Particle and use thereof

Abstract

A particle and a use thereof. The particle comprises activation molecules and costimulatory molecules.

Description

Particles and their applications Technical Field

[0001] This invention relates to the field of cell therapy, and more specifically to a particle and its application. Background Technology

[0002] In the field of cell therapy, vectors such as LNPs, lentiviral vectors, and retroviral vectors are commonly used to transduce T cells and deliver CAR genes to prepare CAR-T cells. Currently, the effects of constructing targeting molecules on the vector surface that can bind to, activate, and stimulate T cells, such as CD3 antibodies and CD80 / CD86, on improving the efficiency of vector-transduced T cells, promoting T cell proliferation, and enhancing the killing efficiency of CAR-T cells still need further improvement. Therefore, the need to effectively improve the efficiency of vector-transduced T cells, promote T cell proliferation, and enhance the killing efficiency of CAR-T cells remains unmet. Summary of the Invention

[0003] In one aspect, the present invention provides a particle comprising activating molecules and co-stimulatory molecules displayed on its surface:

[0004] A. The activating molecule is a first activating polypeptide that can specifically bind to CD3; and / or

[0005] B. The co-stimulatory molecule is a first co-stimulatory polypeptide that can specifically bind to CD137 (4-1BB).

[0006] In some embodiments of the present invention, the particles contain only the first activating molecular polypeptide and the first co-stimulatory molecular polypeptide, and do not contain other activating molecules and / or co-stimulatory molecules.

[0007] In some embodiments of the present invention, CD3 is selected from CD3γ, CD3δ and CD3ε.

[0008] In some embodiments of the present invention, the human CD3 is selected from human CD3γ, human CD3δ, and human CD3ε.

[0009] In some embodiments of the present invention, the first activating molecular polypeptide in A comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

[0010] In some embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment is an anti-CD3 scFv or VHH.

[0011] In some embodiments of the present invention, the anti-CD3 antibody is UCHT1, OKT3, HuM291, SP34, 1452c11 or TR66, or a variant thereof, or a derivative thereof.

[0012] In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3 scFv, the anti-CD3 scFv is UCHT1 (UCHT1-scFv), the LCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:39-41 respectively, and the HCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:36-38 respectively.

[0013] In some embodiments of the present invention, the first co-stimulatory polypeptide in B comprises: a ligand targeting CD137 or its extracellular domain, or a functional fragment thereof; or an anti-CD137 antibody, or its antigen-binding fragment thereof.

[0014] In some embodiments of the present invention, the ligand targeting CD137 is CD137L, and the amino acid sequence of CD137L has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:42.

[0015] In some embodiments of the present invention, the first activating molecular polypeptide and / or the first co-stimulatory molecular polypeptide are membrane-expressed proteins.

[0016] Preferably, the first activating molecular polypeptide and / or the first co-stimulatory molecular polypeptide are recombinant transmembrane proteins.

[0017] In some embodiments of the present invention, the recombinant transmembrane protein includes a transmembrane region;

[0018] Preferably, the recombinant transmembrane protein further comprises a linker domain.

[0019] In some embodiments of the present invention, the recombinant transmembrane protein comprises, from the N-terminus to the C-terminus: a leader signal peptide, the UCHT1-scFv, the linker domain, and the transmembrane region.

[0020] In some embodiments of the present invention, the recombinant transmembrane protein comprises, from the N-terminus to the C-terminus, the CD137L extracellular domain, the linker domain, and the transmembrane region.

[0021] In some embodiments of the present invention, the recombinant transmembrane protein comprises, from the N-terminus to the C-terminus: a leader signal peptide, the CD137L extracellular domain, the linker domain, and the transmembrane region.

[0022] In some embodiments of the present invention, the transmembrane region of the recombinant transmembrane protein 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, 4-1B B. OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B 4. BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1.

[0023] In some embodiments of the present invention, the transmembrane region of the recombinant transmembrane protein is the CD8α transmembrane region; the amino acid sequence of the CD8α transmembrane region has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:29.

[0024] In some embodiments of the present invention, the transmembrane region of the recombinant transmembrane protein is connected to the first activating molecular polypeptide or the first co-stimulatory molecular polypeptide through the linker domain.

[0025] In some embodiments of the present invention, the linker domain of the recombinant transmembrane protein is selected from: a) immunoglobulin hinge regions, wherein the immunoglobulin hinge regions are selected from wild-type or modified hinge regions of IgG1, IgG2, IgG3, IgG4, IgA, and IgD; b) hinge regions, wherein the hinge regions are selected from 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, CD1 34, CD137, ICOS, and CD154; c) all or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2, and CH3 domains; d) the stalk domain of a type II C-lectin, wherein the type II C-lectin is selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and e) a flexible linker peptide; preferably (G4S)n linker peptide and linker 1: GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 74); wherein n = 1 to 4.

[0026] In some embodiments of the present invention, the linker domain of the recombinant transmembrane protein is the CD8α hinge region; the amino acid sequence of the CD8α hinge region has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:28.

[0027] In some embodiments of the present invention, the first activating molecular polypeptide is linked to the first co-stimulatory molecular polypeptide via a first polypeptide linker.

[0028] In some embodiments of the present invention, the particle further comprises a viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof containing modification (Modified-G).

[0029] In some embodiments of the invention, the viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, is linked to the first activating molecular polypeptide or the first co-stimulatory molecular polypeptide via a second polypeptide linker.

[0030] In the particles of the above embodiments, the first and / or dipeptide linker includes one or more of the following:

[0031] a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0032] b) 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;

[0033] c) All or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains;

[0034] d) 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; and

[0035] e) Flexible linker peptide; preferably (G4S)n linker peptide and linker 1: GSTGSGSGKPGSGEGSTKG (SEQ ID NO:74); wherein n=1 to 4.

[0036] Preferably, the polypeptide linker is a (G4S)3 linker peptide, GGGGSGGGGSGGGGS (SEQ ID NO:32).

[0037] The viral glycoproteins in the particles described in the above embodiments are selected from: vesicular stomatitis virus strain 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 choriomeningitis virus (LCMV) glycoprotein;

[0038] The vesicular stomatitis virus (VSV) glycoproteins are selected from: Indiana VSV, Cocal VSV, Maraba VSV, Morreton VSV, Alagoas VSV, New Jersey VSV, Carajas VSV, and Chandipura VSV. The glycoproteins of the following strains are included: Eptesicus vesicular stomatitis virus strain, Isfahan vesicular stomatitis virus strain, Jurona vesicular stomatitis virus strain, Malpais vesicular stomatitis virus strain, Perinet vesicular stomatitis virus strain, Piry vesicular stomatitis virus strain, Radi vesicular stomatitis virus strain, Rhinolopus vesicular stomatitis virus strain, and Yug Bogdanovac vesicular stomatitis virus strain.

[0039] Furthermore, the viral glycoprotein is:

[0040] a) Vesicular stomatitis virus (VSV) Indiana strain glycoprotein (VSV-G), or a variant thereof, or a functional fragment thereof, or a modified version thereof containing the modification (VSV-G modified version); or

[0041] b) Vesicular stomatitis virus genus Cocal strain glycoprotein (Cocal-G), or a variant thereof, or a functional fragment thereof, or a modified version thereof containing the modification (Cocal-G modified version).

[0042] In some embodiments of the present invention, the amino acid sequence of the wild-type VSV-G (excluding its signal peptide) is shown in SEQ ID NO:1.

[0043] 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:9;

[0044] Among them, the amino acid sequence shown in positions 1-16 of SEQ ID NO:9 is as follows:

[0045] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.

[0046] In some embodiments of the present invention, the amino acid sequence of the wild-type Cocal-G (excluding its signal peptide) is shown in SEQ ID NO:2.

[0047] 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:17;

[0048] Among them, the sequence shown in positions 1-17 of SEQ ID NO:17: MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild-type Cocal-G.

[0049] 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 vectors containing VSV-G or Cocal-G can be effectively improved.

[0050] In some embodiments of the present invention,

[0051] a) The ability of the VSV-G variant, VSV-G functional fragment, or VSV-G modifier to specifically bind to its receptor LDL-R is inhibited; or

[0052] b) The ability of the said Cocal-G variant, functional fragment of Cocal-G, or Cocal-G modifier to specifically bind to its receptor LDL-R is inhibited.

[0053] Furthermore, relative to wild-type VSV-G or Cocal-G, the VSV-G or Cocal-G variant contains a first mutation that inhibits its ability to specifically bind LDL-R, the first mutation being selected from one or more of the following mutations:

[0054] 1) 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, or deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353; or

[0055] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the 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, or the deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353, corresponding to SEQ ID NO:1 or SEQ ID NO:2;

[0056] Preferably, the first mutation is selected from one or more of the following mutations:

[0057] 1) The amino acid deletion at positions 331-364, the amino acid deletion at positions 344-354, the substitution of K47, the deletion of K47, the substitution of R354, the substitution of Y209, and the substitution of I182 at positions 331-364, the amino acid deletion at positions 344-354, the substitution of K47, the deletion of K47, the substitution of R354, the substitution of Y209, and the substitution of I182 or V182 at positions 331-364;

[0058] 2) After optimal global alignment with SEQ ID NO:1, the amino acid positions 331-364 and 344-354 corresponding to SEQ ID NO:1 are deleted, or K47 is substituted, or R354 is substituted, or Y209 is substituted, or I182 is substituted; or after optimal global alignment with SEQ ID NO:2, the amino acid positions 331-364 and 344-354 corresponding to SEQ ID NO:2 are deleted, or K47 is substituted, or R354 is substituted, or Y209 is substituted, or I182 or V182 is substituted.

