Non-cellular particles, preparation method therefor, and use thereof

Abstract

The present invention relates to the field of biopharmaceuticals and particularly to non-cellular particles, a preparation method therefor, and use thereof. Provided are non-cellular particles. The non-cellular particles can enable targeted delivery to a target cell and transient expression of a target gene, and can be applied to gene therapy and cell therapy.

Description

A non-cellular particle, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a non-cellular particle, its preparation method, and its application. Background Technology

[0002] In the fields of gene therapy and cell therapy, non-cellular particles, such as exosomes, extracellular vesicles, and viral particles, are often used to deliver target genes (GOI) / transgenes or target proteins (POI).

[0003] However, naturally occurring exosomes and extracellular vesicles have limitations such as size inhomogeneity and structural instability. At the same time, their targeting is also low, making it impossible to deliver transgenes and target proteins to target cells. Viral particles, such as lentiviral particles, have high immunogenicity, and their ability to randomly integrate transgenes into the target cell genome carries the risk of inducing insertional mutagenesis. While adenoviruses and adeno-associated virus vectors (AAVs) can deliver target genes instantaneously to target cells, their targeting is low, and they can carry a low transgene payload. Summary of the Invention

[0004] In view of this, one aspect of the present invention provides a non-cell particle (NCP), the non-cell particle comprising:

[0005] (a) Viral glycoprotein; and

[0006] (b) Targeting molecules that can bind to antigens on the surface of target cells.

[0007] In some embodiments of the present invention, the ability of the viral glycoprotein to bind to its receptor is weakened or inhibited.

[0008] In some embodiments of the present invention, the number of binding sites available for the viral glycoprotein to bind to its receptor is reduced.

[0009] In some embodiments of the present invention, the viral glycoprotein has bound to one or more of its receptor and antibody.

[0010] In some embodiments of the present invention, the viral glycoprotein contains a first mutation that weakens or inhibits the ability of the viral glycoprotein to bind to its receptor.

[0011] In some embodiments of the present invention, any of the aforementioned viral glycoproteins is selected from vesicular stomatitis virus strains glycoprotein, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and T-cell choroid plexus meningitis virus (LCMV) glycoprotein;

[0012] Preferably, the vesicular stomatitis virus glycoprotein is selected from: vesicular stomatitis virus Indiana strain glycoprotein, vesicular stomatitis virus Cocal strain glycoprotein, vesicular stomatitis virus Maraba strain glycoprotein, vesicular stomatitis virus Morreton strain glycoprotein, vesicular stomatitis virus Alagoas strain glycoprotein, and vesicular stomatitis virus New... The following vesicular stomatitis virus (VSV) strains contain glycoproteins: Jersey, Carajas, Chandipura, Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and Yug Bogdanovac.

[0013] In some embodiments of the present invention, the viral glycoprotein is the glycoprotein of the Indiana strain of vesicular stomatitis virus (VSV-G) or the glycoprotein of the Cocal strain of vesicular stomatitis virus (Cocal-G).

[0014] In some embodiments of the present invention, the VSV-G (wild type) comprises an amino acid sequence as shown in SEQ ID NO:1.

[0015] In some embodiments of the present invention, the amino acid sequence of the full-length protein (including the signal peptide) of the wild-type VSV-G is shown in SEQ ID NO:20;

[0016] Among them, the amino acid sequence shown in positions 1-16 of SEQ ID NO:20 is as follows:

[0017] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.

[0018] In some embodiments of the present invention, the Cocal-G (wild type) comprises an amino acid sequence as shown in SEQ ID NO:2.

[0019] In some embodiments of the present invention, the amino acid sequence of the full-length protein (including the signal peptide) of the wild-type Cocal-G is shown in SEQ ID NO:27;

[0020] Among them, the sequence shown in positions 1-17 of SEQ ID NO:27 is as follows:

[0021] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild-type Cocal-G.

[0022] The receptors for VSV-G and Cocal-G, the low-density lipoprotein receptor (LDL-R), are widely expressed on the surface of various cells. Therefore, NCPs containing VSV-G or Cocal-G have broad infectivity but low targeting specificity. By inhibiting the ability of VSV-G or Cocal-G to bind to their receptors, the targeting specificity of NCPs containing VSV-G or Cocal-G can be effectively improved.

[0023] In some embodiments of the present invention, (a) the VSV-G has bound to one or more of its receptor LDL-R and anti-VSV-G antibody; or (b) the Cocal-G has bound to one or more of its receptor LDL-R and anti-Cocal-G antibody.

[0024] In some embodiments of the present invention, the NCP specifically binds to one or more of (a) LDL-R; (b) anti-VSV-G antibody; and (c) anti-Cocal-G antibody within the packaging / host cell and / or on the cell membrane.

[0025] In some embodiments of the present invention, the NCP has been bound to one or more of free and / or purified (a) LDL-R; (b) anti-VSV-G antibody; and (c) anti-Cocal-G antibody.

[0026] In some embodiments of the present invention, one or more of the following: (a) LDL-R; (b) anti-VSV-G antibody; and (c) anti-Cocal-G antibody, are linked to an endoplasmic reticulum retention peptide at their C-terminus.

[0027] In some embodiments of the present invention, the VSV-G or Cocal-G contains a first mutation that weakens or inhibits the ability of the VSV-G or Cocal-G to bind to its receptor LDL-R.

[0028] In some embodiments of the present invention, the first mutation is selected from one or more of the following mutations:

[0029] (a) Substitution or deletion of amino acids at positions 8, 9, 10, 47, 50, 51, 183, 179, 180, 182, 184, 209, 347, 350, 352, 353, and 354 in SEQ ID NO:1 or SEQ ID NO:2; deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353; and

[0030] (b) Substitution or deletion of amino acids at positions 8, 9, 10, 47, 50, 51, 183, 179, 180, 182, 184, 209, 347, 350, 352, 353, and 354, and deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353, after best global alignment with SEQ ID NO:1 or SEQ ID NO:2;

[0031] Preferably, the first mutation is selected from one or more of the following mutations:

[0032] (a) Amino acid deletion at positions 331-364, amino acid deletion at positions 344-354, substitution of K47, deletion of K47, substitution of R354, substitution of Y209, and substitution of I182 located in SEQ ID NO:1;

[0033] (b) After best global alignment with SEQ ID NO:1, the amino acid deletions at positions 331-364, positions 344-354, substitutions for K47, deletions of K47, substitutions for R354, substitutions for Y209, and substitutions for I182 are located at positions corresponding to SEQ ID NO:1.

[0034] (c) Deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354, substitution of Y209, and substitution of V182 in SEQ ID NO:2; and

[0035] (d) After best global alignment with SEQ ID NO:2, the amino acid deletions at positions 331-364, positions 344-354, substitutions for K47, deletions of K47, substitutions for R354, substitutions for Y209, and substitutions for V182 are located at positions corresponding to SEQ ID NO:2.

[0036] More preferably, the first mutation is:

[0037] (a) K47 missing in SEQ ID NO:1 or SEQ ID NO:2; or

[0038] (b) K47 deletion located at the equivalent of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2.

[0039] In some embodiments of the present invention, the first mutation is:

[0040] (a) K47 missing in SEQ ID NO:1 or SEQ ID NO:2; or

[0041] (b) K47 deletion located at the equivalent of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2.

[0042] In some embodiments of the present invention, the viral glycoprotein contains a second mutation that enhances or prevents the viral glycoprotein from being inactivated by complement.

[0043] In some embodiments of the present invention, the viral glycoprotein is VSV-G or Cocal-G;

[0044] Preferably, the second mutation is selected from one or more of the following site mutations:

[0045] (a) The 214th amino acid located in SEQ ID NO:1 or SEQ ID NO:2;

[0046] (b) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at position 214, which corresponds to SEQ ID NO:1 or SEQ ID NO:2;

[0047] (c) The 352nd amino acid located in SEQ ID NO:1 or SEQ ID NO:2;

[0048] (d) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:1 or SEQ ID NO:2;

[0049] (e) The 50th amino acid located in SEQ ID NO:1 or SEQ ID NO:2;

[0050] (f) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:1 or SEQ ID NO:2;

[0051] (g) The amino acid located at position 146 of SEQ ID NO:1 or SEQ ID NO:2; and

[0052] (h) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at position 146, which corresponds to SEQ ID NO:1 or SEQ ID NO:2;

[0053] More preferably, the mutation at the site is selected from amino acid substitutions, deletions, and insertions;

[0054] More preferably, the mutation at the site is an amino acid substitution.

[0055] In some embodiments of the present invention, the second mutation is selected from a combination of mutations at the following sites:

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

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

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

[0059] (d) After best global alignment with SEQ ID NO:2, the substitutions located at (1) K214 and T352, or (2) K214, T352, K50 and S146 of SEQ ID NO:2;

[0060] Preferably, the second mutation is selected from a combination of mutations at the following sites:

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

[0062] (b) After best global alignment with SEQ ID NO:1, it is located at (1) T214N and T352A, or (2) T214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:1;

[0063] (c) K214N and T352A located in SEQ ID NO:2, or (2) K214N, T352A, K50T and S146T; and

[0064] (d) After best global alignment with SEQ ID NO:2, it is located at (1) K214N and T352A, or (2) K214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:2.

[0065] In some embodiments of the present invention, the second mutation is (1) T214N and T352A located in SEQ ID NO:1.

[0066] In some embodiments of the present invention, the second mutation is (1) K214N and T352A located in SEQ ID NO:2.

[0067] In some embodiments of the present invention, viral glycoproteins that have undergone any of the aforementioned first mutations and / or second mutations retain the ability to mediate membrane fusion and lysosomal escape.

[0068] In some embodiments of the present invention, the target cells are lymphocytes;

[0069] Preferably, the lymphocytes are selected from one or more of T cells, NK cells, and B cells;

[0070] More preferably, the antigens on the surface of the T cells are selected from: CD2, CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD47, CD57, CD58, CD62L, CD71, AhR, CD69, CD94, CD95, 4-1BB, C One or more of the following: D103, 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;

[0071] More preferably, the antigen on the surface of the B cells is selected from one or more of the following: CD19, CD20, CD21, CD22, CD23, CD24, CD27, CD32b, CD37, CD38, CD40, CD52, CD72, CD74, CD80, CD84, CD86, IL-7RA (CD127), CD138, CD257, CD267, CD268, CD269, and CD270;

[0072] More preferably, the antigen on the surface of the NK cells is selected from one or more of the following: CD16, CD56, NKp46, KIRs, NKG2D, KLRB1 (CD161), KLRD1 (CD94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R.

