Particle and use thereof

Abstract

A particle and the use thereof. CAR-T cells prepared by means of transducing T cells with the particle not only have an enhanced killing activity, expansion capacity and persistence, but also exhibit an effectively reduced cytokine secretion, and are more suitable for use in in-vivo CAR-T cell therapy.

Description

A particle and its application Technical Field

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

[0002] Currently, CAR-T cell therapy has been widely used to treat hematological malignancies and certain solid tumors. However, the ability of CAR-T cells to expand, kill, and persist in patients still needs improvement to enhance the efficacy of CAR-T cell therapy in treating hematological malignancies and solid tumors. Furthermore, common side effects of CAR-T cell therapy, such as cytokine release syndrome (CRS), also affect its efficacy. One cause of CRS is the release of cytokines such as IFN-γ, TNF-α, and IL-2 when activated CAR-T cells kill target cells, such as cancer cells. Effectively reducing the release of cytokines induced by CAR-T cell therapy is also key to improving its efficacy. Therefore, the need for a CAR-T cell therapy that can effectively improve the expansion, killing, and persistence of CAR-T cells, and more importantly, simultaneously effectively reduce cytokine release, remains unmet. Summary of the Invention

[0003] One aspect of the present invention provides a particle, the particle comprising:

[0004] a) Viral glycoproteins or their variants, or functional fragments thereof, or their modified forms;

[0005] b) One or more targeting molecules that can specifically bind to antigens on the surface of T cells;

[0006] c) A first exogenous polynucleotide encoding an engineered cytokine receptor (eCR); and

[0007] d) The second exogenous polynucleotide encodes a chimeric antigen receptor (CAR).

[0008] In some embodiments of the present invention, the eCR comprises:

[0009] a) One or more intracellular domains of IL-7Rα or functional fragments thereof;

[0010] b) IL-7Rα transmembrane domain; and,

[0011] c) One or more extracellular domains, said extracellular domains comprising the IL-7Rα extracellular domain, or a functional fragment thereof, or a derivative thereof.

[0012] In some embodiments of the present invention, one or more IL-7Rα intracellular domains of the eCR trigger signal transduction via the STAT5 or STAT3 pathway.

[0013] In some embodiments of the present invention, the amino acid sequence of the IL-7Rα intracellular domain or its functional fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:64.

[0014] In some embodiments of the present invention, the transmembrane domains of the eCR are self-oligomery.

[0015] In some embodiments of the present invention, the mutated IL-7Rα transmembrane domain contains one or more mutations that enable the eCR to homodimerize through transmembrane and intracellular domain components.

[0016] In some embodiments of the present invention, the transmembrane domains of the eCR are self-oligomery.

[0017] In some embodiments of the present invention, the mutated IL-7Rα transmembrane domain contains one or more mutations that enable the eCR to homodimerize through transmembrane and intracellular domain components.

[0018] In some embodiments of the present invention, the mutated IL-7Rα transmembrane domain contains one or more mutations that enable structural torsion of the transmembrane and intracellular domains of the eCR, such that the Janus kinase associated with the intracellular domain is directed to allow cross-phosphorylation and activation.

[0019] In some embodiments of the present invention, the mutated IL-7Rα transmembrane domain contains one or more mutations that enable the transmembrane and intracellular domains of the eCR to twist together in a helical manner.

[0020] In some embodiments of the present invention, the eCR polypeptide has constitutive activity because the transmembrane domain of the eCR contains one or more mutations that allow receptor homodimerization, making it unnecessary for external signals (e.g., cytokines) to activate the transmembrane and intracellular domains. In at least some cases, one or more mutations in the transmembrane domain of the eCR confer a transmembrane / intracellular domain conformation that brings the molecule closer to the target molecule required for its signaling to function. In specific examples, the mutations in the transmembrane domain of the eCR are or include the insertion of at least one cysteine ​​residue into the amino acid sequence of the transmembrane domain to allow disulfide bond formation (thus promoting homodimerization of the polypeptide), and / or include the insertion of at least one proline residue to induce a conformational change (e.g., causing the molecule to kink, twist, or rotate around an axis passing through the receptor molecule), both of which will structurally affect the properties of the molecule and thus its signaling.

[0021] In some embodiments of the present invention, the mutated IL-7Rα transmembrane domain contains one or more mutations located in the sequence: PILLTISILSFFSVALLVILACVLW (SEQ ID NO:65).

[0022] In some embodiments of the present invention, the mutation is to introduce at least one cysteine ​​C into the transmembrane domain of the eCR.

[0023] In some embodiments of the present invention, the mutation is the introduction of proline P into the transmembrane domain of the eCR.

[0024] In some embodiments of the present invention, the transmembrane domain of the eCR has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with any amino acid sequence in SEQ ID NO:63 or SEQ ID NO:72-93.

[0025] In some embodiments of the present invention, the transmembrane domain of the eCR has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with PILLTCPTISILSFFSVALLVILACVLW (SEQ ID NO:63).

[0026] In some embodiments of the present invention, the length of the transmembrane domain of the eCR is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids.

[0027] In some embodiments of the present invention, the extracellular domain of the eCR is the IL-7Rα extracellular domain, or a functional fragment thereof, or a derivative thereof.

[0028] In some embodiments of the present invention, the amino acid sequence of the extracellular domain of the IL-7Rα has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:67.

[0029] In some embodiments of the present invention, the amino acid sequence of the eCR (eCR2) has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:70.

[0030] In some embodiments of the present invention, the eCR further comprises a leader signal peptide. The leader signal peptide of the eCR is not particularly limited, as long as it can mediate the exoexpression of the eCR.

[0031] In some embodiments of the present invention, the leader signal peptide of the eCR is selected from CD8α signal peptide, IgA signal peptide, CD28 signal peptide, CD34 signal peptide, and IL-7Rα signal peptide. Preferably, the leader signal peptide is IL-7Rα signal peptide.

[0032] In some embodiments of the present invention, the amino acid sequence of the IL-7Rα 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:66.

[0033] In some embodiments of the present invention, the amino acid sequence of the eCR2 containing the IL-7Rα 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:71.

[0034] In some other embodiments of the present invention, the extracellular domain of the eCR is not the extracellular domain of IL-7Rα.

[0035] In some embodiments of the present invention, the eCR comprises at least one intracellular domain, at least one transmembrane domain, and at least one extracellular domain, and optionally includes one or more extracellular domains. In some embodiments, the intracellular domain of the eCR is wild-type, i.e., functionally active in cytokine signaling. In one specific embodiment, the eCR peptide comprises a normal (wild-type) extracellular domain; in this embodiment, the peptide homodimerizes and transmits signals in the presence or absence of homologous cytokines. In another specific embodiment, the eCR peptide comprises an extracellular domain that is not a wild-type extracellular domain of the intracellular domain of the eCR peptide. In a more specific embodiment, the extracellular domain of the eCR peptide binds ligands that are generally harmful to cells expressing the eCR peptide (e.g., ligands that typically downregulate cells or induce cell anergy or apoptosis). For example, such a non-wild-type extracellular domain can be, or can act as, a camouflaged receptor for, for example, a checkpoint protein, such as a receptor for checkpoint protein PD-1. In some cases, the non-wild-type extracellular domain is the target of an antibody (which, when bound to the extracellular domain, targets cells containing the eCR peptide for destruction). In another specific embodiment, the eCR peptide lacks the extracellular domain. In some embodiments, the cytokine is an interleukin, such as IL-7, IL-21, IL-23, or IL-12.

[0036] In some embodiments of the present invention, the extracellular domain of the eCR is not derived from a cytokine receptor.

[0037] In some embodiments of the invention, the eCR homodimerizes and facilitates downstream signaling without binding to associated cytokines. A common feature of these eCRs is that they are engineered (e.g., in the transmembrane domain) to contain one or more mutations that induce or promote homodimerization and thus signaling in the absence of bound homologous cytokines. Therefore, they are constitutively active (i.e., the mutations are “gain-of-function” mutations) in relation to cytokine signaling.

[0038] In some embodiments of the present invention, the extracellular domain of the eCR is a masquerading receptor lacking signal transduction activity.

[0039] In some embodiments of the present invention, the extracellular domain of the eCR is the extracellular domain of CD34.

[0040] In some embodiments of the present invention, the extracellular domain of the eCR (eCR1) is the extracellular domain of CD34; the transmembrane domain of the eCR is the mutated IL-7Rα transmembrane domain; and the intracellular domain of the eCR is the intracellular domain of IL-7Rα.

[0041] In some embodiments of the present invention, the amino acid sequence of the extracellular domain of CD34 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:62.

[0042] In some embodiments of the present invention, the amino acid sequence of the eCR (eCR1) has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:68.

[0043] In some embodiments of the present invention, the eCR1 further comprises a leader signal peptide.

[0044] In some embodiments of the present invention, the lead signal peptide of eCR1 is not particularly limited, as long as it can mediate the exoexpression of eCR.

[0045] In some embodiments of the present invention, the leader signal peptide of eCR1 is the CD34 signal peptide.

[0046] In some embodiments of the present invention, the amino acid sequence of the CD34 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:61.

[0047] In some embodiments of the present invention, the amino acid sequence of the eCR1 containing the CD34 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:69.

[0048] In some embodiments of the present invention, the masquerading receptor comprises a constitutively active cytokine receptor, wherein the extracellular domain of the eCR is or comprises an extracellular domain derived from PD-1 or B7.

[0049] In some embodiments of the present invention, the extracellular domain of the eCR is at least 70 amino acids in length.