[0059] More preferably, the first mutation is:

[0060] 1) K47 is missing in SEQ ID NO:1 or SEQ ID NO:2; or

[0061] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, K47 is missing at the position equivalent to SEQ ID NO:1 or SEQ ID NO:2.

[0062] In some embodiments of the present invention, the modifications contained in the VSV-G modifier or Cocal-G modifier reduce the number of binding sites available for specific binding of LDL-R.

[0063] In some embodiments of the present invention, when the particle contains any of the aforementioned viral glycoprotein variants or modifiers, the first activating molecular polypeptide anti-CD3 antibody or its antigen-binding fragment is a high-affinity antibody.

[0064] In some embodiments of the present invention, the affinity (Kd value) of the high-affinity antibody for its antigen is...

[0065] 1×10 -12 M-5×10 -7 M, 1×10 -11 M-4×10 -7 M, 1×10 -10 M-3×10 -7 M, 1×10 -9 M-2×10 -7 M, 1×10 -9 M-1×10 -7 M, 1×10 -9 M-9×10 -8 M, 1×10 -9 M-8×10 -8 M, 1×10 -9 M-7×10 -8 M, 1×10 -9 M-6×10 -8 M, 1×10 -9 M-5×10 -8 M, 1×10 -9 M-4×10 -8 M, 1×10 -9 M-3×10 -8 M, 1×10 -9 M-2×10 -8 M, 1×10 -9 M-1×10 -8 M, 1×10-9 M-9×10 -9 M, 1×10 -9 M-8×10 -9 M, 1×10 -9 M-7×10 -9 M or 1×10 -9 M-6×10 -9 M, each containing its end value.

[0066] In some embodiments of the present invention, the affinity (Kd value) of the high-affinity antibody for its antigen is less than 5 × 10⁻⁶. -7 M.

[0067] In some embodiments of the present invention,

[0068] a) The VSV-G variant, VSV-G functional fragment, or VSV-G modifier; or

[0069] b) The Cocal-G variant, functional fragment of Cocal-G, or modified form of Cocal-G;

[0070] The mutation includes a second mutation that enhances its ability to antagonize complement inactivation; this enhanced ability to antagonize complement inactivation is relative to the absence of the second mutation.

[0071] a) with respect to the VSV-G variant, VSV-G functional fragment, or VSV-G modifier; or

[0072] (b) with regard to the Cocal-G variant, Cocal-G functional fragment, or Cocal-G modifier.

[0073] In some embodiments of the present invention,

[0074] When the viral glycoprotein is a VSV-G or Cocal-G variant, a VSV-G or Cocal-G functional fragment, or a VSV-G or Cocal-G modified form, the second mutation is selected from one or more of the following site mutations:

[0075] 1) The 214th amino acid located in SEQ ID NO:1 or SEQ ID NO:2, or the 214th amino acid located in SEQ ID NO:1 or SEQ ID NO:2 after best global alignment;

[0076] 2) The amino acid located at position 352 of SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at position 352 of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2;

[0077] 3) The amino group located at position 50 of SEQ ID NO:1 or SEQ ID NO:2, or, after optimal global alignment of SEQ ID NO:1 or SEQ ID NO:2, the amino group located at position 50 of SEQ ID NO:1 or SEQ ID NO:2; or

[0078] 4) The 146th amino acid located in SEQ ID NO:1 or SEQ ID NO:2; or the 146th amino acid located in SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2;

[0079] Preferably, the mutation at the site is selected from amino acid substitutions, deletions, and insertions;

[0080] More preferably, the mutation at the site is an amino acid substitution.

[0081] In some embodiments of the present invention, when the viral glycoprotein is the VSV-G or Cocal-G variant, a VSV-G or Cocal-G functional fragment, or a VSV-G or Cocal-G modified form, the second mutation is selected from a combination of mutations at the following sites:

[0082] 1) Substitution of (1) T214 and T352 located in SEQ ID NO:1 or SEQ ID NO:2, or (2) Substitution of T214, T352, K50 and S146; or

[0083] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the substitutions of (1) T214 and T352, or (2) T214, T352, K50 and S146, located in the equivalent of SEQ ID NO:1 or SEQ ID NO:2;

[0084] Preferably, the second mutation is selected from a combination of mutations at the following sites:

[0085] 1) (1) T214N and T352A located in SEQ ID NO:1 or SEQ ID NO:2, or (2) T214N, T352A, K50T and S146T; or

[0086] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, it is located at (1) T214N and T352A, or (2) T214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:1 or SEQ ID NO:2.

[0087] In some embodiments of the present invention, any of the aforementioned viral glycoprotein variants or modifiers retain the ability to fuse with membranes and mediate lysosomal escape.

[0088] In some embodiments of the present invention, the particles further comprise exogenous polynucleotides, which include polynucleotides encoding chimeric antigen receptors (CARs) and / or engineered TCRs.

[0089] In some embodiments of the present invention, the exogenous polynucleotide comprises a polynucleotide encoding a CAR.

[0090] In some embodiments of the present invention, the CAR includes an antigen-binding region, a transmembrane region, and an intracellular signal transduction domain.

[0091] In some embodiments of the present invention, the antigen-binding region of the CAR can specifically bind to cancer-related antigens;

[0092] Preferably, the cancer-associated antigens are selected from: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, 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-related 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, BCL-2, CXCR4, CRLF2, NKG2D, and GCC (guanylate cyclase).

[0093] More preferably, the cancer-associated antigen is selected from one or more of CD19, CD20, CD22, CD79A, CD79B, CD30, CD37, CD38, CD52, CD123, CRLF2, BCMA, CD33, CD138, GPRC5D, CD3, CD4, CD8, CD5, CD7, CD25, CD56, NKG2D, TCR, ROR1, MSLN, MUC1, HER2, CEA, Nectin-4, Claudin18.2, and GCC.

[0094] In some embodiments of the present invention, the cancer-related antigen is a hematologic cancer-related antigen; the hematologic cancer is selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (M... One or more of the following: CL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), 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), and embryonic centrifugal T-cell lymphoma (FTCL);

[0095] Preferably, the blood cancer-related antigen is selected from one or more of the following: CD19, CD20, CD22, CD79A, CD79B, CD30, CD37, CD38, CD52, CD123, CRLF2, BCMA, CD33, CD138, GPRC5D, CD3, CD4, CD8, CD5, CD7, CD25, CD56, NKG2D, TCR, and ROR1.

[0096] In some embodiments of the present invention, the cancer-associated antigen is selected from one or more of CD19, CD20, CD22 and BCMA.

[0097] In some embodiments of the present invention, the blood cancer-related antigen is a B-cell malignancy-related antigen, and the B-cell malignancy is selected from one or more of the following: non-Hodgkin lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), LBCL unsuitable for transplantation, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, and Burkitt lymphoma (BL).

[0098] Preferably, the B-cell malignancy-associated antigen is selected from one or more of CD19, CD20, CD22, CD79A, CD79B, CD30, CD37, CD38, CD52, CD123, CRLF2, BCMA, and ROR1.

[0099] In some embodiments of the present invention, the cancer-associated antigen is a solid cancer-associated antigen, wherein the solid cancer is selected from one or more of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer;

[0100] Preferably, the solid tumor-associated antigen is selected from one or more of ROR1, MSLN, MUC1, HER2, CEA, Nectin-4, Claudin18.2, and GCC.

[0101] Preferably, 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, OX4. 0. ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, B TLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1.

[0102] More preferably, the transmembrane region of the CAR is the CD8α transmembrane region.

[0103] 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: 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 the ITAM intracellular signal transduction domain of other proteins containing at least one ITAM intracellular signal transduction domain.

[0104] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR is the intracellular signal transduction domain of CD3ζ.

[0105] In some embodiments of the present invention, the amino acid sequence of the intracellular signal transduction domain of CD3ζ has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:31.

[0106] In some embodiments of the present invention, the CAR further includes a connectivity domain and a co-stimulatory signal transduction domain.

[0107] In some embodiments of the present invention, the CAR's linker domain connects the antigen-binding region of the CAR and the transmembrane region of the CAR.

[0108] In some embodiments of the present invention, the CAR connection structure domain is selected from:

[0109] a) 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] b) 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;

[0111] c) 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] d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D.

[0113] In some embodiments of the present invention, the connection structure domain of the CAR is the CD8α hinge region.