[0073] In some embodiments of the present invention, the target cells are cancer cells; the cancer cells include one or more selected from solid cancer cells and hematopoietic cancer cells.

[0074] In some embodiments of the present invention, the antigens on the surface of the cancer cells are selected from:

[0075] 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-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, soybean protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, spermin 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2 ETS fusion gene / ERG, NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut One or more of the following: hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, Nectin-4, NKG2D, and GCC (guanylate cyclase).

[0076] More preferably, the antigen on the surface of the cancer cells is selected from one or more of MSLN, CD19, CD20, CD33, CD79A, CD79B, HER2, CEA and BCMA.

[0077] Lymphocytes, such as T cells, B cells, and NK cells, contain a variety of antigens on their surface. Constructing one or more targeting molecules on the surface of NCPs can enhance the targeting of NCPs to lymphocytes.

[0078] Furthermore, T cells, B cells, and NK cells contain various antigens such as CD3, CD7, and CD28, which are endocytosis receptors. When the ability of the viral glycoprotein to bind to its receptor is weakened or inhibited, the targeting of NCP can be further improved by constructing one or more targeting molecules on the surface of NCP that can target and bind to the endocytosis receptors on the surface of lymphocytes and induce endocytosis.

[0079] In some embodiments of the present invention, the target molecule is not any of the aforementioned viral glycoproteins.

[0080] In some embodiments of the present invention, the target cell is a T cell, and the antigen on the surface of the T cell is selected from: CD2, CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD47, CD57, CD58, CD62L, CD71, AhR, CD69, CD94, CD95 One or more of the following: 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;

[0081] Preferably, the antigen on the surface of the T cells is selected from one or more of CD3, CD3γ, CD3δ, CD3ε, CD5, CD7, CD28, CD58, TCRγ, TCRδ, TCRα, and TCRβ.

[0082] In some embodiments of the present invention, the antigen on the surface of the T cells is selected from one or more of CD7, CD3 and CD28.

[0083] In some embodiments of the present invention, the targeting molecule includes a targeting binding region comprising one or more of (a) an antibody or an antigen-binding fragment thereof capable of binding to an antigen on the surface of a target cell; and (b) a ligand or a binding fragment thereof.

[0084] Preferably, the binding fragment includes one or more selected from the ligand extracellular domain, functional fragment, and epitope.

[0085] In some embodiments of the present invention, the targeting binding region comprises one or more selected from anti-CD7 antibody or its antigen-binding fragment, anti-CD3 antibody or its antigen-binding fragment, anti-CD28 antibody or its antigen-binding fragment, CD80 or its binding fragment, and CD86 or its binding fragment.

[0086] In some embodiments of the present invention, the targeting binding region comprises one or more selected from anti-CD7 antibody or its antigen-binding fragment, anti-CD3 antibody or its antigen-binding fragment, anti-CD28 antibody or its antigen-binding fragment, CD80 or its extracellular domain, and CD86 or its extracellular domain.

[0087] In some embodiments of the present invention, the anti-CD7 antibody comprises an antigen-binding fragment derived from the monoclonal antibody TH-69.

[0088] In some embodiments of the present invention, the targeting molecule further comprises an activating molecule involved in T cell activation.

[0089] In some embodiments of the present invention, the activating molecule includes a TCR-CD3 binding molecule capable of binding to the TCR-CD3 complex.

[0090] In some embodiments of the present invention, the TCR-CD3 binding molecule includes an anti-CD3 antibody or an antigen-binding fragment thereof.

[0091] In some embodiments of the present invention, the activating molecule further includes a co-stimulatory molecule.

[0092] In some embodiments of the present invention, the co-stimulatory molecules include CD80, CD86, CD40L, GITRL, LTalpha, LIGHT, OX40L, 41BBL, ICOSL, CD27, CD30L, MICA, and MICB, or their binding fragments, and one or more of anti-CD28 antibodies or their antigen-binding fragments.

[0093] In some embodiments of the present invention, the co-stimulatory molecules include CD80 and CD86, or their binding fragments, and one or more of an anti-CD28 antibody or its antigen-binding fragment.

[0094] In some embodiments of the present invention, the co-stimulatory molecule includes an anti-CD28 antibody or an antigen-binding fragment thereof.

[0095] In some embodiments of the present invention, the co-stimulatory molecule includes a single-chain fragment variable region (scFv) of 15E8 derived from an anti-CD28 monoclonal antibody.

[0096] In some embodiments of the present invention, the co-stimulatory molecule includes CD80, or its extracellular domain, or its functional fragment, or its epitope.

[0097] In some embodiments of the present invention, the co-stimulatory molecule includes CD86, or its extracellular domain, or its functional fragment, or its epitope.

[0098] In some embodiments of the present invention, the targeting molecule is a transmembrane protein.

[0099] In some embodiments of the present invention, the targeting molecule is CD80 and / or CD86.

[0100] In some embodiments of the present invention, the targeting molecule is a recombinant transmembrane protein;

[0101] Preferably, the targeting molecule includes a transmembrane region;

[0102] More preferably, the targeting molecule further includes a connecting domain.

[0103] In some embodiments of the present invention, the targeting molecule further includes a transmembrane region, and the targeting binding region of the targeting molecule is directly or indirectly connected to the transmembrane region;

[0104] Preferably, the transmembrane region is selected from the transmembrane regions of the following proteins:

[0105] CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM- 1. CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1;

[0106] More preferably, the transmembrane region is the transmembrane region of CD8α.

[0107] In some embodiments of the present invention, the targeting molecule further includes a connecting domain, and the targeting binding region of the targeting molecule is indirectly connected to the transmembrane region through the connecting domain;

[0108] Preferably, the connection structure domain is selected from:

[0109] Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0110] Hinge region, wherein the hinge region is selected from the wild-type or modified hinge regions of the following proteins: CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD22, CD33, CD37, CD134 and ICOS;

[0111] All or a portion of the Fc domains, wherein the Fc domains are selected from one or more of the CH1, CH2, and CH3 domains; and

[0112] The stalk domain of type II C-lectins, wherein the type II C-lectins are selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A and NKG2D;

[0113] More preferably, the connection structure domain is the hinge region of CD8α.

[0114] In some embodiments of the present invention, the surface of the NCP includes (a) any of the aforementioned viral glycoproteins; and (b) any of the aforementioned targeting molecules.

[0115] In some embodiments of the present invention, any of the aforementioned non-cellular particles contains one or more target proteins (POIs).

[0116] In some embodiments of the present invention, the POI includes one or more of intracellular free proteins and membrane-localized expressed proteins.

[0117] In some embodiments of the present invention, the noncellular particles are virus-like particles (VLPs).

[0118] In some embodiments of the present invention, the non-cellular particles are non-integrating lentiviral vectors (NILs).

[0119] In some embodiments of the present invention, the NIL comprises a disabled / defective / inactivated integrase.

[0120] In some embodiments of the present invention, the inoperable integrase comprises a sequence selected from or located at SEQ ID NO:29, or, after best global alignment with SEQ ID NO:29, located at the position corresponding to SEQ ID NO:29. Amino acid mutations at one or more of the following sites in NO:29: positions 10, 11, 12, 13, 16, 41, 42, 51, 53, 55, 64, 69, 71, 81, 85, 87, 94, 116, 117, 119, 120, 122, 124, 128, 152, 156, 157, 159, 157, 159, 160, 164, 166, 167, 168, 170, 171, 173, 185, 186, 188, 198, 199, 202, 211, 214, 216, 221, 231, 235, 236, 246, 247, 253, 262, 263, 264, and 264;

[0121] Preferably, the disabled integrase comprises an amino acid mutation selected from or located at one or more of the following sites corresponding to SEQ ID NO:29, after optimal global alignment with SEQ ID NO:29:

[0122] D10K, E11K, H12N, H12C, E13K, H16C, H16V, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, D64A, E 69A, K71A, S81R, E85A, E87A, G94D, G94E, G94R, G94K, D116N, D116I, D116E, D116A, N117D, N11 7E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120G, N 120K, N120I, N120E, N120R, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152G, E152A, E152D, K156E, K156A, E157A, K159E, K159A, K160A, D164N, R166A, D167 A. Q168L, Q168A, E170A, E170G, H171A, K173A, F185K, K186Q, K186T, K186E, K188T, E198A, R19 9C, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, R231G, R231K, R231D, R231E, R231S, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, K264R, K264H, K266R, and K273R;

[0123] More preferably, the disability mutation includes one or more selected from D64V, D64E, D116N, D116I, D116A, E152G, and E152A.

[0124] HIV-1 integrase:

[0125] FLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQ SQGVIESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED(SEQ ID NO:29)

[0126] In some embodiments of the present invention, the ability of the 3'LTR and / or 5'LTR of any of the aforementioned NILs to recognize integrase is weakened or inhibited.

[0127] In some embodiments of the present invention, the conserved CA dinucleotides of the 3'LTR U5 and / or 5'LTR U3 of the NIL contain mutations (CA mutations).

[0128] In some embodiments, the CA mutation is selected from one or more of the mutations shown in Table 1 below:

[0129] Table 1

[0130]

[0131] In some embodiments of the present invention, the NCP comprises one or more genetically modified organisms.

[0132] In some embodiments of the present invention, the transgene comprises a polynucleotide encoding a chimeric antigen receptor (CAR).

[0133] In some embodiments of the present invention, the CAR includes an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction domain.

[0134] In some embodiments of the present invention, the extracellular antigen-binding region included in the CAR can bind cancer-associated antigens;

[0135] Preferably, the cancer-associated antigen is selected from: 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, TEM. 1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, Erb B4, 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-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, soybean protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, spermin 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2 ETS fusion gene / ERG, NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut One or more of the following: hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, Nectin-4, NKG2D, and GCC (guanylate cyclase).

[0136] More preferably, the cancer-associated antigen is selected from one or more of MSLN, CD19, CD20, CD33, CD79A, CD79B, HER2, CEA, and BCMA.

[0137] In some embodiments of the present invention, the transmembrane region of the CAR is selected from the transmembrane regions of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4 , BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1;

[0138] Preferably, the transmembrane region of the CAR is the transmembrane region of CD8α.

[0139] In some embodiments of the present invention, the amino acid sequence of the transmembrane region of CD8α has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:11.

[0140] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR is selected from the intracellular signal transduction domains of the following proteins:

[0141] CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FceRly, FceRβ, FcyRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12 and other proteins containing at least one ITAM intracellular signal transduction domain;

[0142] Preferably, the intracellular signal transduction domain of CAR is the intracellular signal transduction domain of CD3ζ.

