, take 8.2×10 8 PBMCs for freezing, that is, 8.2×10 8 PBMCs of the semi-compatible donor D01 contain at least 5×10 8 CD3 + T cells.

[0335] 3. D-7~D-2: S1, administering lymphocyte depletion therapy to the patient

[0336] D-7~D-2:

[0337] S1, administering 30mg / m2 fludarabine and 300mg / m2 cyclophosphamide to the patient per day for 3 days to perform a lymphocyte-depleting preconditioning (lymphodepletion) to the patient; observing the patient for 2 days.

[0338] 4. D0: S2, mixing PBMCs of the semi-allogeneic donor and the lentiviral vector for administration

[0339] D0:

[0340] Recovering 8.2x10 8 PBMCs of the semi-allogeneic donor D01 which are cryopreserved, mixing (composition) in vitro with 2x10 8 TU of the lentiviral vector mG1A3 / 28-CAR19, and then intravenously administering the composition to the patient 01 without in vitro culturing or sorting CD3 + T cells of the PBMCs of the semi-allogeneic donor D01.

[0341] 5. Detecting the expression of CD19 and the preparation of CAR-T cells in the peripheral blood of the patient

[0342] Intravenously collecting the peripheral blood of the patient 01 on D4, D7, D14 and D21, isolating PBMCs, and using flow cytometry to detect the expression of CD19 (a B cell marker) and the preparation of CAR-T cells (detecting the expression of FMC-63) in the isolated PBMCs, and the results are shown in FIG. 2 and FIG. 3, respectively.

[0343] As shown in FIG. 4, after administering the PBMCs of the semi-allogeneic donor 01 and the lentiviral vector mG1A3 / 28-CAR19 to the patient 01, the number of CD19 + target cells in the peripheral blood of the patient 01 significantly decreased on D4, D7, D14 and D21; especially on D21, the peripheral blood of the patient 01 almost did not contain CD19 + target cells;

[0344] As shown in FIG. 5, the CAR-T cells were detected in the peripheral blood of the patient 01 on D7, D14 and D21; on D21, the number of CAR-T cells in the PBMCs of the patient was 5.16%.

[0345] 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 population of cells comprising T cells is harvested from a semi-allogeneic donor to a subject; the subject is an individual to whom the composition is to be administered; wherein the viral vector: (a) comprises a heterologous polynucleotide encoding a chimeric antigen receptor (CAR) that can specifically bind a cancer-associated antigen; 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 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; 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.

5. The composition of claim 4, wherein, The viral glycoprotein of the viral vector is VSV-G or Cocal-G, and the viral glycoprotein receptor is 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; Preferably, the viral glycoprotein is VSV-G or Cocal-G, and the viral glycoprotein receptor is LDL-R; More preferably, the viral glycoprotein comprises a first mutation that inhibits the viral glycoprotein's ability to bind its receptor; Still more preferably, 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, respectively; Still further preferably, 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 1181 of SEQ ID NO:

1. (b) is located at an amino acid corresponding to amino acids 331-364 deletion, amino acids 344-354 deletion, substitution of K47, deletion of K47, substitution of R354, substitution of 1182 of SEQ ID NO: l upon optimal global alignment of SEQ ID NO: l; (c) is located at amino acids 331-364 deletion, amino acids 344-354 deletion, substitution of K47, deletion of K47, substitution of R354, substitution of V182 of SEQ ID NO: 2; and (d) is located at an amino acid corresponding to amino acids 331-364 deletion, amino acids 344-354 deletion, substitution of K47, deletion of K47, substitution of R354, substitution of V182 of SEQ ID NO: 2 upon optimal global alignment of SEQ ID NO: 2; Most preferably, the first mutation comprises a mutation: (a) is located at K47 of SEQ ID NO: l or SEQ ID NO: 2; or (b) is located at K47 of SEQ ID NO: l or SEQ ID NO: 2 upon optimal global alignment of SEQ ID NO: l or SEQ ID NO:

2.