[0050] In some embodiments of the present invention, the extracellular domain of the eCR is no more than 2,000 amino acids in length.

[0051] In some embodiments of the present invention, the length of the extracellular domain of the eCR is 70-2000 amino acids, 100-1000 amino acids, 500-2000 amino acids, 50-500 amino acids, 100-750 amino acids, 200-2000 amino acids, or 500-2000 amino acids.

[0052] In some embodiments of the present invention, the extracellular domain of the eCR has a length of 70-2000 amino acids, 100-1000 amino acids, 150-500 amino acids, or 175-350 amino acids.

[0053] In some embodiments of the present invention, the extracellular domain of the eCR is derived from IL-7Rα, PD-1CD30, HER2, EGFR, CD19, CD34, TGF-β receptor, IL-4 receptor, IL-13 receptor α1 and α2, IL-8 receptor, IL-10 receptor, LAG3, TIGIT, CTLA4, FAS, CD19, CD27, CD28, CD52, CD134, CD137, HER2, EGFR, or NGFR.

[0054] In some embodiments of the invention, the constitutively active cytokine receptor utilizes an extracellular domain that acts as a sink or ligand trap, for example, by binding to one or more molecules that are harmful to cells expressing the constitutively active cytokine receptor. Such ligands can be immunosuppressive, for example, because they typically activate signaling pathways to shut down T cells, as in immunosuppression. In some such cases, the extracellular domain binds the harmful ligand as a dummy receptor, but the transmembrane / intracellular domain is still capable of independently generating positive cytokine signals. In some embodiments, the dummy receptor prevents the corresponding ligand from suppressing T cells (as would normally occur). For example, the extracellular domain of the dummy receptor can bind inhibitory cytokines. Examples of harmful ligands that the dummy receptor can bind include TGF-β, PD-L1, IL-4, IL-13, IL-8, and IL-10. In some embodiments, the extracellular domain of the dummy receptor is one of the inhibitory receptors typically expressed by T cells, such as, but not limited to, LAG3, TIGIT, CTLA4, and FAS.

[0055] In some embodiments of the present invention, the CAR includes an antigen-binding region, a transmembrane region, and an intracellular signal transduction domain.

[0056] In some embodiments of the present invention, the antigen-binding region of the CAR can specifically bind to cancer-associated antigens; preferably, the cancer-associated antigens are selected from:

[0057] One or more of them.

[0058] 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 (MCL), 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, etc. The tumor is selected from one or more of the following: anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, T-large granular lymphoblastic leukemia (T-LGL), and embryonic centrifugal T-cell lymphoma (FTCL); preferably, the blood cancer-associated 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.

[0059] In some embodiments of the present invention, the blood cancer-related antigen is a B-cell malignancy-related antigen, wherein 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-related antigen is selected from one or more of CD19, CD20, CD22, CD79A, CD79B, CD30, CD37, CD38, CD52, CD123, CRLF2, BCMA, and ROR1.

[0060] 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; preferably, the solid cancer-associated antigen is selected from one or more of the following: ROR1, MSLN, MUC1, HER2, CEA, Nectin-4, Claudin18.2, and GCC.

[0061] In some embodiments of the present invention, the transmembrane region of the CAR is selected from the transmembrane regions of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4 , BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

[0062] In some embodiments of the present invention, the transmembrane region of the CAR is the CD8α transmembrane region.

[0063] In some embodiments of the present invention, 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.

[0064] 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.

[0065] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR is the intracellular signal transduction domain of CD3ζ.

[0066] 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.

[0067] In some embodiments of the present invention, the CAR further includes a connectivity domain and a co-stimulatory signal transduction domain.

[0068] 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.

[0069] In some embodiments of the present invention, the CAR connection structure domain is selected from:

[0070] (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;

[0071] (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;

[0072] (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; and

[0073] (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

[0074] (e) Flexible linker peptide; preferably (G4S)n linker peptide, wherein n = 1 to 4; linker 1: GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 60); and linker 2: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 94).

[0075] In some embodiments of the present invention, the connection structure domain of the CAR is the CD8α hinge region.

[0076] In some embodiments of the present invention, 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.

[0077] 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; preferably, the co-stimulatory signal transduction domain of the CAR is selected from one or more of the co-stimulatory signal transduction domains of 4-1BB and CD28.

[0078] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR is a 4-1BB costimulatory signal transduction domain.

[0079] In some embodiments of the present invention, the amino acid sequence of the co-stimulatory signal transduction domain of the 4-1BB has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:30.

[0080] 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; preferably, the leader signal peptide is selected from CD8α signal peptide, CD28 signal peptide, IgG signal peptide and HLA-A signal peptide.

[0081] In some embodiments of the present invention, the leader signal peptide of the CAR is not particularly limited, as long as the leader signal peptide can mediate the expression of the CAR membrane. Preferably, the leader signal peptide is a CD8α signal peptide.

[0082] 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.

[0083] In some embodiments of the present invention, the CAR is bound to a CAR-binding molecule, the CAR-binding molecule comprising one or more of (a) an antigen or a binding fragment thereof capable of binding to the antigen-binding region of the CAR; and (b) an anti-antibody; preferably, the antigen-binding fragment comprises one or more selected from antigen extracellular domains, antigen functional fragments, and antigen epitopes.

[0084] In some embodiments of the present invention, the viral glycoprotein is selected from glycoproteins of vesicular stomatitis virus strains, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), bitropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and T-cell choroid plexus meningitis virus (LCMV) glycoprotein; the vesicular stomatitis virus strain glycoprotein is selected from: vesicular stomatitis virus Indiana strain glycoprotein, vesicular stomatitis virus Cocal strain glycoprotein, vesicular stomatitis virus Maraba strain glycoprotein, vesicular stomatitis virus Morre strain glycoprotein. The glycoproteins of the following strains are listed: ton, Alagoas, New Jersey, Carajas, Chandipura, Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and Yug Bogdanovac.

[0085] In some embodiments of the present invention, the number of binding sites available for specific binding to the receptor of the viral glycoprotein modifier is reduced.

[0086] In some embodiments of the present invention, the viral glycoprotein modifier has specifically bound its receptor and / or its antibody.

[0087] In some embodiments of the present invention, the viral glycoprotein variant includes a first mutation that inhibits the ability of the viral glycoprotein variant to specifically bind to its receptor relative to a viral glycoprotein that does not contain the first mutation.

[0088] In some embodiments of the present invention, the viral glycoprotein is: (a) vesicular stomatitis virus strain Indiana 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 strain Cocal 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 viral glycoprotein receptor is LDL-R.

[0089] 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.

[0090] 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; wherein, the amino acid sequence shown in positions 1-16 of SEQ ID NO:9: MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.

[0091] 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.

[0092] 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; wherein, 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.

[0093] 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.

[0094] By inhibiting the ability of VSV-G or Cocal-G to bind to their receptors, the targeting of particles containing VSV-G or Cocal-G can be effectively improved.

[0095] In some embodiments of the present invention, the VSV-G variant, relative to wild-type VSV-G or wild-type Cocal-G, comprises 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:

[0096] 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 / or 354 in SEQ ID NO:1 or SEQ ID NO:2; deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354 and / or 345-353; or,

[0097] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, substitutions or deletions of amino acids at positions 8, 9, 10, 47, 50, 51, 183, 179, 180, 182, 184, 209, 347, 350, 352, 353 and / or 354, and deletions of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354 and / or 345-353, corresponding to SEQ ID NO:1 or SEQ ID NO:2;

[0098] Preferably, the first mutation is selected from one or more of the following mutations:

[0099] 1) Deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354, substitution of Y209, and substitution of I or V182 located in SEQ ID NO:1 or SEQ ID NO:2; or,

[0100] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid deletions at positions 331-364, amino acid deletions at positions 344-354, substitutions for K47, deletions of K47, substitutions for R354, substitutions for Y209, and substitutions for I182 are located at positions corresponding to SEQ ID NO:1 or SEQ ID NO:2.

[0101] More preferably, the first mutation is:

[0102] 1) K47 is missing in SEQ ID NO:1 or SEQ ID NO:2; or

[0103] 2) After the best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the K47 deletion is located at the equivalent of SEQ ID NO:1 or SEQ ID NO:2.

[0104] In some embodiments of the present invention, the first mutation is selected from one or more of the following mutations: 1) deletion of amino acids 331-364, deletion of amino acids 344-354, K47Q, deletion of K47, R354Q, Y209Q, and V182E / I182E / I182D located at 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, deletion of amino acids 331-364, deletion of amino acids 344-354, K47Q, deletion of K47, R354Q, Y209Q, and V182E / I182E / I182D located at SEQ ID NO:1 or SEQ ID NO:2.

[0105] In some embodiments of the present invention, (a) the amino acid sequence of the VSV-G variant has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:3; or (b) the amino acid sequence of the Cocal-G variant has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:11.

[0106] In some embodiments of the present invention, (a) the VSV-G modifier contains modifications that reduce the number of binding sites available for specific binding of its receptor LDL-R; or (b) the Cocal-G modifier contains modifications that reduce the number of binding sites available for specific binding of its receptor LDL-R.

[0107] In some embodiments of the present invention, (a) the VSV-G modifier has specifically bound its receptor LDL-R and / or anti-VSV-G antibody; or (b) the Cocal-G modifier has specifically bound its receptor LDL-R and / or anti-Cocal-G antibody.

[0108] In some embodiments of the present invention, the targeting molecule is a high-affinity antibody.