[0143] In some embodiments of the present invention, the amino acid sequence of the intracellular signal transduction domain of CD3ζ has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:18.

[0144] In some embodiments of the present invention, the CAR further includes a connection domain; the connection domain connects the extracellular antigen-binding region and the transmembrane region of the CAR;

[0145] Preferably, the connection structure domain of the CAR is selected from:

[0146] Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0147] Hinge region, wherein the hinge region is selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0148] All or a portion of the Fc domains, wherein the Fc domains are selected from one or more of the CH1, CH2, and CH3 domains; and

[0149] The stem region of type II C-lectin, wherein the type II C-lectin is selected from the stem region of CD23, CD69, CD72, CD94, NKG2A and NKG2D;

[0150] More preferably, the connection structure domain of the CAR is the hinge region of CD8α.

[0151] In some embodiments of the present invention, the amino acid sequence of the hinge region of CD8α has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:10.

[0152] In some embodiments of the present invention, the CAR further includes a co-stimulatory signal transduction domain;

[0153] Preferably, the co-stimulatory signal transduction domain of the CAR is selected from one or more of the co-stimulatory signal transduction domains of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAmL, CD244, CD100, ICOS, CD40, and MyD88;

[0154] Preferably, the costimulatory signal transduction domain of the CAR is selected from one or more of the costimulatory signal transduction domains of 4-1BB and CD28.

[0155] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR is a 4-1BB costimulatory signal transduction domain.

[0156] In some embodiments of the present invention, the amino acid sequence of the co-stimulatory signal transduction domain of the 4-1BB has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:17.

[0157] In some embodiments of the present invention, the CAR further comprises a leader signal peptide located at the N-terminus of the extracellular antigen-binding region of the CAR;

[0158] Preferably, the leader signal peptide is selected from CD8α signal peptide, CD28 signal peptide, IgG signal peptide and HLA-A signal peptide;

[0159] More preferably, the leader signal peptide is a CD8α signal peptide.

[0160] In some embodiments of the present invention, the amino acid sequence of the CD8α signal peptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:8.

[0161] In some embodiments of the present invention, the CAR is bound to a CAR-binding molecule, the CAR-binding molecule comprising one or more of (a) an antigen or a binding fragment thereof capable of binding to the extracellular antigen-binding region of the CAR; and (b) an anti-antibody;

[0162] Preferably, the antigen-binding fragment includes one or more selected from the antigen extracellular domain, antigen functional fragment, and antigen epitope.

[0163] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR may bind to one or more of MSLN, CD19, CD20, CD33, CD79A, CD79B, HER2, CEA, and BCMA; the CAR-binding molecule comprises:

[0164] CD19, MSLN, CD19, CD20, CD33, CD79A, CD79B, HER2, CEA, and BCMA, or their combined fragments; and

[0165] One or more anti-antibodies can bind to the extracellular antigen-binding region of the CAR.

[0166] In another aspect, the present invention also provides a host cell comprising:

[0167] Polynucleotides encoding any of the aforementioned viral glycoproteins; and

[0168] Polynucleotides that encode any of the aforementioned target molecules.

[0169] In some embodiments of the present invention, the host cell contains one or more target proteins (POIs).

[0170] In some embodiments of the present invention, the POI includes one or more of intracellular free proteins and membrane-localized expressed proteins.

[0171] In some embodiments of the present invention, the host cell further comprises the genome of a non-integrating lentiviral vector (NIL).

[0172] In some embodiments of the present invention, the host cell further comprises the gag genome.

[0173] In some embodiments of the present invention, the host cell further comprises the pol genome.

[0174] In some embodiments of the present invention, the pol genome contains a disabling mutation of any of the aforementioned integrases.

[0175] In some embodiments of the present invention, the host cell further comprises the rev gene.

[0176] In some embodiments of the present invention, the host cell further comprises the backbone genome of a lentiviral vector or a retroviral vector.

[0177] In some embodiments of the present invention, the backbone genome of the lentiviral vector or retroviral vector further includes any of the aforementioned CA mutations.

[0178] In some embodiments of the present invention, the host cell further comprises any of the aforementioned transgenes.

[0179] In another aspect, the present invention also provides a method for preparing non-cellular particles, comprising culturing any of the aforementioned host cells to collect non-cellular particles.

[0180] In another aspect, the present invention also provides a composition comprising a pharmaceutically acceptable carrier or excipient and any of the aforementioned noncellular particles.

[0181] In another aspect, the present invention also provides the use of any of the aforementioned non-cellular particles in the preparation of cancer therapeutic drugs.

[0182] In another aspect, the present invention also provides a method for transducing lymphocytes in vitro, comprising contacting lymphocytes with any of the aforementioned non-cellular particles.

[0183] In another aspect, the present invention also provides a method for transducing lymphocytes in a subject in need, comprising administering any of the aforementioned non-cellular particles to the subject.

[0184] In another aspect, the present invention also provides a method for treating a subject suffering from cancer or killing cancer cells in said subject, comprising administering any of the aforementioned non-cellular particles to said subject.

[0185] In some embodiments of the present invention, the noncellular particles further comprise polynucleotides encoding CAR.

[0186] In some embodiments of the present invention, the cancer includes those selected from: acute myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, cholangiocarcinoma, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic biliary tract cancer. Tubal cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, common germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, colorectal cancer, intrahepatic cholangiocarcinoma, invasive / invasive breast cancer, islet cell carcinoma, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma Meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, stromal tumor, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal carcinoma, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, oat cell carcinoma, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord-stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, sinus cancer, parathyroid cancer, pelvic cancer Penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pinealoblastoma, pituitary adenoma, primary central nervous system cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, bone sarcoma, soft tissue sarcoma, uterine cancer, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymic cancer, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, renal tubular cancer, undiagnosed cancer, ureteral cancer, ureteral cancer.One or more of the following: uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0187] In some embodiments of the present invention, the cancer includes one or more selected from the following cancers: B-cell non-Hodgkin lymphoma (B-NHL), CD19+ hematologic malignancy, CD20+ hematologic malignancy, multiple myeloma (MM), acute myeloid leukemia (AML), precursor B-cell lymphoma / leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia / small lymphocytic lymphoma. (CLL / SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma (BL), primary plasma cell leukemia (pPCL), peripheral T-cell lymphoma (PTCL-NHL), NK / T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, T-large granular lymphocytic leukemia (T-LGL), embryonic centrifugal T-cell lymphoma (FTCL), colorectal cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, pancreatic cancer, bile duct cancer, and ovarian cancer.

[0188] In some embodiments of the present invention, the administration is selected from one or more of the following methods: intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection, nodular injection, infusion technique, oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, via portal vein, intralesional, continuous release system, and implantation device.

[0189] In another aspect, the present invention also provides the application of any of the aforementioned NCPs in the treatment of autoimmune diseases.

[0190] In some embodiments of the present invention, the autoimmune disease includes at least one of systemic lupus erythematosus, psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Behcet's disease, Sjögren's syndrome, myasthenia gravis, celiac disease, type 1 diabetes mellitus, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis, and scleroderma. Beneficial effects

[0191] The non-cellular particles provided by this invention can achieve targeted delivery to target cells and transient expression of transgenes without inducing insertional mutagenesis, and can carry a larger transgene load than viral vectors such as AAV.

[0192] In this article:

[0193] “Non-integrating lentiviral vector”: also known as Non-integrating Lentiviral Vector (NIL). NIL can be designed by introducing lentiviral vector (LV) integrase or Δatt site (LTR integrase binding site) (Engelman A, et al., Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol. 1995 May;69(5):2729-36.); and the transient expression and safety of NIL can be improved through optimization and modification (Luis A. The Old and the New: Prospects for Non-Integrating Lentiviral Vector Technology. Viruses. 2020 Sep 29;12(10):1103.). Developing NILs to facilitate gene transfer and improve their clinical applications, particularly in cell reprogramming and gene therapy (Gurumoorthy N, et al., Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through. Biomedicines. 2022 Jan 5;10(1):107.). The transient gene expression capabilities of NILs make them ideal for cytotoxic cancer therapy, protective NIL immunization, and extrachromosomal expression (Nordin F, et al., Transient Expression of Green Fluorescent Protein in Integrase-Defective Lentiviral Vector-Transduced 293T Cell Line. Methods Mol Biol. 2016;1448:159-73.) (Gurumoorthy N, et al., Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through. Biomedicines. 2022 Jan 5;10(1):107.).

[0194] "Administration": as used herein, "administration," "administrate," or "administrated" have the same meaning and are used interchangeably. In some embodiments of the present invention, "administration" is selected from one or more of the following: intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection, nodular injection, infusion techniques, oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, via portal vein, intralesional, continuous release system, and implanted device.

[0195] "Activation molecule" is used in this article and includes, but is not limited to, molecules that can bind to or interact directly or indirectly with T cells to induce T cell activation. For example, TCR-CD3 binding molecules that can bind to the TCR-CD3 complex and provide an initial signal for T cell activation include, but are not limited to, anti-CD3 antibodies or their antigen-binding fragments.

[0196] "Activating molecules" may also include "costimulatory molecules," as used in this article, which refers to molecules that can provide costimulatory signals for T cell activation; complete T cell activation usually requires the participation of costimulatory molecules.

[0197] For example, “co-stimulatory molecules” include, but are not limited to, CD80, CD86, CD40L, GITRL, LTalpha, LIGHT, OX40L, 41BBL, ICOSL, CD27, CD30L, MICA and MICB, or their extracellular domains, functional fragments, epitopes, and anti-CD28 antibodies or their antigen-binding fragments.

[0198] In some embodiments of the present invention, exemplarily, the “activating molecule” includes, but is not limited to, the binding domains of OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0199] “Inhibition”: When referring to the ability of the viral glycoprotein to bind to its receptor being “inhibited,” the term “inhibition” includes both the complete elimination of the ability of the viral glycoprotein to bind to its receptor and a significant reduction in the binding ability. In a specific embodiment, “significant reduction” means relative to the viral glycoprotein containing the first mutation and / or having already bound its receptor or antibody; “reduction” is selected from reductions of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2%, and at least 1%.

[0200] "Lymphocyte": The term "lymphocyte" refers to immune cells of lymphoid origin, which are cells that exhibit at least one phenotypic characteristic of a lymphocyte or its precursor or progenitor cells, distinguishing the cells from cells of the erythrocyte or myeloid lineage. The term "lymphocyte" includes T cells, B cells, and natural killer (NK) cells.