[0114] In some embodiments of the present invention, 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;

[0115] 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.

[0116] In some embodiments of the present invention, the co-stimulatory signal transduction domain of the CAR is a 4-1BB co-stimulatory signal transduction domain; the amino acid sequence of the 4-1BB co-stimulatory signal transduction domain has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:30.

[0117] 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;

[0118] Preferably, the leader signal peptide is selected from CD8α signal peptide, CD28 signal peptide, IgG signal peptide and HLA-A signal peptide;

[0119] In some embodiments of the present invention, the leader signal peptide is a CD8α signal peptide.

[0120] In some embodiments of the present invention, the amino acid sequence of the CD8α signal peptide has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:27.

[0121] In some embodiments of the present invention, the CAR has been 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 antigen-binding region of the CAR; and (b) an anti-antibody;

[0122] Preferably, the antigen-binding fragment includes one or more selected from the antigen extracellular domain, antigen functional fragment, and antigen epitope.

[0123] In some embodiments of the present invention, the particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles.

[0124] Preferably, the particle is an enveloped virus particle.

[0125] More preferably, the enveloped viral particles are pseudolentiviral vectors (LVV) and / or retroviral vectors (RVV).

[0126] In some embodiments of the present invention, any of the aforementioned particles comprises any of the aforementioned first activating molecule polypeptides and first co-stimulating molecule polypeptides, but does not contain other activating molecules and / or co-stimulating molecules.

[0127] In another aspect, the present invention also provides a composition comprising a pharmaceutically acceptable carrier or excipient and one of the aforementioned particles.

[0128] In another aspect, the present invention also provides a method for preparing CAR-T cells by in vitro transduction of T cells, comprising contacting T cells with the particles described in any of the above claims.

[0129] In another aspect, the present invention also provides a method for preparing CAR-T cells by transducing T cells in a subject in need, comprising administering the particles described in any of the preceding claims to the subject.

[0130] The present invention also provides a method for improving the expression efficiency of exogenous polynucleotides delivered to T cells, wherein the exogenous polynucleotides are carried by particles as described in any of the preceding claims, the particles are contacted with T cells and the exogenous polynucleotides are delivered.

[0131] The present invention also provides a fusion polypeptide comprising a transmembrane polypeptide, an activating molecule, and a co-stimulatory molecule;

[0132] The activating molecule described in A is a first-activating polypeptide that can specifically bind to CD3; and

[0133] The co-stimulatory molecule described in B is a first co-stimulatory polypeptide that can specifically bind to CD137 (4-1BB).

[0134] The fusion peptides in some embodiments of the present invention,

[0135] A. The first activating molecular polypeptide contains an anti-CD3 antibody or its antigen-binding fragment; and

[0136] B. The first co-stimulatory polypeptide contains the extracellular domain of CD137L, or a functional fragment thereof.

[0137] In some embodiments of the present invention, the fusion polypeptide, wherein the anti-CD3 antibody or its antigen-binding fragment is an anti-CD3 scFv or VHH.

[0138] In some embodiments of the present invention, the fusion polypeptide, wherein the anti-CD3 antibody is UCHT1, OKT3, HuM291 or TR66, or a variant thereof, or a derivative thereof.

[0139] In some embodiments of the present invention, the fusion peptide, wherein the anti-CD3 antibody is an anti-CD3 scFv, the anti-CD3 scFv is UCHT1 (UCHT1-scFv), the LCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:39-41 respectively, and the HCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:36-38 respectively.

[0140] In some embodiments of the present invention, the fusion polypeptide, the first activating molecule, the first co-stimulatory molecule polypeptide, and the transmembrane polypeptide are connected by a polypeptide linker.

[0141] The fusion polypeptide in some embodiments of the present invention comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus:

[0142] a) the first activating polypeptide, the polypeptide linker, the first co-stimulatory polypeptide, the polypeptide linker, and the transmembrane polypeptide; or

[0143] b) The first co-stimulatory polypeptide, the polypeptide linker, the first activating polypeptide, the polypeptide linker, and the transmembrane polypeptide.

[0144] In some embodiments of the present invention, the fusion polypeptides, wherein the linkers are selected from:

[0145] a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0146] b) 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;

[0147] c) All or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains;

[0148] d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and

[0149] e) Flexible linker peptide; preferably (G4S)n linker peptide and linker 1GSTSGSGKPGSGEGSTKG (SEQ ID NO:74); wherein n=1 to 4.

[0150] In some embodiments of the present invention, the fusion peptide, wherein the first peptide linker and the second peptide linker are (G4S)3 linker peptides, GGGGSGGGGSGGGGS (SEQ ID NO:32).

[0151] The present invention also provides an isolated polynucleotide encoding the fusion polypeptide described in any of the preceding claims.

[0152] The present invention also provides a particle comprising the fusion polypeptide described in any of the preceding claims.

[0153] In some embodiments of the present invention, the particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles.

[0154] In some embodiments of the present invention, the particles are enveloped viral particles.

[0155] In some embodiments of the present invention, the enveloped viral particles are pseudotyped LVV and / or RVV.

[0156] In some embodiments of the present invention, the particles further comprise exogenous polynucleotides, which encode CARs and / or engineered TCRs.

[0157] In other embodiments of the present invention, the use of any of the particles described herein in the preparation of cancer therapeutic drugs is discussed.

[0158] In some embodiments of the present invention, the cancer is selected from one or more of hematologic malignancies and solid tumors.

[0159] In some embodiments of the present invention, the cancer is a hematologic malignancy selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), and mantle cell lymphoma (MCL). One or more of the following: follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), 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), and embryonic centrifugal T-cell lymphoma (FTCL).

[0160] In some embodiments of the present invention, the hematologic malignancy is a B-cell malignant tumor, which is selected from one or more of the following: non-Hodgkin lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, and Burkitt lymphoma (BL).

[0161] In some embodiments of the present invention, the cancer is a solid cancer, which is selected from one or more of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer.

[0162] The present invention also provides a method for treating a subject who has or is suspected of having cancer, comprising administering to the subject a therapeutically effective amount of any of the particles described above.

[0163] In some embodiments of the present invention, the cancer is selected from one or more of hematologic malignancies and solid tumors.

[0164] In some embodiments of the present invention, the cancer is a hematologic malignancy selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), and mantle cell lymphoma (MCL). It includes one or more of the following: follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), 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), and embryonic centrifugal T-cell lymphoma (FTCL).

[0165] In some embodiments of the present invention, the blood cancer is a B-cell malignant tumor selected from one or more of the following: non-Hodgkin lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, and Burkitt lymphoma (BL).

[0166] In some embodiments of the present invention, the cancer is a solid cancer selected from one or more of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer.

[0167] In some embodiments of the present invention, the method of administration is selected from one or more of the following: 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. Beneficial effects

[0168] The present invention provides particles containing an activating molecule, namely a first activating polypeptide that specifically binds to CD3, and a co-stimulatory molecule, namely a first co-stimulatory polypeptide that specifically binds to CD137 (4-1BB). This significantly improves the efficiency of T cell transduction, the killing efficiency of CAR-T cell preparation, and the efficiency of activating and / or stimulating T cell proliferation.

[0169] In this article:

[0170] "Activation molecule": as used herein, "activation molecule" includes, but is not limited to, molecules that can bind to or interact directly or indirectly with T cells, thereby triggering T cell activation. Examples include TCR-CD3 binding molecules that can bind to the TCR-CD3 complex, CD3, and TCR, providing an initial signal for T cell activation, including, but not limited to, anti-CD3 antibodies or their antigen-binding fragments. In some embodiments of the present invention, exemplarily, "activation molecule" includes, but is not limited to, the binding domains of OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0171] "Costimulatory molecule," as used in this article, refers to a molecule that can provide a costimulatory signal for T cell activation; complete T cell activation usually requires the participation of costimulatory molecules.

[0172] 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.

[0173] CD137 (also known as 4-1BB) is a member of the tumor necrosis factor (TNF) receptor family. CD137 can be expressed by activated T cells, but it is also expressed by CD8+ receptors. + The expression level on T cells is greater than that on CD4. + CD137 expression levels on T cells. Furthermore, CD137 expression is observed on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. The most representative activity of CD137 is its co-stimulatory activity on activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolysis.

[0174] "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.

[0175] "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.

[0176] "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.

[0177] 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).

[0178] Lentiviral vectors are vectors derived from lentiviruses that contain one or more lentiviral packaging proteins and / or lentiviral proteins essential 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 makes the lentiviral vector biosafety-compliant.

[0179] 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.

[0180] Commonly used pseudotyped lentiviral vectors include the so-called third-generation lentiviral vector packaging system. This system typically includes four types of plasmids: a transfer plasmid containing the gene of interest (GOI), such as a transgenic transfer plasmid, the GagPol plasmid, and the Rev plasmid; and an envelope plasmid containing viral glycoprotein genes such as VSV-G or its variants, or Cocal-G or its variants.