7. The composition according to any one of claims 2-5, characterized in that, the viral glycoprotein of the viral vector comprises a second mutation that enhances or does not inactivate the ability of the viral glycoprotein to be inactivated by complement; Preferably, the viral glycoprotein is VSV-G or Cocal-G; More preferably, the second mutation comprises a mutation at one or more of the following positions: (a) is located at amino acid 214 of SEQ ID NO: l or SEQ ID NO: 2; (b) is located at an amino acid corresponding to amino acid 214 of SEQ ID NO: l or SEQ ID NO: 2 upon optimal global alignment of SEQ ID NO: l or SEQ ID NO: 2; (c) is located at amino acid 352 of SEQ ID NO: l or SEQ ID NO: 2; (d) is located at an amino acid corresponding to amino acid 352 of SEQ ID NO: l or SEQ ID NO: 2 upon optimal global alignment of SEQ ID NO: l or SEQ ID NO: 2; (e) is located at amino acid 50 of SEQ ID NO: l or SEQ ID NO: 2; (f) is located at an amino acid corresponding to amino acid 50 of SEQ ID NO: l or SEQ ID NO: 2 upon optimal global alignment of SEQ ID NO: l or SEQ ID NO: 2; (g) is located at amino acid 146 of SEQ ID NO: l or SEQ ID NO: 2; and (h) is located at an amino acid corresponding to amino acid 146 of SEQ ID NO: l or SEQ ID NO: 2 upon optimal global alignment of SEQ ID NO: l or SEQ ID NO: 2; More preferably, the mutation at the position is selected from the group consisting of a substitution, a deletion, and an insertion of an amino acid; Still more preferably, the mutation at the site is a substitution of an amino acid.

8. The composition of claim 7, wherein, The second mutation comprises a combination of mutations at any one of the following sites: (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; Preferably, the mutation comprises a combination of mutations at any one of the following sites: (a) (1) T214N and T352A of SEQ ID NO: 1; or (2) T214N, T352A, K50T, and S146T of SEQ ID NO: 1; (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; (c) (1) K214N and T352A of SEQ ID NO: 2; or (2) K214N, T352A, K50T, and S146T of SEQ ID NO: 2; and (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.

9. The composition according to any one of claims 1-8, characterized in that, 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.

10. The composition according to any one of claims 1-9, characterized in that, The endocytic receptor on the surface of the T cell comprises CD3. Preferably, the targeting molecule that can specifically bind CD3 comprises an anti-CD3 antibody or antigen-binding fragment thereof.

11. The composition according to any one of claims 1-10, characterized in that, The endocytic receptor on the surface of the T cell comprises CD28; Preferably, the CD28-binding 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; More preferably, 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.

12. The composition according to any one of claims 1-11, characterized in that, 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 specifically bind an endocytic receptor on the surface of a T cell; Preferably, when the ligand is a transmembrane protein, the targeting binding region comprises only a receptor-binding fragment of the ligand.

13. The composition of claim 12, wherein, The targeting molecule further comprises a transmembrane region, the targeting binding region being directly or indirectly linked to the transmembrane region; Preferably, 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; More preferably, the transmembrane region comprises a transmembrane region of CD8a.

14. The composition of claim 13, wherein, The targeting molecule further comprises a linking domain, the targeting binding region being indirectly linked to the transmembrane region via the linking domain; Preferably, the linking 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 portion of a transmembrane domain of a protein selected from the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8a, CD8b, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154. (iv) a stalk region of a Type II C-lectin selected from the group consisting of a stalk region of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the linker domain is a hinge region of CD8a.

15. The composition of any one of claims 1-14, wherein, The CAR comprises an extracellular antigen binding region, a transmembrane region, and an intracellular signaling domain.

16. The composition of claim 15, wherein, The extracellular antigen binding region of the CAR can specifically bind to a cancer-associated antigen; (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, FcERIg, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; Preferably, the transmembrane region of the CAR comprises a transmembrane region of CD8a; (b) the intracellular signaling domain of the CAR is selected from the group consisting of an 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; Preferably, the intracellular signaling domain of the CAR is an intracellular signaling domain of CD3zeta.

17. The composition of claim 16, 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; Preferably, the linker domain of the CAR is selected from the group consisting of: (i) an immunoglobulin hinge region selected from the group consisting of a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region; (ii) a hinge region selected from the group consisting of a 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 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 a stalk region of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; Preferably, the costimulatory signaling domain of the CAR comprises a costimulatory signaling domain of one or more 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.