[0201] "T cells": T cells are one of the important types of white blood cells in the human immune system and play an important role in the acquired immune response. One of the main functions of T cells is immune-mediated cell death, which is mainly accomplished by two T cell subtypes: CD8+ T cells (Cytotoxic T cells) and CD4+ T cells (Helper T cells).

[0202] In some embodiments of the present invention, the T cells are CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8- cells, or combinations thereof. In some embodiments of the present invention, CD4+ T cells produce IL-2, TFN, TNF, or combinations thereof after expressing CAR and binding to target cells such as tumor cells. In some embodiments of the present invention, CD8+ T cells lyse antigen-specific target cells after expressing CAR and binding to target cells.

[0203] "Chimeric Antigen Receptor" (CAR) refers to an artificial cell surface receptor modified to be expressed on immune effector cells, including T cells, and specifically bind to antigens. It comprises at least (1) an extracellular antigen-binding domain, such as scFv or VHH; (2) a transmembrane region anchoring the CAR to the cell membrane; and (3) an intracellular signal transduction domain. The extracellular structure of the CAR may further include a hinge region; the intracellular structure of the CAR may further include a co-stimulatory signal transduction domain. The CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner using its extracellular antigen-binding domain. In some embodiments of the present invention, the CAR also comprises a leader signal peptide.

[0204] "Chimera": The term "chimera" refers to any nucleic acid molecule or protein that is not endogenous and comprises a combination of sequences that are joined or linked together, sequences that are not naturally joined or linked together in nature. For example, a chimeric nucleic acid molecule may contain nucleic acids encoding various domains from multiple different genes. As another example, a chimeric nucleic acid molecule may contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that found naturally.

[0205] “Antigen”: The terms “antigen” and “Ag” refer to molecules capable of inducing an immune response. An induced immune response may include antibody production and / or activation of specific immune-competent cells. Macromolecules, including proteins, glycoproteins, and glycolipids, can be used as antigens. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, antigens do not need to be (i) encoded solely by the full-length nucleotide sequence of a gene or (ii) entirely encoded by a gene. Antigens can be generated or synthesized, or they can be derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0206] "Flexible linkers" are typically used when linked domains require a certain 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 usually composed of small, nonpolar (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 amino acids provide flexibility and allow for the movement of linked functional domains. For commonly used flexible linker peptides, see 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.

[0207] "Antibody" refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or the variable regions of the immunoglobulin light chain, enabling it to specifically bind to an antigen. In this article, "antibody" encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0208] The term "antibody" in this article includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin class consisting of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain, running from its N-terminus to its C-terminus. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain refers to a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL), running from its N-terminus to its C-terminus. The heavy chains are linked to each other and to each other with light chains by disulfide bonds, forming a "Y"-shaped structure.

[0209] In the context of antibodies, the term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable regions of the heavy and light chains of natural antibodies (VH and VL regions, respectively) typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL region can be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using the VH or VL regions of the antibody binding to that specific antigen to screen libraries of complementary VL or VH regions, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0210] The terms “complementarity-determining region” and “CDR”, synonyms for “hypervariant region” or “HVR”, are known in the art to refer to the discontinuous sequence of amino acids within the variable region of an antibody that confers antigen specificity and / or binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0211] The term "antibody" in this article also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by camels (Camelus dromedarius), Bactrian camels (Camelus Bactrianus), llamas (Lama Glama), guanicoes (Lama Guanicoe), and alpacas (Vicugna Pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0212] The terms "VHH domain," "nanoantibody," and "single-domain antibody" (sdAb) used in this article have the same meaning and are used interchangeably. They refer to the cloning of the variable region of a heavy chain antibody to construct a single-domain antibody consisting of only one heavy chain variable region. This is the smallest antigen-binding fragment with complete function. Typically, a naturally occurring heavy chain antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0213] The term "antibody" in this article also includes monoclonal antibodies or their antigen-binding moieties. Monoclonal antibodies or their antigen-binding moieties may be non-human, chimeric, humanized, or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Chapter 14 of *Antibodies: A Laboratory Manual*, edited by Harlow et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0214] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. Non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, alpacas, ostriches, cynomolgus monkeys (e.g., cynomolgus monkeys and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0215] In this article, "antigen-binding fragment" refers to a part or a variant of a complete antibody that does not possess the full structure of the complete antibody, but only contains a part or a variant of the complete antibody, and the part or variant has the ability to bind antigens.

[0216] For example, in this document, "antibody or its antigen-binding fragment" includes, but is not limited to: immunoglobulin (full-length antibody), hapten, Fab, Fab', F(ab')2, Fv fragment, single-chain variable region fragment (scFv), disulfide bond stable antibody (dsFv), heavy chain variable region (VH) or light chain variable region (VL) of antibody, Fd fragment composed of VH and CH1 domains, linear antibody, heavy chain antibody and nanobody (VHH).

[0217] In some embodiments of the present invention, there is no particular limitation on the order in which the scFv contains the VH or VL region from the N-terminus to the C-terminus, such as containing VH-Linker-VL or VL-Linker-VH from the N-terminus to the C-terminus; the linker peptide may be selected from flexible linker peptides.

[0218] "Ligand": In receptor-ligand binding, the ligand is typically a molecule that binds to a site on the receptor to generate a signal. This binding usually leads to a conformational change in a complex structure, thereby inducing related physiological activities.

[0219] "Ligand-binding fragment": refers to a fragment that does not possess the complete structure of the complete ligand, but only contains a portion or a variant of the complete ligand, which has the ability to bind to a receptor. Exemplarily, "ligand-binding fragment" herein includes, but is not limited to, the extracellular domain, functional fragments, epitopes, binding regions, and variable regions of the ligand.

[0220] "Variant": A variant is a mutant that has at least 50% identity with the amino acid sequence of a non-mutant (wild-type) mutant. "At least 50% identity" means that the variant has 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% identity with the amino acid sequence of a non-mutant (wild-type) mutant. Alternatively, a variant is a mutant whose nucleic acid sequence encoding the variant has at least 50% identity with the nucleic acid sequence encoding a non-mutant (wild-type) mutant. "At least 50% identity" means that the nucleic acid sequence encoding the variant has 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% identity with the nucleic acid sequence encoding a non-mutant (wild-type) mutant. In some embodiments of the present invention, variants include mutants containing conserved substitutions relative to non-mutants. “Conservative substitution” is, in the art, considered as the substitution of one amino acid for another having similar properties. Exemplary examples of conserved substitutions are well known in the art (see, for example, WO 97 / 09433, page 10, published March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY: NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8).

[0221] Endocytosis is a process by which substances enter a cell. In endocytosis, the substance to be taken in is surrounded by a region of the plasma membrane, which then buds inside the cell to form a vesicle containing the taken-in substance. Endocytosis can be classified into four types: receptor-mediated endocytosis (also known as clathrin-mediated endocytosis), caveolae, pinocytosis, and phagocytosis (Marsh M, Endocytosis. Oxford University Press. p.vii., 2001).

[0222] "Complement": The complement system is composed of a series of proteins and is part of the innate immune system. Complement is present in the serum, tissue fluid, and cell membrane surface of normal humans and animals. After activation, it possesses enzymatic activity and can undergo complex cascade reactions. The complement system is initiated by a series of enzymes cleaving each other, ultimately forming a pore-like membrane attack complex on the target microorganism, causing the microorganism to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, clearing immune complexes through cytolysis, opsonization, phagocytosis, and mediating inflammatory responses, exhibiting corresponding biological functions. Complement is widely involved in the body's defense responses against microbial infections and immune regulation, and also mediates immunopathological damage responses, making it an important effector system and effector mechanism system in the body. Viral glycoproteins such as VSV-G may be recognized and inactivated by complement after entering serum; therefore, improving the ability of viral glycoproteins to antagonize complement inactivation can effectively improve the survival rate of viral glycoproteins such as VSV-G in the body or blood of subjects.

[0223] "Exogenous" refers to any molecule originating from outside the organism, including nucleic acids, proteins, polypeptides, or small molecule compounds. In contrast, the term "endogenous" refers to any molecule originating from within the organism (i.e., naturally produced by the organism).

[0224] "Viral envelope": This refers to the outermost layer of many viruses (HURLBERT, RONALD E., Fundamentals of Microbiology, 102. Chapter #11: Viruses. Archived from the original on 2008-11-10.). As viruses travel through host cells, the viral envelope protects their genetic material throughout their life cycle. Not all viruses have a viral envelope. Many human pathogenic viruses are encased in a lipid bilayer; they infect target cells by fusing their viral envelope with the cell membrane. Viruses with a viral envelope include retroviruses, among others.

[0225] Lentivirals are complex retroviruses that contain not only the common retroviral genes Gag, Pol, and env, but also other genes with regulatory or structural functions. This high complexity allows viruses to regulate their life cycle, as they do during latent infection. Lentivirals belong to the genus of retroviruses that can infect both 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 simultaneous immunodeficiency virus (SIV).

[0226] Lentiviral vectors are vectors derived from lentiviruses that contain one or more lentiviral packaging proteins and / or lentiviral proteins necessary for the expression of one or more genes carried by the vector. They are produced by repeatedly attenuating the virulence genes of lentiviruses such as HIV through gene editing, genetic engineering, and other techniques. For example, deleting genes such as env, vif, vpr, vpu, and nef can make lentiviral vectors biosafety-compliant.

[0227] Lentiviral vectors or retroviral vectors are generally packaged in packaging cells using lentiviral vector packaging systems or retroviral vector packaging systems. For an exemplary procedure and method for packaging lentiviral vectors, see Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. (2016), 3, 16017, which is incorporated herein by reference in its entirety.

[0228] Commonly used pseudotyped lentiviral vectors include the so-called third-generation lentiviral vector packaging system. This system typically includes four plasmids: a transfer plasmid containing the gene of interest (GOI), such as a transgenic transfer plasmid, GagPol plasmid, Rev plasmid, and an envelope plasmid (containing viral glycoprotein genes such as VSV-G or its variants, or Cocal-G or its variants).