[0109] In some embodiments of the present invention, the activated molecular anti-CD3 antibody or its antigen-binding fragment is a high-affinity antibody.

[0110] In some embodiments of the present invention, the affinity (Kd value) of the high-affinity antibody for its antigen is 1 × 10⁻⁶. -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, 1×10⁻⁹M⁻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 - 9M, 1×10 -9 M-7×10 -9 M or 1×10 -9 M-6×10 -9 M, each containing its end value.

[0111] 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.

[0112] In some embodiments of the present invention, the high-affinity antibody against CD3 is UCHT1 or SP34.

[0113] In some embodiments of the present invention, the viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, includes a second mutation that enhances its ability to antagonize complement inactivation, the enhanced ability to antagonize complement inactivation being relative to a viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, that does not contain the second mutation. Preferably, the viral glycoprotein is VSV-G or Cocal-G.

[0114] In some embodiments of the present invention, the VSV-G or Cocal-G, or variants thereof, or functional fragments thereof, or modified versions thereof, comprising a second mutation selected from one or more of the following site mutations:

[0115] 1) The amino acid located at position 214 of SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at position 214 of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment; or

[0116] 2) The amino acid at position 352 of SEQ ID NO:1 or SEQ ID NO:2, or the amino acid at position 352 of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment; or

[0117] 3) The 50th amino acid located in SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at the 50th amino acid of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment; or

[0118] 4) The 146th amino acid located in SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at the equivalent of the 146th amino acid in SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2;

[0119] Preferably, the mutation at the site is selected from amino acid substitution, deletion, and insertion. More preferably, the mutation at the site is an amino acid substitution.

[0120] In some embodiments of the present invention, the second mutation contained in the VSV-G or Cocal-G, or a variant thereof, or a functional fragment thereof, or a modified version thereof, is selected from a combination of mutations at the following sites:

[0121] 1) Substitution of (i) T / K214 and T352 located in SEQ ID NO:1 or SEQ ID NO:2, or (ii) substitution of T / K214, T352, K50 and S146; or

[0122] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the substitutions located at (i) T / K214 and T352, or (ii) T / K214, T352, K50 and S146, which are equivalent to SEQ ID NO:1 or SEQ ID NO:2;

[0123] Preferably, the second mutation is selected from a combination of mutations at the following sites:

[0124] 1) (i) T / K214N and T352A located in SEQ ID NO:1 or SEQ ID NO:2, or (ii) T / K214N, T352A, K50T and S146T; or

[0125] 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, it is located at (i) T / K214N and T352A, or (ii) T / K214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:1 or SEQ ID NO:2.

[0126] In some embodiments of the present invention, any of the aforementioned viral glycoproteins, or variants thereof, or functional fragments thereof, or modified forms thereof, retain the ability to fuse with membranes and mediate lysosomal escape.

[0127] In some embodiments of the present invention, the antigens on the surface of the T cells are selected from: CD2, CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD57, CD71, CD69, CD94, CD95, 4-1BB, CD103, CD122, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5). R5), PD-1, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, IL6ST, P2RX7, TIGIT, TIM-3, and LAG-3; preferably, the antigen on the surface of the T cells is selected from one or more of CD3, CD3γ, CD3δ, CD3ε, CD5, CD7, CD28, CD2, CD127, 4-1BB, OX40, ICOS, TCRγ, TCRδ, TCRα, and TCRβ.

[0128] T cell surfaces contain various antigens, such as CD5, CD7, and CD3. Constructing one or more targeting molecules on the particle surface can enhance the particle's targeting of T cells. Furthermore, various antigens on the T cell surface, such as CD3, CD7, CD137, and CD28, are endocytosis receptors. When the ability of viral glycoproteins, or their variants, or their functional fragments, or their modifiers to specifically bind to their receptors is weakened or inhibited, constructing one or more targeting molecules on the particle surface that can specifically bind to the endocytosis receptors on the T cell surface and induce endocytosis can further enhance the particle's targeting of T cells.

[0129] In some embodiments of the present invention, the target molecule is not any of the aforementioned viral glycoproteins.

[0130] In some embodiments of the present invention, the targeting molecule includes an activating molecule.

[0131] In some embodiments of the present invention, the activating molecule can specifically bind to CD3, CD7, CD5 and / or TCR.

[0132] In some embodiments of the present invention, the CD3 is human CD3, CD7, CD5 and / or TCR.

[0133] In some embodiments of the present invention, the activating molecule includes anti-CD3 antibody, anti-CD7 antibody, anti-CD5 antibody and / or anti-TCR antibody or antigen-binding fragment thereof.

[0134] In some embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment is an anti-CD3 scFv or VHH.

[0135] In some embodiments of the present invention, the anti-CD3 antibody is UCHT1, SP34, OKT3, HuM291 or TR66, or a variant thereof, or a derivative thereof.

[0136] 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.

[0137] In some embodiments of the present invention, the targeting molecule includes at least one co-stimulatory molecule.

[0138] In some embodiments of the present invention, the at least one co-stimulatory molecule can specifically bind to one or more of CD28, CD137, OX40, and ICOS.

[0139] In some embodiments of the present invention, the co-stimulatory molecule is selected from anti-CD28 antibody or its antigen-binding fragment, CD80, CD86, CD137L, OX40L or ICOSL, or its extracellular domain, or its functional fragment; preferably, the anti-CD28 antibody or its antigen-binding fragment is a 15E8 scFv (15E8-scFv), and 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.

[0140] In some embodiments of the present invention, the targeting molecule is a transmembrane protein that can be membrane-exposed on the surface of the particle.

[0141] In some embodiments of the present invention, the co-stimulatory molecule 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.

[0142] In some embodiments of the present invention, the target molecule is a recombinant transmembrane protein.

[0143] In some embodiments of the present invention, the recombinant transmembrane protein includes a transmembrane region; preferably, the recombinant transmembrane protein further includes a linker domain.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] In some embodiments of the present invention, the transmembrane region of the recombinant transmembrane protein is the CD8α transmembrane region.

[0149] In some embodiments of the present invention, the transmembrane region of the recombinant transmembrane protein is connected to the activating molecule or the co-stimulatory molecule through the linker domain.

[0150] In some embodiments of the present invention, the linker domain of the recombinant transmembrane protein is selected from any of the aforementioned linker domains.

[0151] In some embodiments of the present invention, the linker domain of the recombinant transmembrane protein is the CD8α hinge region.

[0152] In some embodiments of the present invention, the activating molecule is linked to the co-stimulatory molecule via a first polypeptide linker (PolypeptideLinker1).

[0153] In some embodiments of the present invention, the viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, is linked to the activating molecule or the co-stimulatory molecule via a second polypeptide linker.

[0154] In some embodiments of the present invention, (a) the viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, is linked to the activating molecule via the second polypeptide linker; the activating molecule is linked to the co-stimulatory molecule via the first polypeptide linker; or (b) the viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, is linked to the co-stimulatory molecule via the second polypeptide linker; the co-stimulatory molecule is linked to the activating molecule via the first polypeptide linker.

[0155] In some embodiments of the present invention, the first polypeptide linker and the second polypeptide linker are selected from any of the aforementioned linker domains.

[0156] In some embodiments of the present invention, the first polypeptide linker and the second polypeptide linker are (G4S)3 linker peptides, GGGGSGGGGSGGGGS (SEQ ID NO:32).

[0157] In some embodiments of the present invention, the particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles.

[0158] In some embodiments of the present invention, the particles are enveloped viral particles.

[0159] In some embodiments of the present invention, the enveloped viral particles are pseudolentiviral vectors (LVV) and / or retroviral vectors (RVV).

[0160] In another aspect, the present invention also provides a composition comprising a pharmaceutically acceptable carrier or excipient and any of the aforementioned particles.

[0161] In another aspect, the present invention also provides a method for preparing CAR-T cells by transducing T cells in vitro, including contacting T cells with any of the aforementioned particles.

[0162] 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 any of the aforementioned particles to the subject.

[0163] In some embodiments of the present invention, administration of T cells and / or a cell population containing T cells to the subject is also included.

[0164] In another aspect, the present invention also provides a method for reducing or decreasing the release or secretion of cytokines by CAR-T cells while improving, enhancing or strengthening at least one of the amplification capacity, persistence capacity and killing capacity of CAR-T cells prepared in a subject in need, including administering any of the aforementioned particles to the subject in need.

[0165] In some embodiments of the present invention, administration of T cells and / or a cell population containing T cells to the subject is also included.

[0166] In some embodiments of the present invention, the cytokines are selected from one or more of IL-2, IFN-γ, TNF-α, and IL-6.

[0167] In some embodiments of the present invention, the cell population is selected from one or more of the subject's own, allogeneic, and iPSC-derived cell populations.

[0168] In some embodiments of the present invention, the cell population is selected from PBMCs, lymphocytes, and leukocytes.

[0169] 29 In another aspect, the present invention also provides the use of any of the aforementioned particles in cancer treatment drugs.

[0170] In another aspect, 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 aforementioned particles.

[0171] In some embodiments of the present invention, the cancer is selected from one or more of hematologic malignancies and solid tumors.

[0172] 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).

[0173] 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).

[0174] 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.

[0175] In some embodiments of the present invention, the administration is selected from one or more of the following methods: intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection, nodular injection, infusion technique, oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, via portal vein, intralesional, continuous release system, and implantation device.

[0176] In another aspect, the present invention also provides a method for alleviating, preventing or treating a subject who has or is suspected of having CRS, comprising incorporating any of the aforementioned eCR peptides into CAR-T cells in the subject.