[0181] 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 processes of the viral genome. Through these sequences, lentiviruses can integrate transgenes into the host cell's genome. For safety reasons, transfer vectors are usually designed so that the resulting viral vector cannot self-replicate. 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 enabling the virus to "self-inactivate." 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 can be 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 ("IRES") to allow entry of a single mRNA into the viral particle. Simultaneous translation of two or more open reading frames (ORFs) enables multi-gene expression. Some transfer plasmids, such as the lentiviral master plasmid / transfer plasmid 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.

[0182] 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).

[0183] 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 another 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 a Gag polyprotein precursor containing lentiviral structural proteins, including a matrix, capsid, and nucleocapsid; the Pol gene encodes a Pol polyprotein precursor providing 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 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 be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral particles. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0184] 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.

[0185] Compared to pseudolentiviral vector packaging systems, pseudoretroviral vector packaging systems typically do not contain Rev plasmids. This is because the genomic RNA of 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 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 transfer plasmids 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.

[0186] 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.

[0187] 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.

[0188] "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.

[0189] For explanations of other terms used herein, please refer to patents WO 2025209590A and WO 2025011662A. All publications, documents, and patents mentioned herein are hereby incorporated in their entirety by reference, as if each publication, document, or patent not specifically and individually indicated to be incorporated in its entirety by reference were incorporated in its entirety by reference. 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. Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Attached Figure Description

[0190] Figure 1: Detection of CD3 in human inactive PBMCs after transduction of the lentiviral vectors V1-8 into them. + The flow cytometry results of CAR-19 expression efficiency in T cells.

[0191] Figure 2: The lentiviral vectors V1-8 were transduced into inactive human PBMCs, and the CD3 levels were detected relative to Day 0 on Day 5 and Day 7. + Bar chart showing the fold increase of T cells.

[0192] Figure 3: Map of the encapsulated plasmid.

[0193] Figure 4: Transfer plasmid map.

[0194] Figure 5: The lentiviral vector ar described in Example 3 was used to transduce inactive human PBMCs, and the CD3+ content was detected. + The flow cytometry results of CAR-19 expression efficiency in T cells.

[0195] Figure 6: The lentiviral vector af described in Example 4 was used to transduce inactive human PBMCs, and the CD3+ content was detected. + The flow cytometry results of CAR-19 expression efficiency in T cells.

[0196] Figure 7: The lentiviral vector ad described in Example 5 was used to transduce inactive human PBMCs, and the CD3+ content was detected. + The flow cytometry results of CAR-19 expression efficiency in T cells.

[0197] Figure 8: Animal experiment results in Example 6. Detailed Implementation Example 1

[0198] The packaging contains multiple lentiviral vectors V1, V2, V3, V4, V5, V6, V7, and V8 containing different activating and co-stimulating molecules.

[0199] The lentiviral vectors V1-8 contain different activating molecules and co-stimulatory molecules, as shown in Table 1 below:

[0200] LVV activating molecules, co-stimulatory molecules, adhesion molecules, V1UCHT1-scFv15E8-scFv / V2OKT3-scFvCD80CD58V3UCHT1-scFvCD80CD58V4UCHT1-scFvCD86CD58V5UCHT1-scFv15E8-scFvCD58V6UCHT1-scFvCD86 / V7UCHT1-scFvCD137L / V8UCHT1-scFvCD137L+CD86

[0201] The lentiviral vectors V1-8 all contain mutant VSV-G1 and carry polynucleotides encoding a CAR (CAR-19) targeting CD19.

[0202] The amino acid sequence of the mutant VSV-G1 is shown in SEQ ID NO:7; relative to the wild-type VSV-G containing the amino acid sequence shown in SEQ ID NO:1, the mutant VSV-G1 contains the R354Q mutation, which inhibits its ability to specifically bind to LDL-R, and the T214N and T352A mutations, which enhance its ability to antagonize complement inactivation.

[0203] The amino acid sequence of the mutant VSV-G1 (mutant spVSV-G1) containing the VSV-G signal peptide is shown in SEQ ID NO:10.

[0204] The CAR-19, from the N-terminus to the C-terminus, comprises: a CD8α signal peptide, an antigen-binding region targeting CD19, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signal transduction domain, and a CD3ζ intracellular signal transduction domain; the antigen-binding region targeting CD19 is a scFv (FMC63-scFv) derived from the monoclonal antibody FMC63, and the FMC63-scFv comprises the VH region and VL region of FMC-63, wherein the VH region is connected to the VL region via the (G4S)3 linker peptide.

[0205] The amino acid sequence of the CAR-19 is shown in SEQ ID NO:33.

[0206] (1) The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO:27;

[0207] (2) The amino acid sequence of the VH region of the FMC63-scFv is shown in SEQ ID NO:18, the amino acid sequence of the VL region of the FMC63-scFv is shown in SEQ ID NO:22, the amino acid sequence of the FMC63-scFv is shown in SEQ ID NO:26, the amino acid sequences of HCDR1-3 of the FMC63-scFv are shown in SEQ ID NO:19-21, and the amino acid sequences of LCDR1-3 of the FMC63-scFv are shown in SEQ ID NO:23-25, respectively.

[0208] (3) The amino acid sequence of the hinge region of CD8α is shown in SEQ ID NO:28;

[0209] (4) The amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO:29;

[0210] (5) The amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:30;

[0211] (6) The amino acid sequence of the intracellular signal transduction domain of CD3ζ is shown in SEQ ID NO:31;

[0212] (7) The amino acid sequence of the (G4S)3 linker peptide is shown in SEQ ID NO:32.

[0213] 1. Packaging Lentiviral Vector V7

[0214] The surface of the lentiviral vector V7 contains membrane-expressed UCHT1-scFv and membrane protein CD137L (full-length protein).

[0215] The UCHT1-scFv can bind to human CD3ε / CD3E; the amino acid sequence of the human CD3ε is shown in SEQ ID NO:72, Uniprot NO.P07766;

[0216] The membrane protein CD137L can bind to human CD137; the amino acid sequence of the human CD137 is shown in SEQ ID NO:73, Uniprot NO.P41273.

[0217] The membrane-expressed UCHT1-scFv comprises, from the N-terminus to the C-terminus: the CD8α signal peptide, the UCHT1-scFv, the CD8α hinge region, and the CD8α transmembrane region;

[0218] The amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO:35; the amino acid sequences of HCDR1-3 of the UCHT1-scFv are shown in SEQ ID NO:36-38 respectively; the amino acid sequences of LCDR1-3 of the UCHT1-scFv are shown in SEQ ID NO:39-41 respectively.

[0219] The amino acid sequence of CD137L is shown in SEQ ID NO:42.

[0220] A. Prepare the lentiviral vector V7 packaging system

[0221] Prepare the following four plasmids: envelope plasmid 7, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and transfer plasmid CAR-19. The transfer plasmid CAR-19 contains a polynucleotide encoding CAR-19.

[0222] The envelope plasmid 7 contains a polynucleotide encoding the mutant spVSV-G1, a polynucleotide encoding the FT2A peptide, and a polynucleotide encoding a cleavable polypeptide 1; wherein the mutant VSV-G1, the membrane-expressed UCHT1-scFv, and the CD137L can be separately expressed on the cell membrane of the packaging cell via the self-cleaving peptide FT2A peptide.

[0223] The cleavable polypeptide 1 comprises: the CD8α signal peptide, the UCHT1-scFv, the CD8α hinge region, the CD8α transmembrane region, the FT2A peptide, and the CD137L; the amino acid sequence of the cleavable polypeptide 1 is shown in SEQ ID NO:43.

[0224] The amino acid sequence of the FT2A peptide is shown in SEQ ID NO:34.

[0225] Both the envelope plasmid 7 and the transfer plasmid CAR-19 were synthesized using conventional molecular cloning methods.

[0226] B. Transfect packaging cells to package lentiviral vector V7.

[0227] Prepare the HEK-293T cell culture system: Take 56 mL of FBS, filter it into 500 mL of DMEM / high glucose (10% FBS), add 4 mL of P / S (double antibiotic, penicillin × streptomycin), shake well, and place in a carbon dioxide incubator for preheating and neutralization before transfection.

[0228] Day 0, HEK-293T cells were seeded at a density of 4.5 × 10⁴ cells in a 10 cm culture dish. 6 Approximately 48 hours after inoculation, when the cell confluence reached 80-90%, the four plasmids were transfected into HEK-293T packaging cells using PEI reagent, including:

[0229] Add 9 µg of the transfer plasmid CAR-19, 4 µg of pMDLg / pRRE packaging plasmid, 2 µg of pRSV-REV packaging plasmid, and 2 µg of the envelope plasmid 7 to 1 mL of Opti-MEM medium. After shaking well, add 64 µL of PEI reagent, mix well by pipetting, and let stand for 10 minutes. Then add to the HEK-293T cell culture system. Replace the medium after 6 hours. Collect the supernatant 48 hours after transfection, filter it through a 0.45 µm filter membrane, centrifuge at 50,000 g for 2.5 h, discard the supernatant, resuspend the lentiviral vector V7 in 200 µL of F12 medium, and store at -80 °C.