[0229] A transfer vector contains the lentiviral vector backbone genome and transgenes. Transfer vectors typically have one or more transgenes flanked by long terminal repeats (LTRs), which facilitate the integration of the transgenes contained in the transfer vector into the host genome. LTRs are responsible for the reverse transcription and integration of the viral genome. Through these sequences, lentiviruses can integrate transgenes into the host cell's genome. For safety reasons, transfer vectors are usually engineered to prevent the resulting viral vector from self-replicating. For example, transfer vectors lack the genetic elements necessary to produce infectious lentiviral particles in the host cell. Furthermore, transfer plasmids can be engineered to lack the 3'LTR, thereby achieving "self-inactivating" of the virus. Compared to traditional second-generation pseudotyped lentiviral vector packaging systems (typically containing a single packaging plasmid and a separate envelope plasmid encoding nucleic acids for Gag, Pol, Rev, and Tat), 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) to the transfer plasmid. Transfer plasmids typically contain a Ψ sequence (Psi sequence, also known as the Ψ packaging signal) downstream of the 5'LTR, responsible for packaging the transgenic RNA into the viral particle. The Ψ sequence ensures that only transgenic RNA is packaged into the viral particle. Optionally, transfer plasmids may also include an internal ribosome entry site. Sites (“IRES”) are used to allow simultaneous translation of two or more open reading frames (ORFs) on a single mRNA, thereby enabling multi-gene expression. Some transfer plasmids, such as the lentiviral master plasmids / transfer plasmids used in some embodiments of the present invention, may also contain selection marker genes, such as antibiotic resistance genes (e.g., PuroR, encoding puromycin resistance) or fluorescent protein genes (e.g., GFP), for screening or tracking transduced cells.

[0230] For details on transfer plasmids in lentiviral vector packaging systems, please refer to DuLl, et al., J.Virol.72:8463-71 (1998); Miyoshi, et al., J.Virol.72:8150-57 (1998).

[0231] Third-generation lentiviral vector systems typically include three packaging plasmids: the GagPol plasmid, the Rev plasmid, and the envelope plasmid. The envelope plasmid usually carries viral glycoprotein genes; wild-type VSV-G or Cocal-G are commonly used viral glycoproteins. The viral glycoprotein genes are operatively linked to a promoter, typically a CMV promoter, to initiate transcription of the viral glycoprotein genes. Third-generation lentiviral vector systems also include two packaging plasmids: one containing genes encoding the Gag and Pol proteins (GagPol packaging plasmid), and the other containing genes encoding the Rev protein (Rev plasmid) as a further safety feature, representing an improvement over the single packaging plasmid in so-called second-generation packaging systems. The Gag gene encodes the Gag polyprotein precursor, which contains lentiviral structural proteins including the matrix, capsid, and nucleocapsid; the Pol gene encodes the Pol polyprotein precursor, which provides the lentiviral enzyme functions necessary for replication, including a protease, reverse transcriptase, and integrase; the Rev gene encodes the Rev protein, which binds Rev response elements (RREs) to allow the nuclear export of unspliced ​​and single-spliced ​​HIV RNA during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral particle preparation. The Rev protein binds to the Rev response element (RRE) sequence on the viral RNA, facilitating the transport of incompletely cleaved viral RNA from the nucleus to the cytoplasm through interaction with the host cell's nuclear export mechanism. This unspliced ​​RNA can then be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral particles.

[0232] For example, the packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0233] Lentiviral vectors and lentiviral vector backbone genomes are known in the art; see Naldini, et al., (1996) Science 272: 263-7; Zufferey, et al., (1998) J.Virol. 72: 9873-9880; DuLl, et al., (1998) J.Virol. 72: 8463-8471; U.S. Patent Nos. 6,013,516 and 5,994,136, each of which is incorporated herein by reference in its entirety.

[0234] Compared to pseudolentiviral vector packaging systems, pseudoretroviral vector packaging systems typically do not contain the Rev plasmid. This is because the genomic RNA from retroviruses such as Moloney Murine Leukemia Virus (MMLV) can be naturally transported from the nucleus to the cytoplasm for translation and assembly, thus eliminating the need for specific nuclear export mechanisms such as Rev proteins. Pseudoretroviral vector packaging systems typically contain one transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgenic sequence contained in the transfer plasmid is flanked by long terminal repeats (LTRs), which facilitate the integration of the transfer plasmid sequence into the host genome. Generally, during viral transduction, sequences between and including the LTRs are integrated into the host genome. The backbone genomes of MMLV or Murine Stem Cell Virus (MSCV), containing their respective LTRs, are often used to construct the transfer plasmid in pseudoretroviral vector packaging systems. GagPol packaging plasmids contain the Gag and Pol genes; envelope plasmids typically contain polynucleotides encoding viral glycoproteins such as VSV-G or Cocal-G. In some embodiments, packaging plasmids containing the pol gene contain mutations that disable integrase (disabling mutations) and / or transfer plasmids / master plasmids containing transgenes contain LTRs of the lentiviral / retroviral backbone genome that contain CA mutations.

[0235] In some embodiments, production cells are transfected with a defined ratio of transfer plasmid and envelope plasmid. In some embodiments, the ratio of the plasmids is determined by quality and is not particularly limited as long as it can package biologically active non-cellular particles. In some embodiments, the defined ratio of transfer plasmid to envelope plasmid is from about 1:1 to about 9:2; in some embodiments of the invention, the envelope plasmid may contain nucleic acid encoding a target molecule.

[0236] In some embodiments, production cells are transfected with a defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid. In some embodiments, the ratio of each plasmid is determined by mass, and is not particularly limited as long as it can package a biologically active non-integrating lentiviral vector. In some embodiments, the mass of each of the transfer plasmid and GagPol plasmid is higher than the mass of each of the envelope plasmid and Rev plasmid. In some embodiments, the defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid is from about 1:1:1:1 to about 9:4:2:2; in some embodiments of the invention, the envelope plasmid may contain nucleic acid encoding a target molecule.

[0237] In some embodiments, the envelope plasmid contains a tandem expression cassette encoding VSV-G or a variant thereof or Cocal-G or a variant thereof and a target molecule as disclosed herein. In a specific embodiment, the tandem expression cassette contained in the envelope plasmid contains a polynucleotide encoding a first signal peptide, a polynucleotide encoding a target 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 some embodiments, the polynucleotide encoding VSV-G or a variant thereof or Cocal-G or a variant thereof is located at the 5' end of the polynucleotide encoding the target molecule. In other embodiments, the polynucleotide encoding VSV-G or a variant thereof or Cocal-G or a variant thereof is located at the 3' end of the polynucleotide encoding the target molecule. The polynucleotide encoding the target 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 IRES, a furin cleavage site, or a viral 2A peptide, which allows co-expression of both proteins by a single mRNA. In some implementations, the viral 2A peptide is porcine cheshvirus-1 (P2A), Thosea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof.

[0238] The use of lentiviral / retroviral vectors or non-cellular particle packaging systems relies on “packaging cell lines.” Generally, a packaging cell line is a cell line whose cells, upon introduction of a transfer plasmid or one or more packaging plasmids, are capable of producing lentiviral vectors, retroviral vectors, or non-cellular particles (including NILs) that are not capable of self-replication and can infect / transduce target cells. An overview of available packaging lines is provided in Cold Spring Harbour Laboratory Press, 1997, p. 447, by JM Coffin, SM Hughes, et al., and is incorporated herein by reference in its entirety.

[0239] For example, various plasmids can be introduced into packaging cell lines using transfection methods including chemically mediated transfection, physically mediated transfection, or biologically mediated transfection. For instance, chemically mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-glucan, or PEI (polyethylenimine transfection reagent), while physically mediated transfection methods include transfection methods such as electroporation.

[0240] The packaging cells can be genetically engineered to improve the efficiency of non-cellular particles (including NILs) transducing target cells disclosed in this invention in other ways; such other ways include, but are not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0241] Production / host / packaging cells that can be used to prepare the non-cellular particles (including NILs) disclosed in this invention include human embryonic kidney (HEK) 293 cells and their derivatives. Production cells can be adherent cell lines such as HEK293T production cells, or suspension cell lines such as HEK293T / 17SF production cells.

[0242] For example, the packaging cells / host cells are selected from 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;

[0243] Preferably, the packaging cell / host cell is HEK-293T cell.

[0244] "Retrovirus" and "Retroviral Vector": These are the terms Retrovirus and Retroviral Vector. A "retrovirus" is an RNA virus containing a single-stranded, positive-signal RNA molecule. Retroviruses contain reverse transcriptase and integrase. Upon entering a target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the integrase integrates the DNA molecule into the host cell's genome. The sequence derived from the retrovirus after integration into the host cell's genome is called a provirus (e.g., a proviral sequence or proviral vector). A retroviral vector typically refers to a pseudotyped retroviral vector derived from a retrovirus, exemplarily from γ-retrovirus. Unlike lentiviral vectors that can transduce both dividing and non-dividing cells, retroviral vectors can only transduce dividing cells, and the amount of exogenous transgenes they can carry is generally relatively small. For a comparison and discussion of lentiviral vectors and retroviral vectors, please see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, KK, Vorburger, SA, Swisher, SG(eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0245] "Viral glycoproteins" are glycoproteins that coat the outer layer of a virus. They play an important role in viral adsorption and penetration into host cells, pathogenicity, downregulation of host surface protein expression, and increased viral packaging and budding processes.

[0246] "Pharmaceutically acceptable excipients or carriers": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration used.

[0247] “Subject”: As used herein, the terms “subject,” “patient,” and “individual” are used synonymously, including, but not limited to, mammals, such as humans or non-human mammals, such as livestock, agricultural animals, or wild animals, as well as birds and aquatic animals. “Patient” is a subject who suffers from a disease, condition, or illness, or is at risk of developing a disease, condition, or illness, or otherwise requires any of the noncellular particulate (NIL) compositions or treatments provided herein.

[0248] “Treatment”: As used herein, “treatment” includes any beneficial or desired effect associated with treatment. “Treatment” does not necessarily indicate the complete eradication or cure of a disease or condition, or its associated symptoms. In this document, “treatment” includes administering to a subject the noncellular particles (including NILs), compositions, or treatment / prevention methods described herein provided by the invention to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). Effective treatment methods for patients can vary depending on various factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0249] "Prevention": As used herein, "prevention" and similar terms, such as "avoidance," indicate methods used to prevent, suppress, or reduce the likelihood of the occurrence or recurrence of a condition. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition before its onset or recurrence.

[0250] "Stable integration": also known as "stable transduction" or "stable gene expression", refers to the integration of exogenous nucleic acids into the host cell genome after they are introduced into the host cell, and their stable expression in the host cell over a long period of time.