18. The composition of any one of claims 1-17, wherein, The cell population is selected from the group consisting of white blood cells, PBMCs, and CD3 + one or more of T cells.

19. The composition of claim 18, wherein, The haploidentical donor of the subject is a healthy donor; more preferably, the subject is a human.

20. The composition of any one of claims 1-20, wherein, The composition further comprises a pharmaceutically acceptable carrier or excipient.

21. A method of making a chimeric antigen receptor-expressing T cell (CAR-T cell), characterized in that, The method comprises the following steps: S1, administering a lymphodepleting therapy to a subject having a cancer for conditioning; and S2, administering the composition of claims 1-20 to the subject.

22. The method of claim 21, wherein, In S2, mixing the composition, administering the composition to the subject; Preferably, mixing the population of cells and the viral vector for 1 minute to 2 hours, administering the composition to the subject.

23. The method of claim 21, wherein, In S2, after administering the population of cells comprising T cells or the viral vector in the composition to the subject (first administration), administering the viral vector or the population of cells comprising T cells to the subject (second administration); When the first administration is administering the population of cells to the subject, the second administration is administering the viral vector to the subject; When the first administration is administering the viral vector to the subject, the second administration is administering the population of cells to the subject.

24. The method of claim 23, wherein, In S2, the second administration is performed within 1 minute to 7 days after the completion of the first administration; Preferably, the second administration is performed within 1 minute to 24 hours after the completion of the first administration; More preferably, the second administration is performed within 1 minute to 60 minutes after the completion of the first administration.

25. The method of claim 21, wherein, In S2, the population of cells and the viral vector are simultaneously administered to the subject using a dual-lumen catheter (dual-lumen catheter administration); wherein the population of cells is administered using one lumen of the dual-lumen catheter, and the viral vector is administered using the other lumen of the dual-lumen catheter.

26. The method of any one of claims 22-25, wherein, In S2, after administering 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 dual-lumen catheter administration, the administration of the composition, the administration of the population of cells, and the administration of the viral vector.

27. The method of any one of claims 21-26, wherein, In S1, the lymphodepleting therapy comprises administering to the subject an immunosuppressive agent; Preferably, the immunosuppressive agent is administered to the subject daily for 2-4 days; more preferably, for 3 days.

28. The method of claim 27, wherein, The immunosuppressive agent comprises one or more of cyclophosphamide and fludarabine; Preferably, the immunosuppressive agent comprises fludarabine and cyclophosphamide; More preferably, the immunosuppressive agent comprises 20-40 mg / m2 fludarabine and 200-400 mg / m2 cyclophosphamide; Further more preferably, the immunosuppressive agent comprises 30 mg / m2 fludarabine and 300 mg / m2 cyclophosphamide.

29. The method of any one of claims 21-28, wherein, In S1, the administering of the lymphodepleting therapy is completed within 2 to 7 days prior to performing S2.

30. The method of any one of claims 21-29, wherein, S2, administering to the subject the cell population comprising 5 x 10 6 9 + T cells, administering to the subject 1 x 10 3 9 TU / KG of the viral vector.​​​ 31. The method of any one of claims 21-30, wherein, The subject has an abnormal number and / or function of T cells.

32. Use of the composition of any one of claims 1-20 in the manufacture of CAR-T cells.

33. Use of the composition of any one of claims 1-20 in the manufacture of a medicament for treating a cancer selected from one or more of a hematological cancer and a solid cancer; Preferably, the hematological cancer is a CD19-expressing hematological cancer; More preferably, the CD19-expressing hematological cancer 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- cell rich large B-cell lymphoma; and acute B-lymphoblastic leukemia.

34. A method for treating cancer, characterized in that, The method comprises the steps of the method of claim 21-31 for making chimeric antigen receptor-expressing T cells (CAR-T cells).

35. The method of claim 54, wherein, The cell population and the viral vector make CAR-T cells in vivo in the subject, killing cancer cells. The method comprises the steps of the method of claim 21-31 for making chimeric antigen receptor-expressing T cells (CAR-T cells).

36. The method of claim 38, wherein, The subject was given an injection containing 5×10 6 5 x 10 9 CD3 + The T cell population; administered 1 × 10 to the subject 3 ~1×10 9 The viral vector described in TU / KG.

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