[0230] Opti-MEM alpha serum-reduced culture medium: Brand: GIBCO, Catalog No.: #SP0272;

[0231] DMEM culture medium: Brand: GIBCO, Product No.: #C12430500BT;

[0232] FBS: Brand: EXCELL, Item No.: #FSP500;

[0233] F12 medium: Brand: GIBCO, Product No.: #C11330500BT;

[0234] Needle filter: Brand: SORFA, Item No.: #622120.

[0235] 2. Packaging lentiviral vectors V1, V6, and V8

[0236] Using the same method described above for packaging lentiviral vector V7, lentiviral vectors V1, V6, and V8 are packaged.

[0237] A. Packaging Lentiviral Vector V1

[0238] The surface of the lentiviral vector V1 contains membrane-expressed UCHT1-scFv and membrane-expressed anti-CD28 antibody 15E8-scFv;

[0239] The amino acid sequence of the membrane-expressed 15E8-scFv is shown in SEQ ID NO:44; the 15E8-scFv is an scFv derived from the monoclonal antibody 15E8; the amino acid sequences of HCDR1-3 of the 15E8-scFv are shown in SEQ ID NO:45-47, respectively; the amino acid sequences of LCDR1-3 of the 15E8-scFv are shown in SEQ ID NO:48-50, respectively.

[0240] The membrane-expressed 15E8-scFv comprises, from the N-terminus to the C-terminus, the CD8α signal peptide, the 15E8-scFv, the CD8α hinge region, and the CD8α transmembrane region. The following four plasmids are prepared: envelope plasmid 1, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the transfer plasmid CAR-19.

[0241] The envelope plasmid 1 contains a polynucleotide encoding the mutant spVSV-G1, a polynucleotide encoding the FT2A peptide, and a polynucleotide encoding a cleavable polypeptide 2; the amino acid sequence of the cleavable polypeptide 2 is shown in SEQ ID NO:51; wherein the mutant VSV-G1, the membrane-expressed UCHT1-scFv, and the membrane-expressed 15E8-scFv can be separately expressed on the cell membrane of the packaging cell via the self-cleaving peptide FT2A peptide.

[0242] The cleavable polypeptide 2 comprises: the CD8α signal peptide, the UCHT1-scFv, the CD8α hinge region, the CD8α transmembrane region, the FT2A peptide, the CD8α signal peptide, the 15E8-scFv, the CD8α hinge region, and the CD8α transmembrane region.

[0243] The lentivirus vector V1 is packaged using the same method as described for packaging the lentivirus vector V7.

[0244] B. Packaging Lentiviral Vector V6

[0245] The surface of the lentiviral vector V6 contains the membrane-expressed UCHT1-scFv and membrane protein CD86-ECD+TM;

[0246] The membrane protein CD86-ECD+TM includes its extracellular domain and transmembrane region, but does not include its intracellular region; the amino acid sequence of the membrane protein CD86-ECD+TM is shown in SEQ ID NO:52.

[0247] The amino acid sequence of the membrane protein CD86-sp+ECD+TM, which includes its signal peptide, extracellular domain, and transmembrane region, is shown in SEQ ID NO:53.

[0248] Prepare the following four plasmids: envelope plasmid 6, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the transfer plasmid CAR-19.

[0249] The envelope plasmid 6 contains a polynucleotide encoding the mutant spVSV-G1, a polynucleotide encoding the FT2A peptide, and a polynucleotide encoding the cleavable polypeptide 3; wherein the mutant spVSV-G1, the membrane-expressed UCHT1-scFv, and the membrane protein CD86-ECD+TM can be separately expressed on the cell membrane of the packaging cell via the self-cleaving peptide FT2A peptide.

[0250] The cleavable polypeptide 3 comprises: the CD8α signal peptide, the UCHT1-scFv, the CD8α hinge region, the CD8α transmembrane region, the FT2A peptide, and the membrane protein CD86-sp+ECD+TM; the amino acid sequence of the cleavable polypeptide 3 is shown in SEQ ID NO:54.

[0251] The lentivirus vector V6 is packaged using the same method as described for packaging the lentivirus vector V7.

[0252] C. Packaging Lentiviral Vector V8

[0253] The surface of the lentiviral vector V8 contains the membrane-expressed UCHT1-scFv, the membrane protein CD86-ECD+TM, and the membrane protein CD137L;

[0254] Prepare the following four plasmids: envelope plasmid 8, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the transfer plasmid CAR-19.

[0255] The envelope plasmid 8 contains a polynucleotide encoding the mutant spVSV-G1, a polynucleotide encoding the FT2A peptide, and a polynucleotide encoding the cleavable polypeptide 4; wherein the mutant spVSV-G1, the membrane-expressed UCHT1-scFv, the membrane protein CD86-ECD+TM, and the membrane protein CD137L can be separately expressed on the cell membrane of the packaging cell via the self-cleaving peptide FT2A peptide.

[0256] The cleavable polypeptide 4 comprises: the CD8α signal peptide, the UCHT1-scFv, the CD8α hinge region, the CD8α transmembrane region, the FT2A peptide, the membrane protein CD86-sp+ECD+TM, the FT2A peptide, and the membrane protein CD137L; the amino acid sequence of the cleavable polypeptide 4 is shown in SEQ ID NO:55.

[0257] The lentivirus vector V8 is packaged using the same method as described for packaging the lentivirus vector V7.

[0258] 3. Packaging lentiviral vector V2-5

[0259] The packaging surface contains the lentiviral vector V2-5 in MDF (multi-domain fusion).

[0260] MDF is a fusion protein containing multiple polypeptides.

[0261] In this embodiment:

[0262] The lentiviral vector V2 contains MDF1, which comprises, from the N-terminus to the C-terminus: the adhesion molecule CD58 signal peptide, the CD58 extracellular domain, OKT3-scFv, a linker, and the membrane protein CD80-ECD+TM+ED; the amino acid sequence of MDF1 is shown in SEQ ID NO:56.

[0263] The lentiviral vector V3 contains MDF2, which comprises, from the N-terminus to the C-terminus: the adhesion molecule CD58 signal peptide, the CD58 extracellular domain, the UCHT1-scFv, a linker, and the membrane protein CD80-ECD+TM+ED; the amino acid sequence of the MDF2 is shown in SEQ ID NO:57.

[0264] The lentiviral vector V4 contains MDF3, which comprises, from the N-terminus to the C-terminus: the adhesion molecule CD58 signal peptide, the CD58 extracellular domain, the UCHT1-scFv, a linker, and the membrane protein CD86-ECD+TM+ED; the amino acid sequence of the MDF3 is shown in SEQ ID NO:58.

[0265] The lentiviral vector V5 contains MDF4, which comprises, from the N-terminus to the C-terminus: an adhesion molecule CD58 signal peptide, a CD58 extracellular domain, the UCHT1-scFv, a linker, the 15E8-scFv, a CD8α hinge region, and a CD8α transmembrane region; the amino acid sequence of the MDF4 is shown in SEQ ID NO:59.

[0266] The OKT3-scFv is an scFv derived from the monoclonal antibody OKT3, and the amino acid sequence of the OKT3-scFv is shown in SEQ ID NO:60; the HCDR1-3 of the OKT3-scFv are shown in SEQ ID NO:61-63, and the LCDR1-3 of the OKT3-scFv are shown in SEQ ID NO:64-66.

[0267] (1) The amino acid sequence of the CD58 signal peptide is shown in SEQ ID NO:67;

[0268] (2) The amino acid sequence of the extracellular domain of CD58 is shown in SEQ ID NO:68;

[0269] (3) The amino acid sequence of the linker is shown in SEQ ID NO:69;

[0270] (4) The amino acid sequence of the membrane protein CD80-ECD+TM+ED is shown in SEQ ID NO:70; the membrane protein CD80-ECD+TM+ED contains its extracellular domain, transmembrane region and intracellular domain, but does not contain its signal peptide;

[0271] (5) The amino acid sequence of the membrane protein CD86-ECD+TM+ED is shown in SEQ ID NO:71; the membrane protein CD86-ECD+TM+ED contains its extracellular domain, transmembrane region and intracellular domain, but does not contain its signal peptide.

[0272] Adhesion molecules can specifically bind to conjugated molecules and enhance the binding of particles such as lentiviral vectors to target cells such as T cells with sufficient affinity. The adhesion molecule CD58 can specifically bind to the T cell surface antigen CD2, and theoretically, this could enhance the binding of CD58-containing lentiviral vectors to T cells.

[0273] Packaging Lentiviral Vector V2:

[0274] Prepare the following four plasmids: envelope plasmid 2, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the transfer plasmid CAR-19;

[0275] The envelope plasmid 2 contains a polynucleotide encoding the mutant spVSV-G1, a polynucleotide encoding the FT2A peptide, and a polynucleotide encoding the MDF1; the mutant VSV-G1 and the MDF1 can be separately expressed on HEK-293T cells via the self-cleaving peptide FT2A.