[0251] "Specific binding": As used herein, the term "specific binding" refers to binding that occurs between paired molecular species (e.g., receptor and ligand, antibody and antigen). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, specific binding between one or more species is direct. In some embodiments of the invention, the affinity of specific binding is 1, 1.5, 2, 5, 10, 20, 50, 100, or 1000 times or more of background binding (non-specific binding).

[0252] "Sequence identity": Generally speaking, "sequence identity" or "sequence homology" refers to the precise correspondence between nucleotides or amino acids of two nucleic acid sequences or protein / peptide sequences. Typically, techniques used to determine sequence identity involve identifying the nucleotide sequence of the nucleic acid and / or the amino acid sequence it encodes, and comparing these sequences to control nucleotide or amino acid sequences. Two or more sequences (nucleic acids or amino acids) can be compared by determining their "percentage of identity." Whether it's a nucleic acid or amino acid sequence, the percentage of identity between two sequences is the number of precise matches between the two aligned sequences divided by the length of the shorter sequence, and then multiplied by 100. For example, the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percentage of identity. The BLAST procedure is based on the following alignment methods: Karlin and Altschu L, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and discussed in Altschu L et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschu L, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschu L et al., Nucleic Acids Res. 25: 3389-3402 (1997). In short, the BLAST procedure defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of shorter symbols in both sequences. The procedure can be used to determine the percentage of identity across the entire length of the compared protein / peptide.

[0253] "MOI" stands for "Multiplicity of Infection (MOI)," which refers to the number of viral particles added to each cell during viral infection or transduction. For example, when one million viral particles are added to one million cells, MOI = 1.

[0254] “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. 2A peptides are 18 to 22 residue-long viral oligopeptides that mediate the “cleavage” of polypeptides during translation in eukaryotic cells. “2A peptide” can refer to peptides with different amino acid sequences. In this disclosure, it should be understood that in the case where non-cellular particles (including NILs) contain two or more 2A peptides, the 2A peptides may be identical or different from each other. Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) ColdSpring Harb. Protoc. 2012:199-204.

[0255] "Self": As used in this article, the term "self" means any material derived from the same individual that is subsequently reintroduced into that individual.

[0256] "Allogeneic" as used in this article refers to grafts that are derived from different individuals of the same species.

[0257] "Therapeutic Effective Amount": As used herein, "therapeutic Effective Amount" is the amount of a composition or active substance thereof administered to an individual that is sufficient to provide a beneficial effect or otherwise reduce harmful, non-beneficial events, such as the amount of noncellular particles (including NILs) provided by the present invention. "Therapeutic Effective Dose" herein means the dose that produces one or more desired or anticipated (e.g., beneficial) effects as a result of its administration, given once or more times over a specified period of time. The exact dose will depend on the therapeutic purpose and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0258] “Transduction”: As used herein, the terms “transfection,” “transformation,” and “transduction” are used synonymously to refer to the process of transferring or introducing exogenous nucleic acids into host / packaging cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0259] Methods for introducing vectors such as non-cellular particles (NILs) or isolated polynucleotides into mammalian cells are known in the art. The described vectors can be transferred to immune effector cells by physical, chemical, or biological methods.

[0260] Physical methods for introducing vectors or isolated polynucleotides into immune effector cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, the vector is introduced into cells via electroporation. In some embodiments of the present invention, the vector is introduced into cells via a PEI transfection reagent.

[0261] All publications, documents, and patents mentioned herein are hereby incorporated in their entirety by reference, as are each publication, document, or patent not specifically and individually indicated to be incorporated herein by reference in its entirety. In case of conflict, this application (including any definitions herein) shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or recommendation of any kind.

[0262] The section headings used in this article are for organizational purposes only and are not intended to limit the topics described. Attached Figure Description

[0263] Figure 1: A plasmid map of packaging plasmid 1 containing the integrase disabling mutation D64V in Example 1;

[0264] Figure 2: A plasmid map of transfer plasmid 1 containing the CA mutation in Example 1;

[0265] Figure 3: Flow cytometry results of GFP positivity rate detected on Day 2, Day 4 and Day 6 after the NIL-A3mG1-GFP and the control group targeted LVV-GFP were respectively infected with LDL-R+CD3-Nalm-6 cells and LDL-R+CD3+Jurkat cells in Example 1.

[0266] Figure 4: Flow cytometry results of GFP positivity rate after NCP-A3mG1-GFP infection of Jurkat cells in Example 2;

[0267] Figure 5: Flow cytometry results of CAR-19 positivity rate after NCP-A3mG1-CAR19 infects Jurkat cells in Example 3;

[0268] Figure 6: A comparison of the GFP positivity rate after NCP-A3mG1-GFP infection of Jurkat / Nalm-6 in Example 4;

[0269] Figure 7: A comparison of the GFP positivity rate after NCP-A3mG1-GFP infection of Jurkat / Nalm-6 in Example 5; Embodiments of the present invention

[0270] The present invention and its technical effects will be clearly and completely described below with reference to embodiments, so as to fully understand the technical solution, the technical problem solved, and the beneficial effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments; other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0271] Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions known in the art, or as selected according to the product instructions. Reagents and raw materials not specifically named in this invention are commercially available. Example 1

[0272] The package contains (a) a mutated viral glycoprotein; (b) an anti-CD3 antibody targeting molecule; and (c) a noncellular particle (NCP) of the GFP gene, and a non-integrating lentiviral vector NIL-A3mG1-GFP.

[0273] 1. Constructing anti-CD3 antibody targeting molecules

[0274] In this example, a membrane-based anti-CD3 antibody targeting molecule is constructed, whose structure from the N-terminus to the C-terminus is as follows: CD8α signal peptide, anti-CD3 antibody (scFv derived from the anti-CD3 monoclonal antibody UCHT1, scFv-UCHT1), CD8α hinge region, and CD8α transmembrane region.

[0275] (1) The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO:8;

[0276] (2) The amino acid sequence of the anti-CD3 antibody (scFv) UCHT1 is shown in SEQ ID NO:9;

[0277] (3) The amino acid sequence of the hinge region of CD8α is shown in SEQ ID NO:10;

[0278] (4) The amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO:11.

[0279] 2. Packaging NIL-A3mG1-GFP

[0280] A. Preparing the NIL Packaging System

[0281] Prepare the following four plasmids: 1 encapsulation plasmid, 1 packaging plasmid, 1 pRSV-REV packaging plasmid, and 1 transfer plasmid;

[0282] The envelope plasmid 1 contains a polynucleotide encoding a mutant VSV-G1 and a polynucleotide encoding the expression of the anti-CD3 antibody in the membrane; the mutant VSV-G1 contains the amino acid sequence shown in SEQ ID NO:3; relative to SEQ ID NO:1, SEQ ID NO:3 contains a K47 deletion;

[0283] The packaging plasmid 1 (plasmid map shown in Figure 1) contains a polynucleotide encoding a disabled integrase, the disabled integrase containing the D64V mutation located at SEQ ID NO:29;

[0284] The transfer plasmid 1 (plasmid map shown in Figure 2) contains a lentiviral backbone genome with the CA mutation of U3 in 5'LTR and 3'LTR replaced by TG.

[0285] The plasmids described above were synthesized using conventional molecular cloning techniques known in the art.

[0286] B. Plasmid transfection and packaging of cells to package NIL-A3mG1-GFP

[0287] Prepare the HEK-293T cell culture system: DMEM / high glucose (brand: GIBCO, catalog number: C12430500BT) + 10% FBS (brand: EXCELL, catalog number: FSP500) + 1× penicillin-streptomycin mixture;

[0288] Day 2: 4 × 10⁶ HEK-293T cells were seeded in a 10 cm culture dish. Approximately 48 hours after seeding, the cells were cultured until the cell confluence reached 80-90%.

[0289] On Day 0, 9 μg of the transfer plasmid 1, 2 μg of the envelope plasmid 1, 4 μg of the packaging plasmid 1, and 2 μg of the pRSV-REV packaging plasmid were added to 1 mL of Opti-MEM medium. After shaking well, 64 μL of PEI reagent was added, and the mixture was allowed to stand for 10 minutes. Then, it was added to the HEK-293T cell culture system. The medium was replaced after 6 hours. 48 hours after transfection, the supernatant was collected, filtered through a 0.45 μm filter membrane, centrifuged at 50,000 g for 2.5 h, the supernatant was discarded, and the NIL-A3mG1-GFP was resuspended in 200 μL of F12 medium and stored at -80 °C.

[0290] Opti-MEM alpha serum-reduced culture medium, brand: GIBCO, catalog number: #SP0272;

[0291] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog No.: #C12430500BT; FBS: Brand: EXCELL, Catalog No.: #FSP500;

[0292] F12 medium: Brand: GIBCO, Product No.: #C11330500BT;

[0293] Needle filter: Brand: SORFA, Item No.: #622120.

[0294] C. Packaging control group targeted lentiviral vector (LVV)

[0295] Following the packaging method for NIL-A3mG1-GFP, a control group targeting LVV-GFP was packaged, wherein the packaging plasmid 1 was replaced with a pMDLg / pRRE packaging plasmid that does not contain the integrase disabling mutation D64V; the transfer plasmid 1 was replaced with a lentiviral transfer plasmid (pGClenti-GFP) that does not contain the CA mutation in its LTRs; the control group targeting LVV-GFP has the ability to integrate the GFP gene it carries into the target cell genome and express it stably.

[0296] D.NIL Infection Test

[0297] 100 μL of NIL-A3mG1-GFP and the control group targeting LVV-GFP were co-incubated with 1×105 LDL-R+CD3+Jurkat cells and LDL-R+CD3-Nalm-6 cells, respectively. On Day 2, Day 4 and Day 6, the expression of GFP in each group of cells was detected by flow cytometry. The results are shown in Figure 3.

[0298] As shown in Figure 3, the NIL-A3mG1-GFP cannot effectively infect LDL-R+CD3-Nalm-6 cells, but it can effectively infect LDL-R+CD3+Jurkat cells. This proves that the NIL-A3mG1-GFP can target and deliver the GFP gene to CD3+Jurkat cells.

[0299] Furthermore, based on the observations on Day 4 and Day 6, the GFP gene remained stably expressed in the control group Jurkat cells infected with LVV-GFP (the GFP positivity rate remained above 30%), indicating that the control group targeting LVV-GFP can stably integrate the GFP gene into the genome of LDL-R+CD3+ Jurkat cells for continuous expression. In contrast, in the Jurkat cells infected with NIL-A3mG1-GFP, the GFP gene expression efficiency was significantly reduced on Day 4 and Day 6, especially on Day 6, where only a GFP positivity rate of 1.91% was detected. This demonstrates that NIL-A3mG1-GFP has the ability to deliver the GFP gene to target cells and transiently express it.