[0177] Beneficial effects: Any of the particles provided by this invention can effectively improve the expansion capacity, persistence and / or killing capacity of CAR-T cells, and can reduce the release or secretion of cytokines by CAR-T cells, making them more suitable for application in in vivo CAR-T cells.

[0178] In this article:

[0179] "Engineered Cytokine Receptors (eCRs)": This invention discloses eCRs and their applications. An eCR comprises at least one extracellular domain, a transmembrane domain, and at least one intracellular domain, wherein the intracellular domain is derived from a cytokine receptor. In some embodiments, the transmembrane domain, intracellular domain, and extracellular domain originate from the same molecule. In some embodiments, the transmembrane domain and extracellular domain originate from the same molecule, and the intracellular domain originates from a different molecule, but the eCR is constitutively active because the molecule includes mutations torturing the transmembrane domain and / or intracellular domain. In certain cases, eCRs are constitutively active (constitutively active cytokine receptor molecules) because their transmembrane domain and / or intracellular domain components are configured to deliver activation signals without receiving corresponding signals from their operatively connected extracellular domains. That is, in certain embodiments of the eCR, no ligand requirement exists for the cytokine receptor. In some cases, the transmembrane and / or intracellular domains of the constitutively active cytokine receptors of this disclosure can be configured such that they can homodimerize in a non-natural manner or under non-natural conditions or environments, thereby allowing the extracellular domains to remain in a state that transmits activation signals to their corresponding entities downstream in the signal transduction pathway. In specific embodiments, the transmembrane / intracellular domains of the constitutively active cytokine receptors independently generate positive cytokine signals in the absence of binding of cytokines to their operatively linked extracellular domains. In embodiments, specific constitutively active cytokine receptors are used in the methods of this disclosure.

[0180] "Extracellular domains of the eCR": In specific embodiments, the eCR / constitutive active cytokine receptor comprises one, two, or more extracellular domains. In specific embodiments, the extracellular domains are capable of binding ligands and transmitting the corresponding signals. In specific embodiments, the extracellular domains are capable of binding ligands, but the signal itself is not transmitted, for example, due to structural or other reasons. In specific embodiments, the extracellular domains may or may not originate from the same natural molecule as their corresponding transmembrane and / or intracellular domains. The extracellular domains may or may not originate from the same natural molecule as their operatively linked intracellular domains.

[0181] In some cases, the extracellular domains of the eCR are spherical, resembling the three-dimensional geometry of the IL7-receptor extracellular domain and the CD34 extracellular domain. One feature provided by the extracellular domains of the eCR can be the provision of protein stabilization, as at least some data suggest that at least certain external domains allow for high protein expression and associated high signal activation (e.g., pSTAT5 activation). In some cases, the extracellular domains of the eCR contain high glycosylation, which stabilizes proteins and can enhance signal transduction. In some embodiments, the extracellular domains of the eCR allow for the identification of transduced cells. For example, in cases where the extracellular domains are not typically expressed on T cells, the extracellular domains allow for the identification of transduced cells, for example, by gating during flow cytometry analysis and, for example, by magnetic selection for enrichment (e.g., using magnetic beads conjugated to the corresponding antibody).

[0182] In some embodiments, the eCR / constitutive active cytokine receptor utilizes an extracellular domain that confers library or ligand capture functionality, for example, by binding one or more molecules that are harmful to cells expressing constitutive active cytokine receptors. In some embodiments, the ligand is immunosuppressive, for example, because it normally activates signaling pathways to shut down T cells (immunosuppression). In some such cases, the extracellular domain binds the harmful ligand as a dummy receptor, but the transmembrane / intracellular domain is still capable of independently generating positive cytokine signaling. In some embodiments, the dummy receptor prevents the corresponding ligand from suppressing T cells (as would normally occur). In certain cases, for example, the dummy receptor extracellular domain is capable of binding inhibitory cytokines. Examples of harmful ligands include TGF-β, PD-L1, IL-4, IL-13, IL-8, and IL-10. Furthermore, the dummy receptor extracellular domain is capable of encoding the extracellular domain of inhibitory receptors normally expressed by T cells, such as, but not limited to, LAG3, TIGIT, CTLA4, and FAS. In some embodiments, the receptor is constitutive signaling, and signaling is further enhanced in the presence of a designated trigger. In alternative embodiments, the extracellular domain is not derived from a cytokine receptor, for example, it may include domains not derived from the IL-4 or IL-13 cytokine receptor. In some cases, the extracellular domain may or may not contain an antibody, such as scFv.

[0183] In certain respects, the extracellular domain of the eCR is a target for cell destruction. For example, the extracellular domain can be used as a target for molecules that directly or indirectly induce apoptosis in cells expressing the receptor. For instance, the extracellular domain can be targeted with a corresponding antibody that binds to it, leading to the eventual destruction of the cell.

[0184] In some cases, the constitutively acting receptor has an extracellular domain that acts as a camouflaged receptor for inhibitory ligands, and also has a domain for suicide targeting, although in some cases the camouflaged receptor extracellular domain and the suicide-targeting extracellular domain are one and the same.

[0185] In specific embodiments, the extracellular domain of the eCR includes extracellular domains of CD30, HER2, EGFR, CD19, CD34, TGF-β receptor, IL-4 receptor, IL-13 receptor α1 and α2, IL-8 receptor, IL-10 receptor, PD-1, LAG3, TIGIT, CTLA4, FAS, CD19, CD27, CD28, CD52, CD134, CD137, HER2, EGFR, or NGFR. Furthermore, the extracellular domain can contain a monoclonal antibody or its derivative (such as scFV) or a dimer domain (such as FKBP).

[0186] In some cases, the extracellular domain of the eCR contains podocyte protein (also known as podocyte protein-like protein 1; PODXL (also known as PCLP1); thrombosin; gp135; GCTM2; TRA-1-60; TRA-1-81; or endoglycan (also known as podocyte protein-like protein 2, PODXL2, or PCLP2). In some embodiments, the extracellular domain component of the eCR is derived from a naturally occurring molecule and is wild-type, but in other cases, the extracellular domain contains one or more mutations compared to the corresponding wild-type naturally occurring molecule. For example, these one or more mutations may serve to further stabilize the receptor. Where the extracellular domain contains one or more mutations compared to the corresponding wild-type sequence, the mutated form may have a percentage identity with the corresponding wild-type protein / nucleic acid sequence in part or all of the sequence, such as at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity.

[0187] In some embodiments, the extracellular domain has a certain length. In specific embodiments, the length of the extracellular domain is 10-275 amino acids, 10-225 amino acids, 10-200 amino acids, 10-175 amino acids, 10-150 amino acids, 10-125 amino acids, 10-100 amino acids, 10-50 amino acids, 50-250 amino acids, 50-225 amino acids, 50-200 amino acids, 50-175 amino acids, 50-150 amino acids, 50-100 amino acids, 90-250 amino acids, 90-225 amino acids, 90-200 amino acids, 90-175 amino acids, 90-150 amino acids, 90-125 amino acids, 90-100 amino acids. Amino acids, 100-250 amino acids, 100-225 amino acids, 100-200 amino acids, 100-175 amino acids, 100-150 amino acids, 100-125 amino acids, 125-250 amino acids, 34125-225 amino acids, 125-200 amino acids, 125-175 amino acids, 125-150 amino acids, 150-250 amino acids, 150-225 amino acids, 150-200 amino acids, 150-175 amino acids, 175-250 amino acids, 175-225 amino acids, 175-200 amino acids, 200-250 amino acids, 200-225 amino acids, etc. In alternative embodiments, eCR lacks an extracellular domain.

[0188] "Transmembrane domain of eCR": In specific embodiments, the eCR / constitutively active cytokine receptor comprises a transmembrane domain operatively linked to both an extracellular and intracellular domain. The transmembrane domain may or may not originate from the same native molecule as its operatively linked intracellular domain. In certain embodiments, the transmembrane domain is not derived from the same native molecule as its operatively linked extracellular domain. In certain embodiments, the transmembrane domain is not native because it requires a mutation that causes molecular torsion. In at least some embodiments, the transmembrane domain contains one or more mutations compared to its corresponding wild-type molecule, which allow the transmembrane domain to be self-active. As used herein, the term "self-active" refers to a transmembrane domain that, together with its operatively linked intracellular domain, transduces signaling to the cell in the absence of a corresponding activation signal from its operatively linked extracellular domain. In some embodiments, the transmembrane domain contains one or more mutations that induce / promote homodimerization.

[0189] In certain contexts, the transmembrane domain of the eCR endows the eCR with a functional conformation that allows for self-activation, such as allowing downstream intracellular domains to orient themselves relative to each other in a manner conducive to signal transduction, for example, allowing Janus kinases associated with the intracellular domains to interact with and cross-activate each other. In certain aspects, the transmembrane domain contains one or more mutations that allow the transmembrane and intracellular domains to function in a non-transient binding manner when they do not naturally do so. In at least some aspects, the mutations enable the transmembrane and extracellular domains to artificially homodimerize. For example, the transmembrane domain may contain one or more mutations that allow for the homodimerization of two separate molecules, each containing a transmembrane domain and at least one intracellular domain, wherein homodimerization of these molecules does not occur in nature. In specific embodiments, for example, one or more mutations in the transmembrane domain induce structural torsion of the transmembrane and intracellular domains of the receptor homodimer to form a self-activating helical structure. In specific embodiments, one or more mutations in the transmembrane domain induce partial or complete helical torsion of the receptor molecule (or one or more components thereof) about a vertical axis. The ability to determine whether a specific mutation will induce a structural conformation that enables the self-activation of transmembrane and intracellular domains can be determined by conventional methods, such as measuring STAT3 or STAT5 phosphorylation after cell growth carrying the specific receptor being tested in the absence of growth factors.