[0276] The lentivirus vector V2 is packaged using the same method as described for packaging the lentivirus vector V7.

[0277] Following the same method used to package lentiviral vector V2, lentiviral vectors V3, V4, and V5 were packaged separately. Example 2

[0278] In this embodiment, the lentiviral vector V1-8 was used to transduce human non-activated PBMCs, and the expression efficiency of CAR-19 and the efficiency of T cell proliferation were detected.

[0279] Resuscitate 8 groups of 1×10⁸ PBMCs from cryopreserved inactive PBMCs of Donor1 (healthy person). 5 Individual inactive PBMCs (the method of thawing and freezing PBMCs is well known to those skilled in the art); each group of human inactive PBMCs was resuspended in 200 µL of PBMC culture medium, wherein the PBMC culture medium included XVT medium, IL-7 at a final concentration of 20 ng / mL and IL-15 at a final concentration of 20 ng / mL;

[0280] On Day 0, the lentiviral vectors V1-8 were added to each group of PBMCs according to MOI=0.5.

[0281] Day 5, CD3 levels in 8 groups of PBMCs were detected by flow cytometry. + The expression of CAR-19 in cells is shown in Figure 1.

[0282] As shown in Figure 1, on Day 5, CD3 of PBMCs transduced by the lentiviral vector V7 containing the membrane-expressed UCHT1 and membrane protein CD137L... + In cells, the CAR-19 exoexpression efficiency was the highest, at approximately 32.10%; followed by the lentiviral vector V8, which contains the membrane-expressed UCHT1, CD86, and membrane protein CD137L, at approximately 29.44%; then the lentiviral vectors V6, V3, V1, V4, V2, and V5, at approximately 22.65%, 22.41%, 14.87%, 8.01%, 1.72%, and 0.51%, respectively.

[0283] On Day 0, Day 5, and Day 7, the number of cells in each group was counted using a cell counter (brand: COUNTERSTAR, model: Rigel S2), and CD3 expression was detected by flow cytometry to calculate the CD3 content. + The proportion of T cells; where the number of T cells on Day 0 was set to 1, the fold increase of T cells on Day 5 and Day 7 was calculated relative to Day 0; the results are shown in Figure 2.

[0284] As shown in Figure 2, relative to Day 0, on Day 5, among the PBMCs transduced by the lentiviral vector V7 containing the membrane-expressed UCHT1 and membrane protein CD137L, and the lentiviral vector V8 containing the membrane-expressed UCHT1, CD86, and membrane protein CD137L, CD3... + The T cell proliferation efficiency was the highest, reaching approximately 9-fold on Day 5; significantly superior to the lentiviral vector V1-6 described above;

[0285] Day 7, in the PBMCs transduced by the lentiviral vector V7, CD3 + T cells proliferated to approximately 25-fold, while in the PBMCs transduced by the lentiviral vector V8, CD3... + T cells proliferated to approximately 17-fold; in the PBMCs transduced by the lentiviral vector V6, CD3... + T cells proliferated to 16-fold; it can be seen that on Day 7, the efficiency of the lentiviral vector V7 in stimulating the activation and proliferation of non-activated T cells was significantly better than that of other lentiviral vectors.

[0286] In summary, the lentiviral vector V7, containing the activating molecule UCHT1-scFv and the co-stimulatory molecule CD137L, exhibits significantly higher efficiency in transduction and activation-stimulating T cell proliferation than other lentiviral vectors V1-6 and V8, which contain different activating and co-stimulatory molecules. Example 3

[0287] A. Packaging contains multiple lentiviral vectors with different activating and co-stimulating molecules.

[0288] Using molecular cloning techniques, a lentiviral envelope plasmid was constructed based on the pMD2.G plasmid backbone. A fragment expressing activating molecules and / or co-stimulatory molecules was inserted downstream of VSV-G (plasmid map shown in Figure 3), including the following from the N-terminus to the C-terminus:

[0289] a. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region;

[0290] b. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD8α signal peptide, anti-CD28 (15E8-scFv), CD8α hinge region, CD8α transmembrane region;

[0291] c. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD8α signal peptide, anti-CD28 (15E8-scFv), CD8α hinge region, CD8α transmembrane region, FT2A, CD137L;

[0292] d. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD137L;

[0293] e.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD86;

[0294] f. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD80;

[0295] g. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD137L, FT2A, CD8α signal peptide, IL2, CD8α hinge region, CD8α transmembrane region;

[0296] h.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, B7H2;

[0297] i. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD8α signal peptide, aOX40(XD), CD8α hinge region, CD8α transmembrane region;

[0298] j. CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, OX40L;

[0299] k.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD70;

[0300] l.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, HVEM;

[0301] m.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD40;

[0302] n.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, TL1A;

[0303] o.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, GITRL;

[0304] p.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, SLAM;

[0305] q.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD48;

[0306] r.CD8α signal peptide, UCHT1-scFv, CD8α hinge region, CD8α transmembrane region, FT2A, CD58;

[0307] Among them, VSV-G contains the same mutation as in Example 1, which inhibits its ability to specifically bind to LDL-R.

[0308] B. Construction of CAR-CD19 plasmid (transfer plasmid)

[0309] Using molecular cloning technology, a CAR molecular fragment targeting CD19 was inserted into the middle of the pGClenti-GFP plasmid. The structure from the N-terminus to the C-terminus is as follows: CD8sp-FMC63(scFv)-CD8Hinge-CD8TM-41BB-CD3Z, thereby constructing a transfer plasmid (same as in Example 1, the plasmid map is shown in Figure 4).

[0310] C. Packaging of lentiviral vectors

[0311] Referring to the viral packaging method in Example 1, lentiviral expression vectors were constructed using different envelope plasmids constructed in step A: HEK-293T cells were transfected with 9µg of the transfer plasmid in B, 4µg of the pMDLg / pRRE packaging plasmid, 2µg of the pRSV-REV packaging plasmid, and 2µg of the envelope plasmid in A, and the lentiviral vectors were collected after culturing.

[0312] D. Lentiviral infection experiment

[0313] Resuscitate inactive PBMCs from cryopreserved healthy individuals and collect 8 groups of 1×10⁻⁶ samples. 5 Individual inactive PBMCs (the method of thawing and freezing PBMCs is well known to those skilled in the art); each group of human inactive PBMCs was resuspended in 200 µL of PBMC culture medium, wherein the PBMC culture medium included XVT medium, IL-7 at a final concentration of 20 ng / mL and IL-15 at a final concentration of 20 ng / mL;

[0314] Day 0, add the above lentiviral vectors to each group of PBMCs according to MOI=5;

[0315] Day 5, CD3 levels in 18 groups of PBMCs were detected using flow cytometry. + The expression of CAR-19 in cells is shown in Figure 5. The CAR positivity rate of lentiviral vectors expressing co-stimulatory molecules containing the CD137L structural region after infection with PBMCs was higher than that of lentiviral vectors without the CD137L structural region. Compared with viruses that only express activating molecules but not co-stimulatory molecules, viruses that express both activating molecules and co-stimulatory molecules have different effects on the infection efficiency of PBMCs. Example 4

[0316] Referring to Example 3, various lentiviral vectors containing different activating and co-stimulating molecules were packaged. The difference from Example 3 is that a fragment expressing the activating and / or co-stimulating molecules was inserted downstream of VSV-G (plasmid map shown in Figure 3), comprising, from the N-terminus to the C-terminus:

[0317] a. CD8α signal peptide, SP34, CD8α hinge region, CD8α transmembrane region;

[0318] b. CD8α signal peptide, SP34, CD8α hinge region, CD8α transmembrane region, FT2A, CD8α signal peptide, anti-CD28 (15E8-scFv), CD8α hinge region, CD8α transmembrane region;

[0319] c. CD8α signal peptide, SP34, CD8α hinge region, CD8α transmembrane region, FT2A, CD137L;

[0320] d. CD8α signal peptide, OKT3, CD8α hinge region, CD8α transmembrane region;

[0321] e.CD8α signal peptide, OKT3, CD8α hinge region, CD8α transmembrane region, FT2A, CD8α signal peptide, anti-CD28 (15E8-scFv), CD8α hinge region, CD8α transmembrane region;

[0322] f. CD8α signal peptide, OKT3, CD8α hinge region, CD8α transmembrane region, FT2A, CD137L.