[0300] In summary, the NIL-A3mG1-GFP can target and deliver the GFP gene to Jurkat cells for transient expression. Example 2

[0301] The package contains (a) a mutant viral glycoprotein, the mutant VSV-G1; (b) the targeting molecule, membrane-expressing an anti-CD3 antibody; and (c) a non-cellular particle of the intracellular free protein GFP, NCP-A3mG1-GFP.

[0302] 1. Packaging NCP-A3mG1-GFP

[0303] Prepare the following two plasmids: the envelope plasmid 1 and the pGClenti-GFP plasmid;

[0304] Following the method described in Example 1 for packaging NIL-A3mG1-GFP, the two plasmids were transfected into HEK-293T cells, and the non-cellular particles NCP-A3mG1-GFP were packaged and collected.

[0305] 2. NCP infection test

[0306] 100 μL and 200 μL of NCP-A3mG1-GFP were co-incubated with 1×105 CD3+Jurkat cells, respectively. After 24 h, the expression of GFP was detected by flow cytometry. The results are shown in Figure 4.

[0307] As shown in Figure 4, the NCP-A3mG1-GFP can effectively infect CD3+Jurkat cells and deliver intracellular free target protein GFP. Example 3

[0308] The package contains (a) a mutant viral glycoprotein, said mutant VSV-G1; (b) an anti-CD3 antibody targeting molecule; and (c) a noncellular particle NCP-A3mG1-CAR19 of the membrane-localized expressed protein CAR-19.

[0309] 1. Constructing the membrane-localized expression protein CAR-19

[0310] In this embodiment, a chimeric antigen receptor CAR-19 targeting CD19 is constructed, the structure of which from the N-terminus to the C-terminus is as follows: the CD8α signal peptide, the extracellular antigen-binding region targeting CD19, the CD8α hinge region, the CD8α transmembrane region, the 4-1BB co-stimulatory signal transduction domain, and the CD3ζ intracellular signal transduction domain; wherein, the extracellular antigen-binding region targeting CD19 is scFv (scFv-FMC63) derived from FMC63, the scFv-FMC63 includes the VH region and VL region of FMC-63, and the VH region is connected to the VL region through the (G4S)3 linker peptide.

[0311] (1) The amino acid sequence of the VH region of FMC-63 is shown in SEQ ID NO:14, and the amino acid sequence of the VL region of FMC-63 is shown in SEQ ID NO:15;

[0312] (2) The amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:17;

[0313] (3) The amino acid sequence of the intracellular signal transduction domain of CD3ζ is shown in SEQ ID NO:18;

[0314] (4) The amino acid sequence of the (G4S)3 linker peptide is shown in SEQ ID NO:16.

[0315] 2. Packaging: NCP-A3mG1-CAR19

[0316] NCP-A3mG1-CAR19 was packaged according to the method for packaging NCP-A3mG1-GFP in Example 2; wherein, the pGClenti-GFP plasmid was replaced with transfer plasmid 2, and the transgene carried by the transfer plasmid 2 was a polynucleotide encoding the CAR-19, relative to the transgenic GFP gene carried by the pGClenti-GFP plasmid.

[0317] 3. NCP infection test

[0318] 10 μL and 20 μL of NCP-A3mG1-CAR19 were co-incubated with 1×105 Jurkat cells. After 24 h, the expression of CAR-19 was detected by flow cytometry. The results are shown in Figure 5.

[0319] As shown in Figure 5, the NCP-A3mG1-CAR19 can effectively infect CD3+Jurkat cells and deliver the target protein, CAR-19 expressed on the membrane. Example 4

[0320] 1. Construct an envelope plasmid containing an anti-CD19 antibody targeting molecule according to Example 1, replacing the anti-CD3 antibody element with an anti-CD19 antibody element; wherein: the amino acid sequence of the anti-CD19 antibody (scFv) is as shown in SEQ ID NO:30: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0321] 2. Using the envelope plasmid constructed in step 1, package the NIL-A3mG1-GFP virus according to Example 1.

[0322] 3. Referring to Example 1, the virus prepared in step 2 was co-incubated with 1×10⁵ Jurkat cells and Nalm-6 cells, respectively. On Day 2, Day 4 and Day 6, the expression of GFP in each group of cells was detected, and the results are shown in Figure 6.

[0323] As shown in Figure 6, the NIL-A3mG1-GFP could not effectively infect Jurkat cells, but it could effectively infect Nalm-6 cells. This proves that the virus constructed in step 2 can target and deliver the GFP gene to CD19+ Nalm-6 cells. Furthermore, a significant decrease in GFP gene expression efficiency was detected on Day 4 and Day 6. This proves that the NIL-A3mG1-GFP has the ability to deliver the GFP gene to target cells and express it transiently. Example 5

[0324] 1. Construct an envelope plasmid containing an anti-CD7 antibody targeting molecule according to Example 1, replacing the anti-CD3 antibody element with an anti-CD7 antibody element; wherein: the amino acid sequence of the anti-CD7 antibody (scFv) is as shown in SEQ ID NO:31: AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSS.

[0325] 2. Using the envelope plasmid constructed in step 1, package the NIL-A3mG1-GFP virus according to Example 1.

[0326] 3. Referring to Example 1, the virus prepared in step 2 was co-incubated with 1×10⁵ Jurkat cells and Nalm-6 cells, respectively. On Day 2, Day 4 and Day 6, the expression of GFP in each group of cells was detected, and the results are shown in Figure 7.

[0327] As shown in Figure 7, the NIL-A3mG1-GFP can effectively infect Jurkat cells but cannot effectively infect Nalm-6 cells, which proves that the virus constructed in step 2 can target and deliver the GFP gene to CD7+ Jurkat cells; furthermore, a significant decrease in GFP gene expression efficiency was detected on Day 4 and Day 6, which proves that the NIL-A3mG1-GFP has the ability to deliver the GFP gene to target cells and express it transiently.

Claims

1. A non-cell particle (NCP), characterized in that, The noncellular particles comprise: (a) Viral glycoprotein; and (b) Targeting molecules that can bind to antigens on the surface of target cells.

2. The acellular particles of claim 1, wherein, The viral glycoprotein's ability to bind to its receptor is weakened or inhibited.

3. The non-cellular particles according to claim 1 or 2, characterized in that, The number of binding sites available for the viral glycoprotein to bind to its receptor has decreased.

4. The non-cellular particle according to any one of claims 1-3, characterized in that, The viral glycoprotein has bound to one or more of its receptors and antibodies.

5. The non-cellular particle according to any one of claims 1-4, characterized in that, The viral glycoprotein contains a first mutation that weakens or inhibits the viral glycoprotein's ability to bind to its receptor.

6. The non-cellular particle according to any one of claims 1-5, characterized in that, The viral glycoproteins are selected from vesicular stomatitis virus strains, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and T-cell choroid plexus meningitis virus (LCMV) glycoprotein. Preferably, the vesicular stomatitis virus glycoprotein is selected from: vesicular stomatitis virus Indiana strain glycoprotein, vesicular stomatitis virus Cocal strain glycoprotein, vesicular stomatitis virus Maraba strain glycoprotein, vesicular stomatitis virus Morreton strain glycoprotein, vesicular stomatitis virus Alagoas strain glycoprotein, vesicular stomatitis virus New Jersey strain glycoprotein, vesicular stomatitis virus Carajas strain glycoprotein, and vesicular stomatitis virus Chandipura strain glycoprotein. The glycoproteins of the following strains of vesicular stomatitis virus (VSV) are: Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and YugBogdanovac.

7. The non-cellular particle according to any one of claims 1-6, characterized in that, The viral glycoprotein is either the Indiana strain glycoprotein of vesicular stomatitis virus (VSV-G) or the Cocal strain glycoprotein of vesicular stomatitis virus (Cocal-G).

8. The non-cellular particles according to claim 7, characterized in that, (a) The VSV-G has bound to one or more of its receptor LDL-R and anti-VSV-G antibodies; or (b) The Cocal-G has been bound to one or more of its receptor LDL-R and anti-Cocal-G antibodies.

9. The non-cellular particles according to claim 7, characterized in that, The VSV-G or Cocal-G contains a first mutation that weakens or inhibits the ability of the VSV-G or Cocal-G to bind to its receptor LDL-R.

10. The non-cellular particles according to claim 9, characterized in that, The first mutation is selected from one or more of the following mutations: (a) Substitution or deletion of amino acids at positions 8, 9, 10, 47, 50, 51, 183, 179, 180, 182, 184, 209, 347, 350, 352, 353, and 354 in SEQ ID NO:1 or SEQ ID NO:2; deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353; and (b) Substitution or deletion of amino acids at positions 8, 9, 10, 47, 50, 51, 183, 179, 180, 182, 184, 209, 347, 350, 352, 353, and 354, and deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353, after best global alignment with SEQ ID NO:1 or SEQ ID NO:2; Preferably, the first mutation is selected from one or more of the following mutations: (a) Amino acid deletion at positions 331-364, amino acid deletion at positions 344-354, substitution of K47, deletion of K47, substitution of R354, substitution of Y209, and substitution of I182 located in SEQ ID NO:1; (b) After best global alignment with SEQ ID NO:1, the amino acid deletions at positions 331-364, positions 344-354, substitutions for K47, deletions of K47, substitutions for R354, substitutions for Y209, and substitutions for I182 are located at positions corresponding to SEQ ID NO:

1. (c) Deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354, substitution of Y209, and substitution of V182 in SEQ ID NO:2; and (d) After best global alignment with SEQ ID NO:2, the amino acid deletions at positions 331-364, positions 344-354, substitutions for K47, deletions of K47, substitutions for R354, substitutions for Y209, and substitutions for V182 are located at positions corresponding to SEQ ID NO:

2. More preferably, the first mutation is: (a) K47 missing in SEQ ID NO:1 or SEQ ID NO:2; or (b) K47 deletion located at the equivalent of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:

2.

11. The noncellular granules according to any one of claims 1-10, characterized in that, The viral glycoprotein contains a second mutation that enhances or prevents the viral glycoprotein from being inactivated by complement.