[0190] In certain embodiments, transmembrane domain components having one or more such gain-of-function mutations can be used in the methods and compositions of this disclosure. For example, the mutation can be a substitution, insertion, deletion, or a combination thereof. In specific embodiments, one or more mutations include at least one cysteine ​​and / or at least one proline. In some cases, the transmembrane domain utilizes mutations identified in cancer patients as gain-of-function mutations in the transmembrane domain. In at least some cases, the mutant form includes a cysteine ​​insertion that induces disulfide bond formation in the transmembrane domain. However, in other embodiments, one or more mutations lack the cysteine ​​insertion. For example, transmembrane domain derivatives that do not have a cysteine ​​insertion (and therefore no disulfide bond) but still signal and are constitutively active can be used, for example, because the mutation causes a conformational change in the transmembrane domain compared to its native form, thereby allowing induced signal transduction. For example, the insertion of amino acids such as proline generates a “knot” in the transmembrane domain, which induces signal transduction (see, for example, Shochat et al., 2011, J Exp Med. 2011 May 9; 208(5):901-8; Zenatti et al., 2011, Nat Genet. 2011 Sep 4; 43(10):932-9).

[0191] In specific embodiments, the transmembrane domain of the eCR is derived from the IL-7Rα receptor, and the mutation in the transmembrane domain is in the sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO:30). In some embodiments, the mutation is or includes inserting one or more cysteine ​​residues and / or one or more proline residues into the amino acid sequence of SEQ ID NO:30, wherein the mutation enables or promotes homodimerization of the receptor. In some cases, the mutation includes inserting a trimer peptide of cysteine, proline, and threonine (CPT) into the transmembrane domain. This mutation confers disulfide bond formation between the -SH (thiol) groups of the cysteine ​​residues of two molecules (for example, two IL7RP2 receptor α chains), allowing the formation of a homodimer between them (in a specific embodiment, the proline immediately following the cysteine ​​helps to steer the homodimer in the correct direction). In specific embodiments, the threonine inserted into the CPT is not threonine but another amino acid, and in at least certain cases, the other amino acid is or is not cysteine ​​or proline.

[0192] In embodiments in which one or more amino acids are inserted into SEQ ID NO:30 for use as a receptor, the insertion may be between any two amino acids in SEQ ID NO:30. In specific embodiments, the insertion is located after amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 in SEQ ID NO:30. Examples of mutant TM sequences used in exemplary eCR constructs described herein are shown below, wherein the sequence is mutated by adding an underlined sequence: m1-IL7Rα-TM: PILLTCPTISILSFFSVALLVILACVLW (SEQ ID NO:63). Mutant TM sequences in other constitutively active IL-7Rα are shown in Table 1.

[0193] Table 1

[0194]

[0195] In some embodiments of the present invention, the eCR comprises, from the N-terminus to the C-terminus, the extracellular domain of CD34, the mutated IL-7Rα transmembrane region, and the IL-7Rα intracellular domain; the amino acid sequence of the mutated IL-7Rα transmembrane region is shown as any amino acid sequence in SEQ ID NO:63 or SEQ ID NO:72-93.

[0196] In some embodiments of the present invention, the eCR comprises an extracellular domain of IL-7Rα, a mutated IL-7Rα transmembrane region, and an intracellular domain of IL-7Rα from the N-terminus to the C-terminus; the amino acid sequence of the mutated IL-7Rα transmembrane region is shown in any of the amino acid sequences in SEQ ID NO:63 or SEQ ID NO:72-93.

[0197] In a specific embodiment, one or more transmembrane domain mutations structurally alter the dimerized α38 chain (for example IL-7Rα) to orient it so that the bound Janus kinase is now close, thereby allowing cross-phosphorylation and activation; in a specific embodiment, this structural change originates from a twisting of the dimerized chain (Shochat et al., 2011; Durum, 2014).

[0198] In certain embodiments, the transmembrane domain contains one or more mutations compared to its corresponding wild-type component, and thus has a certain percentage identity with the wild type. In certain cases, the transmembrane domain is at least 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the corresponding wild-type transmembrane domain.

[0199] The transmembrane domain of the eCR can have any suitable length, but in specific embodiments, its length is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids.

[0200] "Intracellular Domains of eCRs": eCRs / constitutively active cytokine receptors contain one or more intracellular domains. In some cases, the intracellular domains originate from the same molecule as the transmembrane domains, but in others this is not the case. In specific embodiments, the intracellular domains originate from cytokine receptors, including immunostimulatory cytokine receptors. In specific embodiments, the cytokine receptors function in signaling pathways including STAT5, STAT3, etc. Immunostimulatory cytokine intracellular domains of receptors that can be used in this disclosure include, for example, IL-7Rα, CD122 (a common β receptor for IL-2 and IL-15), IL-21Rα, IL-23Rα, IL-12Rα, and IL-6R.

[0201] In some embodiments, intracellular domains are selected based on the desired downstream pathways. For example, intracellular domains may be selected based on the need to transmit signals via JAK1, STAT5, STAT4, JAK3, STAT3, etc.

[0202] In some cases, signaling pathways involve STAT5. STAT5 is a major downstream signaling node of the immunostimulatory cytokines IL-15 and IL-7, both of which are known to activate T cells in the context of immunotherapy. Several publications have used preclinical models to demonstrate that bypassing cytokine and cytokine receptor interactions and directly activating STAT5 (using constitutively active STAT5 mutants) enhances CD8+ T cell function through increased in vivo persistence and enhanced in vivo antitumor efficacy. The same concept can be extended to activating other STAT proteins downstream of known immunostimulatory cytokines, such as STAT4, which is downstream of IL-12.

[0203] In certain embodiments, the intracellular domain of the eCR comprises an intracellular domain derived from the IL7-Rα chain, which may or may not contain one or more mutations. In some cases, it is operatively linked to a transmembrane domain containing mutations that allow for or promote homodimerization.

[0204] In some embodiments, the intracellular domain of the eCR has a certain length. In specific embodiments, the length of the intracellular domain is 70-250 amino acids, 70-225 amino acids, 70-200 amino acids, 70-175 amino acids, 70-150 amino acids, 70-125 amino acids, 70-100 amino acids, 80-250 amino acids, 80-225 amino acids, 80-200 amino acids, 80-175 amino acids, 80-150 amino acids, 80-100 amino acids, 90-250 amino acids, 90-225 amino acids, 90-200 amino acids, 90-175 amino acids, 90-150 amino acids, 90-125 amino acids, 90-100 amino acids, 100 -250 amino acids, 100-225 amino acids, 100-200 amino acids, 100-175 amino acids, 100-150 amino acids, 100-125 amino acids, 125-250 amino acids, 125-225 amino acids, 125-200 amino acids, 125-175 amino acids, 125-150 amino acids, 150-250 amino acids, 150-225 amino acids, 150-200 amino acids, 150-175 amino acids, 175-250 amino acids, 175-225 amino acids, 175-200 amino acids, 200-250 amino acids, 200-225 amino acids, etc. In some embodiments, these fragments of certain lengths retain signal transduction activity.

[0205] "Activation molecule" is used in this article and includes, but is not limited to, molecules that can bind to or interact directly or indirectly with T cells to induce T cell activation. For example, TCR-CD3 binding molecules that can bind to the TCR-CD3 complex and provide an initial signal for T cell activation include, but are not limited to, anti-CD3 antibodies or their antigen-binding fragments.

[0206] "Costimulatory molecule" 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.

[0207] CD28 is one of the proteins expressed on T cells, providing the co-stimulatory signal required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 can provide a potent signal for the production of various interleukins, particularly IL-6. CD134 (also known as OX40), a member of the TNFR superfamily of receptors, is not constitutively expressed on resting naive T cells, unlike CD28. OX40 is a secondary co-stimulatory molecule, expressed 24 to 72 hours after activation; its ligand OX40L is also not expressed on resting antigen-presenting cells, but is expressed after their activation. OX40 expression depends on complete T cell activation; in the absence of CD28, OX40 expression is delayed and its level is four-fold lower.

[0208] CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family. CD137 can be expressed by activated T cells, but its expression level is higher on CD8 T cells than on CD4 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.

[0209] Exemplary examples 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, or epitopes, and one or more of anti-CD28 antibodies or their antigen-binding fragments. In some embodiments, exemplarily, "activating molecules" include, but are not limited to, the binding domains of OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0210] “Inhibition”: When referring to the ability of the viral glycoprotein variant or modifier to specifically bind to its receptor being “inhibited,” the term “inhibition” includes both the complete elimination of the ability of the viral glycoprotein variant or modifier to specifically bind to its receptor and a significant reduction in the binding ability. In a specific embodiment, “significant reduction” means relative to the wild-type viral glycoprotein; “reduction” is selected from reductions of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2%, and at least 1%.

[0211] “Antigen”: The terms “antigen” and “Ag” refer to molecules capable of inducing an immune response. An induced immune response may include antibody production and / or activation of specific immune-competent cells. Macromolecules, including proteins, glycoproteins, and glycolipids, can be used as antigens. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, antigens do not need to be (i) encoded solely by the full-length nucleotide sequence of a gene or (ii) entirely encoded by a gene. Antigens can be generated or synthesized, or they can be derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0212] 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.