[0323] The rest is the same as in Example 3, and the results are shown in Figure 6: When constructing lentiviral vectors, regardless of which CD3 antibody is used as the activating molecule, the infection efficiency of lentiviral vectors constructed using CD137L as the co-stimulatory molecule is higher than that of lentiviral vectors using anti-CD28 as the co-stimulatory molecule, and higher than that of lentiviral vectors without using co-stimulatory molecules. Example 5

[0324] Referring to Example 3, various lentiviral vectors containing different activating molecules and co-stimulatory molecules were packaged. The difference from Example 3 is that a fragment expressing the activating molecule and / or co-stimulatory molecule was inserted downstream of VSV-G, from the N-terminus to the C-terminus, including:

[0325] a. CD8α signal peptide, UCHT1, CD8α hinge region, CD8α transmembrane region;

[0326] b. CD8α signal peptide, UCHT1, CD8α hinge region, CD8α transmembrane region, FT2A, CD8α signal peptide, anti-CD28 (15E8-scFv), CD8α hinge region, CD8α transmembrane region;

[0327] c. CD8α signal peptide, UCHT1, CD8α hinge region, CD8α transmembrane region, FT2A, CD86;

[0328] d. CD8α signal peptide, UCHT1, CD8α hinge region, CD8α transmembrane region, FT2A, CD137L;

[0329] VSV-G does not contain any mutations that inhibit its ability to specifically bind to LDL-R compared to Example 3; the rest are the same as Example 3. The results are shown in Figure 7: the viral vector with CD137L as the co-stimulatory molecule has the highest infection rate. Example 6

[0330] In vivo animal experiments were conducted using the lentiviral vector constructed in group d of Example 5.

[0331] Nine NSG immunodeficient female mice aged 4-8 weeks were selected and divided into experimental groups 1-3, with 3 mice in each group;

[0332] Day 1, mice in each group were injected via tail vein with 1×10 6 One Nalm-6 cell, the Nalm-6 cell carrying Luciferase, was used; and 200 µL of sodium luciferase at a concentration of 15 mg / mL was injected intraperitoneally into each group of mice, and in vivo imaging was performed on the three groups of mice respectively.

[0333] Day 0, mice in experimental groups 2 and 3 were injected intravenously with 1×10⁻⁶ mg / L of water. 7 Individual non-activated PBMCs; after an interval of 5-10 minutes, experimental group 3 was injected via tail vein with a dose of 5E5 TU of InVivo Virus (the same lentiviral vector as group d in Example 5).

[0334] In vivo imaging was performed on the three groups of mice on days 1, 3, 7, 14 and 21 to detect the growth of Nalm-6 tumor cells and fluorescence values ​​in each group of mice. The results are shown in Figure 8.

[0335] The results showed that the virus (CD3 antibody + CD137L) prepared according to the present invention could effectively transduce inactive T cells in human inactive PBMCs in mice, prepare CAR-T cells, and efficiently kill Nalm-6 tumor cells in mice.

Claims

A type of particle, characterized in that, The particles contain activating molecules and co-stimulatory molecules displayed on their surface: A. The activating molecule is a first activating polypeptide that can specifically bind to CD3; and / or B. The co-stimulatory molecule is a first co-stimulatory polypeptide that can specifically bind to CD137 (4-1BB). The particles according to claim 1 are characterized in that, In A, the first activating molecular polypeptide contains an anti-CD3 antibody or its antigen-binding fragment. The particles according to claim 2 are characterized in that, The anti-CD3 antibody or its antigen-binding fragment is an anti-CD3 scFv or VHH. The particles according to claim 3 are characterized in that, The anti-CD3 antibody is UCHT1, OKT3, HuM291, SP34, 1452c11, or TR66, or a variant thereof, or a derivative thereof. The particles according to claim 4 are characterized in that, The anti-CD3 antibody is an anti-CD3 scFv, the anti-CD3 scFv is UCHT1 (UCHT1-scFv), the LCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:39-41 respectively, and the HCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:36-38 respectively. The particles according to any one of claims 1 to 5 are characterized in that, B. The first co-stimulatory polypeptide comprises: a ligand or extracellular domain of CD137 targeting CD137, or a functional fragment thereof; or an anti-CD137 antibody, or an antigen-binding fragment thereof. The particles according to claim 6 are characterized in that, The ligand targeting CD137 is CD137L, and the amino acid sequence of CD137L has at least 85% identity with SEQ ID NO:

42. The particle according to any one of claims 1 to 7 is characterized in that, The first activating molecular polypeptide and / or the first co-stimulatory molecular polypeptide are membrane-expressed proteins. The particles according to claim 8 are characterized in that, The first activating polypeptide is linked to the first co-stimulatory polypeptide via a first polypeptide linker. The particles according to any one of claims 1 to 9 are characterized in that, The particle also contains viral glycoproteins, or variants thereof, or functional fragments thereof, or modified versions thereof containing modifications (Modified-G). The particles according to claim 10 are characterized in that, The viral glycoprotein, or its variants, or its functional fragments, or its modifications, are linked to the first activating molecular polypeptide or the first co-stimulatory molecular polypeptide via a second polypeptide linker. The particles according to claim 11 are characterized in that, The first and / or dipeptide linker includes one or more of the following: a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions; b) 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; c) All or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains; d) 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; and e) Flexible linker peptide; preferably (G4S)n linker peptide and linker 1: GSTGSGSGKPGSGEGSTKG (SEQ ID NO:74); wherein n=1 to 4. The particles according to claim 12 are characterized in that, The polypeptide linker is a (G4S)3 linker peptide, GGGGSGGGGSGGGGS (SEQ ID NO:32). The particles according to claim 10 are characterized in that, The viral glycoproteins are 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), biphilic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and T-cell choriomeningitis virus (LCMV) glycoprotein; The vesicular stomatitis virus (VSV) glycoproteins are selected from: Indiana VSV, Cocal VSV, Maraba VSV, Morreton VSV, Alagoas VSV, New Jersey VSV, Carajas VSV, and Chandipura VSV. The glycoproteins of the following strains are included: Eptesicus vesicular stomatitis virus strain, Isfahan vesicular stomatitis virus strain, Jurona vesicular stomatitis virus strain, Malpais vesicular stomatitis virus strain, Perinet vesicular stomatitis virus strain, Piry vesicular stomatitis virus strain, Radi vesicular stomatitis virus strain, Rhinolopus vesicular stomatitis virus strain, and Yug Bogdanovac vesicular stomatitis virus strain. The particles according to claim 14 are characterized in that, The viral glycoprotein is: a) Vesicular stomatitis virus (VSV) Indiana strain glycoprotein (VSV-G), or a variant thereof, or a functional fragment thereof, or a modified version thereof containing the modification (VSV-G modified version); or b) Vesicular stomatitis virus genus Cocal strain glycoprotein (Cocal-G), or a variant thereof, or a functional fragment thereof, or a modified version thereof containing the modification (Cocal-G modified version). The particles according to claim 15 are characterized in that, a) The ability of the VSV-G variant, VSV-G functional fragment, or VSV-G modifier to specifically bind to its receptor LDL-R is inhibited; or b) The ability of the said Cocal-G variant, functional fragment of Cocal-G, or Cocal-G modifier to specifically bind to its receptor LDL-R is inhibited. The particles according to claim 16 are characterized in that, Compared to wild-type VSV-G or Cocal-G, the VSV-G or Cocal-G variant contains a first mutation that inhibits its ability to specifically bind LDL-R, the first mutation being selected from one or more of the following mutations: 1) 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, or deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353; or 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the 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, or the deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353, corresponding to SEQ ID NO:1 or SEQ ID NO:2; Preferably, the first mutation is selected from one or more of the following mutations: 1) The amino acid deletion at positions 331-364, the amino acid deletion at positions 344-354, the substitution of K47, the deletion of K47, the substitution of R354, the substitution of Y209, and the substitution of I182 at positions 331-364, the amino acid deletion at positions 344-354, the substitution of K47, the deletion of K47, the substitution of R354, the substitution of Y209, and the substitution of V182 at positions 344-354 in SEQ ID NO:2; 2) After optimal global alignment with SEQ ID NO:1, the amino acid positions 331-364 and 344-354 corresponding to SEQ ID NO:1 are deleted, or K47 is substituted, or R354 is substituted, or Y209 is substituted, or I182 is substituted; or after optimal global alignment with SEQ ID NO:2, the amino acid positions 331-364 and 344-354 corresponding to SEQ ID NO:2 are deleted, or K47 is substituted, or R354 is substituted, or Y209 is substituted, or I182 or V182 is substituted. More preferably, the first mutation is: 1) K47 is missing in SEQ ID NO:1 or SEQ ID NO:2; or 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, K47 is missing at the position equivalent to SEQ ID NO:1 or SEQ ID NO:

2. The particles according to claim 16 are characterized in that, The modifications contained in the VSV-G or Cocal-G modifiers reduce the number of binding sites available for specific binding of LDL-R. The particles according to any one of claims 14 to 18 are characterized in that, a) The VSV-G variant, VSV-G functional fragment, or VSV-G modifier; or b) The Cocal-G variant, functional fragment of Cocal-G, or modified form of Cocal-G; It contains a second mutation that enhances its ability to antagonize complement inactivation. The particles according to claim 19 are characterized in that, When the viral glycoprotein is a VSV-G or Cocal-G variant, a VSV-G or Cocal-G functional fragment, or a VSV-G or Cocal-G modified form, the second mutation is selected from one or more of the following site mutations: 1) The 214th amino acid located in SEQ ID NO:1 or SEQ ID NO:2, or the 214th amino acid located in SEQ ID NO:1 or SEQ ID NO:2 after best global alignment; 2) The amino acid located at position 352 of SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at position 352 of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2; 3) The amino group located at position 50 of SEQ ID NO:1 or SEQ ID NO:2, or, after optimal global alignment of SEQ ID NO:1 or SEQ ID NO:2, the amino group located at position 50 of SEQ ID NO:1 or SEQ ID NO:2; or 4) The 146th amino acid located in SEQ ID NO:1 or SEQ ID NO:2; or the 146th amino acid located in SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2; Preferably, the mutation at the site is selected from amino acid substitutions, deletions, and insertions; More preferably, the mutation at the site is an amino acid substitution. The particles according to claim 19 or 20 are characterized in that, When the viral glycoprotein is a VSV-G or Cocal-G variant, a VSV-G or Cocal-G functional fragment, or a VSV-G or Cocal-G modified form, the second mutation is selected from a combination of mutations at the following sites: 1) Substitution of (1) T214 and T352 located in SEQ ID NO:1 or SEQ ID NO:2, or (2) Substitution of T214, T352, K50 and S146; or 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the substitutions of (1) T214 and T352, or (2) T214, T352, K50 and S146, located in the equivalent of SEQ ID NO:1 or SEQ ID NO:2; Preferably, the second mutation is selected from a combination of mutations at the following sites: 1) (1) T214N and T352A located in SEQ ID NO:1 or SEQ ID NO:2, or (2) T214N, T352A, K50T and S146T; or 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, it is located at (1) T214N and T352A, or (2) T214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:1 or SEQ ID NO:

2. The particles according to any one of claims 1-21 are characterized in that, The particles also contain exogenous polynucleotides, which include polynucleotides encoding chimeric antigen receptors (CARs) and / or engineered TCRs. The particles according to claim 22 are characterized in that, The exogenous polynucleotide includes a polynucleotide encoding a CAR. The particles according to claim 23 are characterized in that, The CAR includes an antigen-binding region, a transmembrane region, and an intracellular signal transduction domain. The particles according to claim 24 are characterized in that, The antigen-binding region of the CAR can specifically bind to cancer-related antigens; Preferably, the cancer-associated antigen is selected from: One or more of them. The particles according to claim 25 are characterized in that, The cancer-related antigen is a hematologic cancer-related antigen; the hematologic cancers are selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), and filtration... One or more of the following: alveolar lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), 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), and embryonic centrifugal T-cell lymphoma (FTCL); Preferably, the blood cancer-related antigen is selected from one or more of the following: CD19, CD20, CD22, CD79A, CD79B, CD30, CD37, CD38, CD52, CD123, CRLF2, BCMA, CD33, CD138, GPRC5D, CD3, CD4, CD8, CD5, CD7, CD25, CD56, NKG2D, TCR, and ROR1. The particles according to claim 26 are characterized in that, The blood cancer-related antigen is a B-cell malignancy-related antigen, and the B-cell malignancy is selected from one or more of the following: non-Hodgkin lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), LBCL unsuitable for transplantation, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, and Burkitt lymphoma (BL). Preferably, the B-cell malignancy-associated antigen is selected from one or more of CD19, CD20, CD22, CD79A, CD79B, CD30, CD37, CD38, CD52, CD123, CRLF2, BCMA, and ROR1. The particles according to claim 27 are characterized in that, The cancer-associated antigen is a solid cancer-associated antigen, and the solid cancer is selected from one or more of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer. Preferably, the solid tumor-associated antigen is selected from one or more of ROR1, MSLN, MUC1, HER2, CEA, Nectin-4, Claudin18.2, and GCC. The particles according to any one of claims 24-28 are characterized in that, The CAR also includes a connectivity domain and a co-stimulatory signal transduction domain. The particles according to any one of claims 1-29 are characterized in that, The particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles. The particles according to claim 30 are characterized in that, The particles are enveloped viral particles. The particles according to claim 31 are characterized in that, The enveloped viral particles are pseudolentiviral vectors (LVV) and / or retroviral vectors (RVV). A composition, characterized in that, The composition comprises a pharmaceutically acceptable carrier or excipient and particles according to any one of claims 1-32. A method for preparing CAR-T cells by in vitro transduction of T cells, characterized in that, This includes contacting T cells with the particles according to any one of claims 22-32. A method for preparing CAR-T cells by transducing T cells in a subject in need, characterized in that, This includes administering the particles according to any one of claims 22-32 to the subject. A method for improving the expression efficiency of exogenous polynucleotides delivered to T cells, characterized in that, Using the particle according to any one of claims 1-21 carrying the exogenous polynucleotide, the particle is contacted with T cells and the exogenous polynucleotide is delivered. A fusion polypeptide, characterized in that, The fusion peptide comprises a transmembrane polypeptide, an activating molecule, and a co-stimulatory molecule; A. The activating molecule is a first-activating polypeptide that can specifically bind to CD3; and B. The co-stimulatory molecule is a first co-stimulatory polypeptide that can specifically bind to CD137 (4-1BB). The fusion polypeptide according to claim 37, characterized in that, In A, the first activating molecular polypeptide contains an anti-CD3 antibody or its antigen-binding fragment; and The first co-stimulatory polypeptide in B contains the extracellular domain of CD137L, or a functional fragment thereof. The fusion polypeptide according to claim 38, characterized in that, The anti-CD3 antibody or its antigen-binding fragment is an anti-CD3 scFv or VHH. The fusion polypeptide according to claim 39, characterized in that, The anti-CD3 antibody is UCHT1, OKT3, HuM291, or TR66, or a variant thereof, or a derivative thereof. The fusion polypeptide according to claim 40 is characterized in that, The anti-CD3 antibody is an anti-CD3 scFv, the anti-CD3 scFv is UCHT1 (UCHT1-scFv), the LCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:39-41 respectively, and the HCDR1-3 of the UCHT1-scFv are shown as SEQ ID NO:36-38 respectively. The fusion polypeptide according to any one of claims 37-41 is characterized in that, The first activating molecule, the first co-stimulatory polypeptide, and the transmembrane polypeptide are connected by polypeptide linkers. The fusion polypeptide according to claim 42 is characterized in that, The fusion polypeptide comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus, the following: a) the first activating polypeptide, the polypeptide linker, the first co-stimulatory polypeptide, the polypeptide linker, and the transmembrane polypeptide; or b) The first co-stimulatory polypeptide, the polypeptide linker, the first activating polypeptide, the polypeptide linker, and the transmembrane polypeptide. The fusion polypeptide according to claim 42 or 43 is characterized in that, The connector is selected from: a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions; b) 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; c) All or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains; d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and e) Flexible linker peptide; preferably (G4S)n linker peptide and linker 1GSTSGSGKPGSGEGSTKG (SEQ ID NO:74); wherein n=1 to 4. The fusion polypeptide according to claim 44 is characterized in that, The first and second polypeptide linkers are (G4S)3 linker peptides, GGGGSGGGGSGGGGS (SEQ ID NO:32). An isolated polynucleotide, characterized in that, The isolated polynucleotide encodes the fusion polypeptide according to any one of claims 37-45. A type of particle, characterized in that, The particles comprise any one of the fusion polypeptides according to claims 37-45. The particles according to claim 47 are characterized in that, The particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles. The particles according to claim 48 are characterized in that, The particles are enveloped viral particles. The particles according to claim 49 are characterized in that, The enveloped viral particles are pseudotyped LVV and / or RVV. The particles according to any one of claims 47-50 are characterized in that, The particles also contain exogenous polynucleotides, which include polynucleotides encoding CARs and / or engineered TCRs. The use of the particles according to any one of claims 1-32 or 47-51 in the preparation of cancer therapeutic drugs. According to the application described in claim 52, the characteristic is that, The cancer is selected from one or more of hematologic malignancies and solid tumors. According to the application described in claim 53, the characteristic is that, The cancer in question is a blood cancer, specifically selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), and follicular lymphoma. One or more of the following: lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), 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), and embryonic centrifugal T-cell lymphoma (FTCL). According to the application described in claim 54, the characteristic is that, The hematologic malignancy is a B-cell malignancy selected from one or more of the following: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, and Burkitt lymphoma (BL). According to the application described in claim 55, the characteristic is that, The cancer is a solid tumor, selected from one or more of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer. A method for treating a subject who has or is suspected of having cancer, characterized in that, The granules include those for which a therapeutically effective amount of any one of claims 1-32 or 47-51 are administered to the subject. The method according to claim 57 is characterized in that, The cancer is selected from one or more of hematologic malignancies and solid tumors. The method according to claim 58 is characterized in that, The cancer in question is a blood cancer, specifically selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), and follicular lymphoma. One or more of the following: lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), 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), and embryonic centrifugal T-cell lymphoma (FTCL). The method according to claim 59 is characterized in that, The hematologic malignancy is a B-cell malignancy selected from one or more of the following: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, and Burkitt lymphoma (BL). The method according to claim 60, characterized in that, The cancer is a solid tumor, selected from one or more of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer. The method according to any one of claims 57-61 is characterized in that, The administration method is selected from one or more of the following: 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 implantable device.

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