12. The particles according to claim 11, characterized in that, The viral glycoprotein is either VSV-G or Cocal-G; Preferably, the second mutation is selected from one or more of the following site mutations: (a) The 214th amino acid located in SEQ ID NO:1 or SEQ ID NO:2; (b) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at position 214, which corresponds to SEQ ID NO:1 or SEQ ID NO:2; (c) The 352nd amino acid located in SEQ ID NO:1 or SEQ ID NO:2; (d) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:1 or SEQ ID NO:2; (e) The 50th amino acid located in SEQ ID NO:1 or SEQ ID NO:2; (f) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:1 or SEQ ID NO:2; (g) The amino acid located at position 146 of SEQ ID NO:1 or SEQ ID NO:2; and (h) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at the 146th position corresponding to SEQ ID NO:1 or SEQ ID NO:2; more preferably, the mutation at the site is selected from amino acid substitution, deletion and insertion; even more preferably, the mutation at the site is an amino acid substitution.

13. The non-cellular particles according to claim 12, characterized in that, The second mutation is selected from a combination of mutations at the following sites: (a) Substitution of (1) T214 and T352 located in SEQ ID NO:1, or (2) Substitution of T214, T352, K50 and S146; (b) Substitutions of (1) T214 and T352, or (2) T214, T352, K50 and S146, located at the equivalent of SEQ ID NO:1 after best global alignment with SEQ ID NO:1; (c) Substitution of (1) K214 and T352 at SEQ ID NO:2, or (2) Substitution of K214, T352, K50 and S146; and (d) After best global alignment with SEQ ID NO:2, the substitutions located at (1) K214 and T352, or (2) K214, T352, K50 and S146 of SEQ ID NO:2; Preferably, the second mutation is selected from a combination of mutations at the following sites: (1) T214N and T352A located in SEQ ID NO:1, or (2) T214N, T352A, K50T and S146T; (b) After best global alignment with SEQ ID NO:1, it is located at (1) T214N and T352A, or (2) T214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:1; (c) K214N and T352A located in SEQ ID NO:2, or (2) K214N, T352A, K50T and S146T; and (d) After best global alignment with SEQ ID NO:2, it is located at (1) K214N and T352A, or (2) K214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:

2.

14. The noncellular granules according to any one of claims 1-13, characterized in that, The target cells are lymphocytes; Preferably, the lymphocytes are selected from one or more of T cells, NK cells, and B cells; More preferably, the antigens on the surface of the T cells are selected from: CD2, CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD47, CD57, CD58, CD62L, CD71, AhR, CD69, CD94, CD95, 4-1BB, C One or more of the following: D103, 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; More preferably, the antigen on the surface of the B cells is selected from one or more of the following: CD19, CD20, CD21, CD22, CD23, CD24, CD27, CD32b, CD37, CD38, CD40, CD52, CD72, CD74, CD80, CD84, CD86, IL-7RA (CD127), CD138, CD257, CD267, CD268, CD269, and CD270; More preferably, the antigen on the surface of the NK cells is selected from one or more of the following: CD16, CD56, NKp46, KIRs, NKG2D, KLRB1 (CD161), KLRD1 (CD94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R.

15. The noncellular granules according to any one of claims 1-14, characterized in that, The targeting molecule includes a targeting binding region comprising one or more of (a) an antibody or an antigen-binding fragment thereof capable of binding to an antigen on the surface of a target cell; and (b) a ligand or a binding fragment thereof. Preferably, the binding fragment includes one or more selected from ligand extracellular domains, functional fragments, and epitopes.

16. The non-cellular particles according to claim 15, characterized in that, The target binding region comprises one or more selected from anti-CD7 antibody or its antigen-binding fragment, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, anti-CD28 antibody or its antigen-binding fragment, CD80 or its binding fragment, and CD86 or its binding fragment.

17. The noncellular granules according to any one of claims 1-16, characterized in that, The target molecule is a transmembrane protein.

18. The non-cellular particles according to claim 17, characterized in that, The target molecule is a recombinant transmembrane protein; Preferably, the targeting molecule includes a transmembrane region; More preferably, the targeting molecule further includes a connecting domain.

19. The noncellular granules according to any one of claims 1-18, characterized in that, The non-cellular particles contain one or more target proteins (POIs).

20. The non-cellular particles according to claim 19, characterized in that, The POI includes one or more of intracellular free proteins and membrane-localized expressed proteins.

21. The noncellular granules according to any one of claims 1-20, characterized in that, The noncellular particles are virus-like particles (VLPs).

22. The noncellular granules according to any one of claims 1-20, characterized in that, The non-cellular particles are non-integrating lentiviral vectors (NILs).

23. The non-cellular particles according to claim 22, characterized in that, The NIL contains a disabled integrase.

24. The non-cellular particles according to claim 23, characterized in that, The disabled integrase comprises a sequence selected from or located at SEQ ID NO:29, or, after optimal global alignment with SEQ ID NO:29, located at the equivalent of SEQ ID NO:

29. Amino acid mutations at one or more of the following sites in NO:29: positions 10, 11, 12, 13, 16, 41, 42, 51, 53, 55, 64, 69, 71, 81, 85, 87, 94, 116, 117, 119, 120, 122, 124, 128, 152, 156, 157, 159, 157, 159, 160, 164, 166, 167, 168, 170, 171, 173, 185, 186, 188, 198, 199, 202, 211, 214, 216, 221, 231, 235, 236, 246, 247, 253, 262, 263, 264, and 264; Preferably, the disabled integrase comprises an amino acid mutation selected from or located at one or more of the following sites corresponding to SEQ ID NO:29, after optimal global alignment with SEQ ID NO:29: D10K, E11K, H12N, H12C, E13K, H16C, H16V, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, D64A, E 69A, K71A, S81R, E85A, E87A, G94D, G94E, G94R, G94K, D116N, D116I, D116E, D116A, N117D, N11 7E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120G, N 120K, N120I, N120E, N120R, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152G, E152A, E152D, K156E, K156A, E157A, K159E, K159A, K160A, D164N, R166A, D167 A. Q168L, Q168A, E170A, E170G, H171A, K173A, F185K, K186Q, K186T, K186E, K188T, E198A, R19 9C, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, R231G, R231K, R231D, R231E, R231S, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, K264R, K264H, K266R, and K273R; More preferably, the disability mutation includes one or more selected from D64V, D64E, D116N, DI161, D116A, E152G, and E152A.

25. The noncellular granules according to any one of claims 22-24, characterized in that, The ability of the NIL's 3'LTR and / or 5'LTR to recognize integrase is weakened or inhibited.

26. The non-cellular particles according to claim 25, characterized in that, The conserved CA dinucleotides of the 3'LTR U5 and / or 5'LTR U3 of the NIL contain mutations (CA mutations).

27. The noncellular particles according to any one of claims 1-26, characterized in that, The NCP contains one or more genetically modified organisms.

28. The non-cellular particles according to claim 27, characterized in that, The transgene contains a polynucleotide encoding a chimeric antigen receptor (CAR).

29. The non-cellular particles according to claim 28, characterized in that, The CAR contains an extracellular antigen-binding region that can bind to antigens associated with cancer or autoimmune diseases.

30. A host cell, characterized in that, The host cell contains: (a) A polynucleotide encoding a viral glycoprotein in a noncellular particle according to any one of claims 1-13; and (b) A polynucleotide encoding a target molecule in a non-cellular particle according to any one of claims 1-18.

31. The host cell according to claim 30, characterized in that, The host cell contains one or more target proteins (POIs).

32. The host cell according to claim 31, characterized in that, The POI includes one or more of intracellular free proteins and membrane-localized expressed proteins.

33. The host cell according to any one of claims 30-32, characterized in that, The host cell also contains the genome of a non-integrating lentiviral vector (NIL).

34. The host cell according to claim 33, characterized in that, The genome of the NIL contains a polynucleotide encoding a disabling integrase; Preferably, the inoperable integrase comprises a sequence selected from SEQ ID NO:29 or, after optimal global alignment with SEQ ID NO:29, located at the position corresponding to SEQ ID NO:

29. Amino acid mutations at one or more of the following sites in NO:29: positions 10, 11, 12, 13, 16, 41, 42, 51, 53, 55, 64, 69, 71, 81, 85, 87, 94, 116, 117, 119, 120, 122, 124, 128, 152, 156, 157, 159, 157, 159, 160, 164, 166, 167, 168, 170, 171, 173, 185, 186, 188, 198, 199, 202, 211, 214, 216, 221, 231, 235, 236, 246, 247, 253, 262, 263, 264, and 264; More preferably, the disabled integrase comprises an amino acid mutation selected from or corresponding to SEQ ID NO:29 and located at one or more of the following sites after optimal global alignment with SEQ ID NO:29: D10K, E11K, H12N, H12C, E13K, H16C, H16V, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, D64A, E69A, K71A, S81R, E85A, E87A, G94D, G94E, G94R, G94K, D116N, D116I, D116 E, D116A, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K , N120D, N120G, N120K, N120I, N120E, N120R, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A 124R, A124K, A128T, E152G, E152A, E152D, K156E, K156A, E157A, K159E, K159A, K160A, D164N, R1 66A, D167A, Q168L, Q168A, E170A, E170G, H171A, K173A, F185K, K186Q, K186T, K186E, K188T, E198 A. R199C, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, R231G, R231K, R231D, R231E, R231S , W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, K264R, K264H, K266R, and K273R; More preferably, the disability mutation comprises one or more selected from D64V, D64E, D116N, D116I, D116A, E152G, and E152A.

35. The host cell according to claim 33 or 34, characterized in that, The ability of the 3'LTR and / or 5'LTR of the NIL to recognize integrase is weakened or inhibited; preferably, the conserved CA dinucleotides of the U5 of the 3'LTR and / or the U3 of the 5'LTR of the NIL contain mutations (CA mutations).

36. A method for preparing non-cellular particles, characterized in that, This includes culturing the host cells according to any one of claims 30-35 to collect non-cellular particles.

37. A composition, characterized in that, The composition comprises a pharmaceutically acceptable carrier or excipient and noncellular particles according to any one of claims 1-29.

38. The use of noncellular particles according to any one of claims 1-29 in the preparation of cancer therapeutic drugs.

39. A method for in vitro transduction of lymphocytes, characterized in that, This includes contacting lymphocytes with non-cellular particles as described in any one of claims 1-29.

40. A method for transducing lymphocytes in a subject in need, characterized in that, This includes administering the noncellular particles of any one of claims 1-29 to the subject.

41. A method for treating a subject suffering from cancer or killing cancer cells in said subject, characterized in that, This includes administering the noncellular particles of any one of claims 1-29 to the subject.

42. The method according to claim 40 or 41, characterized in that, The noncellular particles also contain polynucleotides encoding CAR.

43. The method according to any one of claims 40-42, characterized in that, The subjects had cancer or were at risk of developing cancer.

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