[0213] Compared to pseudolentiviral vector packaging systems, pseudoretroviral vector packaging systems typically do not contain the Rev plasmid. This is because the genomic RNA from retroviruses such as Moloney Murine Leukemia Virus (MMLV) can be naturally transported from the nucleus to the cytoplasm for translation and assembly, thus eliminating the need for specific nuclear export mechanisms such as Rev proteins. Pseudoretroviral vector packaging systems typically contain one transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgenic sequence contained in the transfer plasmid is flanked by long terminal repeats (LTRs), which facilitate the integration of the transfer plasmid sequence into the host genome. Generally, during viral transduction, sequences between and including the LTRs are integrated into the host genome. The backbone genomes of MMLV or Murine Stem Cell Virus (MSCV), containing their respective LTRs, are often used to construct the transfer plasmid in pseudoretroviral vector packaging systems. GagPol packaging plasmids contain the Gag and Pol genes; envelope plasmids typically contain polynucleotides encoding viral glycoproteins such as VSV-G or Cocal-G. In some embodiments, packaging plasmids containing the pol gene contain mutations that disable integrase (disabling mutations) and / or transfer plasmids / master plasmids containing transgenes contain LTRs of the lentiviral / retroviral backbone genome that contain CA mutations.

[0214] In some embodiments, production cells are transfected with a defined ratio of transfer plasmid and envelope plasmid. In some embodiments, the ratio of the plasmids is determined by quality and is not particularly limited as long as it can package a biologically active vector. In some embodiments, the defined ratio of transfer plasmid to envelope plasmid is from 1:1 to 9:2; in some embodiments of the invention, the envelope plasmid may contain nucleic acid encoding a target molecule.

[0215] 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 1:1:1:1 to 9:4:2:2; in some embodiments of the invention, the envelope plasmid may contain nucleic acid encoding a target molecule.

[0216] In some embodiments, the envelope plasmid comprises 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 comprises 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 embodiments, the viral 2A peptide is porcine chevron virus-1 (P2A), Thosea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof.

[0217] The use of lentiviral / retroviral vectors or vector packaging systems relies on "packaging cell lines." Generally, a packaging cell line is a cell line whose cells, upon introduction of a transfer plasmid or one or more packaging plasmids, are capable of producing lentiviral vectors, retroviral vectors, or vectors (including LVV and NIL) that are non-self-replicating and capable of infecting / transducing target cells. An overview is provided in Cold Spring Harbour Laboratory Press, 1997, page 447, by JM Coffin, SM Hughes, et al.

[0218] For example, various plasmids can be introduced into packaging cell lines using transfection methods including chemically mediated transfection, physically mediated transfection, or biologically mediated transfection. For instance, chemically mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-glucan, or PEI (polyethylenimine transfection reagent), while physically mediated transfection methods include transfection methods such as electroporation.

[0219] The packaging cells can be genetically engineered to improve the efficiency of target cell transduction by the vectors (including LVV and NIL) disclosed in this invention in other ways; said other ways include, but are not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0220] Production / host / packaging cells that can be used to prepare the vectors (including LVV and NIL) disclosed in this invention include human embryonic kidney (HEK) 293 cells and their derivatives. Production cells can be adherent cell lines such as HEK293T production cells, or suspension cell lines such as HEK293T / 17SF production cells.

[0221] For example, the packaging cells / host cells are selected from CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, and 211 cells; preferably, the packaging cells / host cells are HEK-293T cells.

[0222] "Retrovirus" and "Retroviral Vector": These are the terms Retrovirus and Retroviral Vector. A "retrovirus" is an RNA virus containing a single-stranded, positive-signal RNA molecule. Retroviruses contain reverse transcriptase and integrase. Upon entering a target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the integrase integrates the DNA molecule into the host cell's genome. The sequence derived from the retrovirus after integration into the host cell's genome is called a provirus (e.g., a proviral sequence or proviral vector). A retroviral vector typically refers to a pseudotyped retroviral vector derived from a retrovirus, exemplarily from γ-retrovirus. Unlike lentiviral vectors that can transduce both dividing and non-dividing cells, retroviral vectors can only transduce dividing cells, and the amount of exogenous transgenes they can carry is generally relatively small. For a comparison and discussion of lentiviral vectors and retroviral vectors, please see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, KK, Vorburger, SA, Swisher, SG(eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0223] For explanations of other terms used in this article, please refer to patents WO 2025209590A and WO 2025011662A.

[0224] All publications, documents, and patents mentioned herein are hereby incorporated in their entirety by reference, as are each publication, document, or patent not specifically and individually indicated to be incorporated 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

[0225] Figure 1: Map of enveloped plasmids.

[0226] Figure 2: Transfer plasmid map.

[0227] Figure 3: Flow cytometry results of CAR-19 expression efficiency detected on Day 5 after the lentiviral vector V1 and the control group CTR1V transduced non-activated PBMCs of Donor1 in Example 1.

[0228] Figure 4: A bar chart showing the detection results of CAR-T cells prepared by non-activated PBMCs of Donor1 transduced by the lentiviral vector V1 and the control group CTR1V killing Nalm-6 cells and releasing cytokine IFN-γ in Example 2.

[0229] Figure 5: A graph showing the detection results of the killing efficiency of CAR-T cells prepared by transducing Donor1 with the lentiviral vector V1 and the control group CTR1V in Example 2, which continuously kills Nalm-6 cells.

[0230] Figure 6: A graph showing the fold increase of T cells in non-activated PBMCs transduced with Donor1 by the lentiviral vector V1 and the control group CTR1V in Example 2.

[0231] Figure 7: In Example 3, the CAR-T cells prepared by transducing non-activated PBMCs with the lentiviral vectors V6 and V7 showed sustained CD20 killing activity. + A graph showing the results of the Raji cell killing efficiency test. Detailed Implementation

[0232] The following detailed description of the invention's concept and technical effects, in conjunction with specific embodiments, provides a clear and complete understanding of the invention's technical solution, the technical problems it solves, and its beneficial effects. Obviously, the described embodiments are only some, not all, of the embodiments of the invention; other embodiments obtained by those skilled in the art based on the embodiments of the invention without creative effort are all within the scope of protection of the invention. Experimental methods in the following embodiments that do not specify specific conditions are performed according to conventional methods and conditions known in the art, or according to the product instructions. Reagents and raw materials not specified in this invention are commercially available. Example 1

[0233] 1. Construction of engineered cytokine receptor eCR1:

[0234] eCR1 was constructed, wherein the structure of eCR1 from N-terminus to C-terminus sequentially comprises: CD34 signal peptide, CD34 extracellular domain, mutated IL-7Rα transmembrane region, and IL-7Rα intracellular domain;

[0235] (1) The amino acid sequence of the CD34 signal peptide is shown in SEQ ID NO:61;

[0236] (2) The amino acid sequence of the extracellular domain of CD34 is shown in SEQ ID NO:62;

[0237] (3) The amino acid sequence of the transmembrane region of the mutated IL-7Rα (m1-IL7Rα-TM) is shown in SEQ ID NO:63;

[0238] (4) The amino acid sequence of the intracellular domain of the IL-7Rα is shown in SEQ ID NO:64.

[0239] CD34 signal peptide:

[0240] MLVRRGARAGPRMPRGWTALCLLSLLPSGFM (SEQ ID NO:61)

[0241] CD34 extracellular domain:

[0242] SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTTSLATSPTKPYT SSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT (SEQ ID NO:62)

[0243] Mutated IL-7Rα transmembrane region:

[0244] PILLTCPTISILSFFSVALLVILACVLW (SEQ ID NO:63)

[0245] Wild-type IL-7Rα transmembrane region:

[0246] PILLTISILSFFSVALLVILACVLW(SEQ ID NO:65)

[0247] Compared to the wild-type IL-7Rα transmembrane region (SEQ ID NO:65), the mutated IL-7Rα transmembrane region, m1-IL7Rα-TM (SEQ ID NO:63), contains an insertion of the amino acid sequence CPT (cysteine-proline-threonine) between amino acids 5 (T) and 6 (I) of SEQ ID NO:65.

[0248] IL-7Rα intracellular domain:

[0249] KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ (SEQ ID NO:64)

[0250] 2. Constructing a targeted molecular membrane expression system for anti-CD3 antibody × anti-CD28 antibody:

[0251] In this example, a membrane-expressing anti-CD3 antibody × anti-CD28 antibody (biantibody) is constructed. The structure of the membrane-expressing biantibody from the N-terminus to the C-terminus is as follows: anti-CD3 antibody region (CD8α signal peptide, the UCHT1-scFv, CD8α hinge region, CD8α transmembrane region), FT2A peptide, and co-stimulatory region (CD8α signal peptide, the 15E8-scFv, CD8α hinge region, CD8α transmembrane region).

[0252] (1) The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO:27;

[0253] (2) The amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO:35;

[0254] (3) The amino acid sequence of the hinge region of CD8α is shown in SEQ ID NO:28;

[0255] (4) The amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO:29;

[0256] (5) The amino acid sequence of the FT2A peptide is shown in SEQ ID NO:34;

[0257] (6) The amino acid sequence of the 15E8-scFv is shown in SEQ ID NO:44.

[0258] 3. Constructing CAR-19:

[0259] In this embodiment, a chimeric antigen receptor CAR-19 targeting CD19 is constructed, the structure of which from the N-terminus to the C-terminus is as follows: the CD8α signal peptide, the antigen-binding region targeting CD19, the CD8α hinge region, the CD8α transmembrane region, the 4-1BB co-stimulatory signal transduction domain, and the CD3ζ intracellular signal transduction domain; wherein, the antigen-binding region targeting CD19 is a scFv derived from FMC63 (FMC63-scFv), the FMC63-scFv contains the VH region and VL region of FMC-63, and the VH region is connected to the VL region through the (G4S)3 linker peptide;

[0260] The amino acid sequence of the CAR-19 is shown in SEQ ID NO:33.

[0261] (1) The amino acid sequence of the VH region of FMC-63 is shown in SEQ ID NO:18; the amino acid sequence of the VL region of FMC-63 is shown in SEQ ID NO:22; the amino acid sequences of the HCDR1-3 regions of FMC-63 are shown in SEQ ID NO:19-21; the amino acid sequences of the LCDR1-3 regions of FMC-63 are shown in SEQ ID NO:23-25; and the amino acid sequence of FMC63-scFv is shown in SEQ ID NO:26.

[0262] (2) The amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:30;

[0263] (3) The amino acid sequence of the intracellular signal transduction domain of CD3ζ is shown in SEQ ID NO:31;

[0264] (4) The amino acid sequence of the (G4S)3 linker peptide is shown in SEQ ID NO:32.

[0265] 4. Packaging pseudo-lentiviral vector V1:

[0266] The packaging virus envelope contains the membrane expressing bispecific antibodies and mutant VSV-G1, and contains a lentiviral vector V1 encoding a first exogenous polynucleotide encoding the eCR1 and a second exogenous polynucleotide encoding the CAR-19;

[0267] The UCHT1-scFv can specifically bind to human CD3ε / CD3E (Uniprot NO. P07766).

[0268] The 15E8-scFv can specifically bind to human CD28 (Uniprot NO. P10747).

[0269] Prepare the following four plasmids: envelope plasmid 1, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and transfer plasmid 1;

[0270] The envelope plasmid 1 (as shown in Figure 1) comprises: a polynucleotide encoding mutant VSV-G1 and a polynucleotide encoding the membrane-expressing bispecific antibody; the mutant VSV-G1 comprises the amino acid sequence shown in SEQ ID NO:7; relative to SEQ ID NO:1, SEQ ID NO:7 comprises R354Q, T214N, and T352A; the amino acid sequence of the mutant VSV-G1 containing the VSV-G signal peptide is shown in SEQ ID NO:10.

[0271] The transfer plasmid 1 (as shown in Figure 2) comprises: a polynucleotide encoding the CAR-19 (a second exogenous polynucleotide) and a polynucleotide encoding the eCR1 (a first exogenous polynucleotide); the second exogenous polynucleotide and the first exogenous polynucleotide can be operatively linked by a polynucleotide encoding a self-cleaving peptide (e.g., the FT2A peptide used in this embodiment);

[0272] The above-mentioned envelope plasmid 1 and transfer plasmid 1 were synthesized using conventional molecular cloning methods.

[0273] 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.

[0274] 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:

[0275] 9 µg of transfer plasmid 1, 4 µg of pMDLg / pRRE packaging plasmid, 2 µg of pRSV-REV packaging plasmid, and 2 µg of the envelope plasmid 1 were added to 1 mL of Opti-MEM medium. After shaking well, 64 µL of PEI reagent was added, and the mixture was incubated for 10 minutes. Then, it was added to the HEK-293T cell culture system. The medium was replaced after 6 hours. 48 hours after transfection, the supernatant was collected, filtered through a 0.45 µm filter membrane, centrifuged at 50,000 g for 2.5 h, the supernatant was discarded, and the lentiviral vector V1 was resuspended in 200 µL of F12 medium and stored at -80 °C.

[0276] Opti-MEM alpha serum-depleted medium, brand: GIBCO, catalog number: #SP0272; DMEM: brand: GIBCO, catalog number: #C12430500BT; FBS: brand: EXCELL, catalog number: #FSP500; F12 medium: brand: GIBCO, catalog number: #C11330500BT; Syringe filter: brand: SORFA, catalog number: #622120.

[0277] 5. Packaging control group CTR1V: The control group CTR1V was packaged using the same method as described above for packaging the lentiviral vector V1. Compared to the lentiviral vector V1, the control group CTR1V does not contain the polynucleotide encoding the eCR1, but only contains the polynucleotide encoding the CAR-19. Example 2

[0278] 1. Using the lentiviral vectors V1 and CTR1V prepared in Example 1 to transduce non-activated PBMCs, the expression efficiency of CAR-19 was detected:

[0279] Resuscitate inactive PBMCs cryopreserved from Donor1 (healthy individual) and take 2 groups of 1×10 5 Personal non-activated PBMCs (methods for reviving frozen PBMCs are well known to those skilled in the art);

[0280] Each group of inactive human PBMCs was resuspended in 200 μL of PBMC culture medium (PBMC culture system); the PBMC culture medium was XVT medium (IRVINE, #91154) containing IL-7 (Essential, #11821-HNAE) and IL-15 (Essential, #10360-H07E) at a final concentration of 20 ng / mL.

[0281] On Day 0, at MOI=1, the lentiviral vector V1 and the control group CTR1V were added to the PBMCs culture system of each group, and the cells were cultured at 37°C and 5% CO2 to prepare experimental CAR-T cells expressing CAR-19 and eCR1, and control CAR-T cells expressing only CAR-19 and not eCR1.

[0282] On Day 5, flow cytometry was used to detect the expression of CAR-19 in T cells in the two PBMC culture systems. The results are shown in Figure 3.

[0283] As shown in Figure 3, compared to the control group CAR-T cells that only expressed CAR-19 and did not express the eCR1, the experimental group CAR-T cells that simultaneously expressed CAR-19 and the eCR1 showed higher CAR-19 expression efficiency. This demonstrates that the lentiviral vector V1 transduces inactive PBMCs to express CAR-19 with significantly higher efficiency than the control group CTR1V.

[0284] 2. Detection of in vitro killing and T cell expansion:

[0285] Day 0, 2×10 5 One Donor1 person inactive PBMCs and 1×10 5 CD19 + Nalm-6 cells (human B-lymphoblastic leukemia cells) were mixed; three groups of the mixed cells were prepared, and each group of mixed cells was resuspended in 200µL of 1640 medium containing 10% FBS; according to MOI=1, the lentiviral vector V1 and the control group CTR1V were added to two of the mixed cells respectively.

[0286] On Day 5, cell supernatants from each group were collected, and the release of cytokine IFN-γ was detected using ELISA. The P-values ​​were compared and calculated: P = 0.018 < 0.05; the bar chart of the detection results is shown in Figure 4.

[0287] As shown in Figure 4, the CAR-T cells prepared by transducing PBMCs with the lentiviral vector V1 released IFN-γ from Nalm-6 cells when killing them were significantly lower than those in the control group CTR1V.

[0288] Starting from Day 5, add 3×10 to each group of mixed cells every 2 days. 5 We collected 10 Nalm-6 cells and tested the killing efficiency of Nalm-6 cells in each group of mixed cells. The results are shown in Figure 5.

[0289] As shown in Figure 5, on Days 5, 7, 9 and 11, the killing efficiency of CAR-T cells prepared by transducing human PBMCs with the lentiviral vector V1 and the control group CTR1V was comparable in killing Nalm-6 cells.

[0290] However, on Day 13 and 15, the CAR-T cells in the experimental group prepared by transducing human PBMCs with the lentiviral vector V1 and expressing both CAR-19 and eCR1 showed better killing efficiency than the control group CAR-T cells prepared by transducing human PBMCs with CTR1V and expressing only CAR-19 and not eCR1; especially on Day 15, the killing efficiency of the experimental group CAR-T cells was significantly better than that of the control group CAR-T cells.

[0291] Meanwhile, on Days 0, 5, 7, and 9, 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 was calculated, and the T cell proliferation was assessed. The results are shown in Figure 6.

[0292] As shown in Figure 6, the CAR-T cells prepared by transducing human PBMCs with the lentiviral vector V1 had significantly better expansion efficiency and persistence than the CAR-T cells prepared by transducing human PBMCs with the control group CTR1V.

[0293] In summary, compared with the control group CTR1V, CAR-T cells prepared by transducing PBMCs with the lentiviral vector V1 have enhanced killing ability, expansion ability and persistence, while reducing cytokine release, making them more suitable for CAR-T cell therapy, especially in vivo CAR-T cell therapy. Example 3

[0294] Two sets of lentiviral vectors were prepared according to Example 1, the difference being that the transfer plasmid of group 1 encodes CAR-20 but not eCR; the transfer plasmid of group 2 encodes both CAR-20 and eCR, and is operatively linked via the FT2A peptide. The amino acid sequence of CAR-20 is shown in SEQ ID NO:95 (CAR molecule targeting CD20).

[0295] Then, on Day 0, 2×10 5 Personal inactive PBMCs and 1×10 5 CD20 + Raji cell hybridization; prepare 3 groups of hybrid cells, and resuspend each group of hybrid cells in 200µL of 1640 medium containing 10% FBS; according to MOI=5, add the lentiviral vector from group 1 (V6) and group 2 (V7) to 2 of the hybrid cells respectively; starting from Day 5, add 1×10 to each group of hybrid cells every 2 days. 5 Figure 7 shows the results of killing Raji cells and detecting the killing efficiency of Raji cells in each group of mixed cells.

[0296] As shown in Figure 7, on Days 5, 7, and 9, the killing efficiency of CAR-CD20 T cells prepared by transducing human PBMCs with lentiviral vectors V6 and V7 in killing Raji cells was comparable. However, by Day 11, a significant difference in killing efficiency began to appear between the two groups, with V7 being higher than V6. This indicates that when constructing CAR-T cells targeting CD20, adding the membrane-expressed eCR1 component can prolong the killing effect of CAR-T cells, a trend consistent with that in Example 2.

Claims

1. A type of particle, characterized in that, The particles comprise: a) Viral glycoproteins or their variants, or functional fragments thereof, or their modified forms; b) One or more targeting molecules that can specifically bind to antigens on the surface of T cells; c) A first exogenous polynucleotide encoding an engineered cytokine receptor (eCR); and d) The second exogenous polynucleotide encodes a chimeric antigen receptor (CAR).

2. The particles according to claim 1, characterized in that, The eCR includes: a) One or more intracellular domains of IL-7Rα or functional fragments thereof; b) IL-7Rα transmembrane domain; and, c) One or more extracellular domains, said extracellular domains comprising the IL-7Rα extracellular domain, or a functional fragment thereof, or a derivative thereof.

3. The particles according to claim 2, characterized in that, The amino acid sequence of the intracellular domain of the IL-7Rα or its functional fragment has at least 85% identity with SEQ ID NO:

64.

4. The particles according to claim 3, characterized in that, The transmembrane domain of the eCR is self-oligomery.

5. The particles according to claim 4, characterized in that, One or more mutations in the IL-7Rα transmembrane domain enable the eCR to homodimerize through transmembrane and intracellular domain components.

6. The particles according to claim 5, characterized in that, One or more mutations in the IL-7Rα transmembrane domain enable structural torsion of the transmembrane and intracellular domains of the eCR, thereby directing the Janus kinase associated with the intracellular domain to allow cross-phosphorylation and activation.

7. The particles according to claim 6, characterized in that, The mutated IL-7Rα transmembrane domain contains one or more mutations that enable the transmembrane and intracellular domains to twist together in a helical manner.

8. The particles according to claim 7, characterized in that, The mutated IL-7Rα transmembrane domain contains one or more mutations located in the sequence shown in SEQ ID NO:

65.

9. The particles according to claim 8, characterized in that, The mutation is to introduce at least one cysteine ​​C into the transmembrane domain of the eCR.

10. The particles according to claim 8, characterized in that, The mutation involves introducing proline P into the transmembrane domain of the eCR.

11. The particles according to claim 8, characterized in that, The transmembrane domain of the eCR has at least 85% identity with any amino acid sequence in SEQ ID NO:63 or SEQ ID NO:72-93.

12. The particles according to any one of claims 2-11, characterized in that, The transmembrane domain of the eCR is 21 to 33 amino acids in length.

13. The particles according to any one of claims 2-11, characterized in that, The extracellular domain of the eCR is the IL-7Rα extracellular domain, or a functional fragment thereof, or a derivative thereof.

14. The particles according to claim 13, characterized in that, The extracellular domain of the eCR has at least 85% identity with SEQ ID NO:

67.

15. The particles according to claim 14, characterized in that, The amino acid sequence of the eCR has at least 85% identity with SEQ ID NO:

70.

16. The particles according to any one of claims 2-11, characterized in that, The extracellular domain of the eCR is not the extracellular domain of IL-7Rα.

17. The particles according to any one of claims 2-11, characterized in that, The extracellular domain of the eCR is not derived from cytokine receptors.

18. The particles according to any one of claims 2-11, characterized in that, The extracellular domain of the eCR is a masquerade receptor lacking signal transduction activity.

19. The particles according to any one of claims 2-11, characterized in that, The extracellular domain of the eCR is the extracellular domain of CD34.

20. The particles according to claim 19, characterized in that, The amino acid sequence of the eCR has at least 85% identity with SEQ ID NO:

68.

21. The particles according to claim 18, characterized in that, The masquerading receptor comprises a constitutively active cytokine receptor, and the extracellular domain of the eCR is or comprises an extracellular domain derived from PD-1 or B7.

22. The particles according to any one of claims 2-11, characterized in that, The extracellular domain of the eCR is derived from IL-7Rα, PD-1CD30, HER2, EGFR, CD19, CD34, TGF-βR, IL-4R, IL-13Rα1, IL-13Rα2, IL-8R, IL-10R, LAG3, TIGIT, CTLA4, FAS, CD19, CD27, CD28, CD52, CD134, CD137, HER2, EGFR, or NGFR.

23. The particles according to any one of claims 1-22, characterized in that, The CAR includes an antigen-binding region, a transmembrane region, and an intracellular signal transduction domain.

24. The particles according to claim 23, characterized in that, The antigen-binding region of the CAR can specifically bind to cancer-associated antigens; preferably, the cancer-associated antigens are selected from: One or more of them.

25. The particles according to claim 24, characterized in that, The cancer-related antigens mentioned are hematologic cancer-related antigens; the hematologic cancers mentioned are selected from: non-Hodgkin's lymphoma, acute B-cell lymphoblastic leukemia, chronic lymphocytic leukemia, large B-cell lymphoma, LBCL unsuitable for transplantation, diffuse LBCL, high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, precursor B-cell lymphoma / leukemia, Burkitt lymphoma, multiple myeloma, acute myeloid leukemia, primary plasma cell leukemia, peripheral T-cell lymphoma, NK / T-cell lymphoma. The blood cancer-associated antigen is selected from one or more of the following: anaplastic large cell lymphoma, intestinal T-cell lymphoma, T-large granular lymphocytic leukemia, and germinal center T-cell lymphoma; preferably, the blood cancer-associated 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.

26. The particles according to claim 25, 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's lymphoma, acute B-cell lymphoblastic leukemia, chronic lymphocytic leukemia, large B-cell lymphoma, LBCL unsuitable for transplantation, diffuse LBCL, high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, precursor B-cell lymphoma / leukemia, and Burkitt lymphoma; preferably, the B-cell malignancy-related antigen is selected from one or more of CD19, CD20, CD22, CD79A, CD79B, CD30, CD37, CD38, CD52, CD123, CRLF2, BCMA, and ROR1.

27. The particles according to claim 24, 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 cancer-associated antigen is selected from one or more of the following: ROR1, MSLN, MUC1, HER2, CEA, Nectin-4, Claudin18.2, and GCC.

28. The particles according to any one of claims 23-27, characterized in that, The CAR also includes a connectivity domain and a co-stimulatory signal transduction domain.

29. The particles according to any one of claims 1-28, characterized in that, The viral glycoproteins are selected from vesicular stomatitis virus strains, Nipah virus glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein, gibberish leukemia virus glycoprotein, bitropic murine leukemia virus glycoprotein, feline endogenous virus glycoprotein, avian plague virus glycoprotein, Ebola virus glycoprotein, and T-cell choroid plexus meningitis virus 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.

30. The particles according to claim 29, characterized in that, The viral glycoprotein modifier has fewer binding sites available for specific binding to its receptor.

31. The particles according to claim 29, characterized in that, The viral glycoprotein modifier has specifically bound to its receptor and / or its antibody.

32. The particles according to claim 30, characterized in that, The viral glycoprotein variant contains a first mutation that inhibits the ability of the viral glycoprotein variant to specifically bind to its receptor relative to viral glycoproteins that do not contain the first mutation.

33. The particles according to any one of claims 30 to 32, 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 VSV-G variant thereof; or b) Vesicular stomatitis virus genus Cocal strain glycoprotein Cocal-G, or a variant thereof, or a functional fragment thereof, or a modified Cocal-G thereof; The viral glycoprotein receptor is LDL-R.

34. The particles according to claim 33, characterized in that, Compared to wild-type VSV-G or wild-type Cocal-G, the VSV-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 / or 354 in SEQ ID NO:1 or SEQ ID NO:2; deletion of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354 and / or 345-353; or, 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, substitutions or deletions of amino acids at positions 8, 9, 10, 47, 50, 51, 183, 179, 180, 182, 184, 209, 347, 350, 352, 353 and / or 354, and deletions of amino acids at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354 and / or 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) Deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354, substitution of Y209, and substitution of I or V182 located 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, the amino acid deletions at positions 331-364, amino acid deletions at positions 344-354, substitutions for K47, deletions of K47, substitutions for R354, substitutions for Y209, and substitutions for I182 are located at positions corresponding to SEQ ID NO:1 or SEQ ID NO:

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

2.

35. The particles according to claim 33, characterized in that, (a) The amino acid sequence of the VSV-G variant has at least 85% identity with SEQ ID NO:3; or (b) The amino acid sequence of the Cocal-G variant has at least 85% identity with SEQ ID NO:

11.

36. The particles according to claim 33, characterized in that, The modifications contained in the VSV-G or Cocal-G modifiers reduce the number of binding sites available for specific binding to their receptor LDL-R.

37. The particles according to claim 33, characterized in that, The VSV-G or Cocal-G modifier has specifically bound its receptor LDL-R and / or anti-VSV-G antibody.

38. The particles according to any one of claims 29-37, characterized in that, The viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, contains a second mutation that enhances its ability to antagonize complement inactivation, the enhanced ability to antagonize complement inactivation being relative to a viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, that does not contain the second mutation.

39. The particles according to claim 38, characterized in that, The VSV-G or Cocal-G, or a variant thereof, or a functional fragment thereof, or a modified version thereof, contains a second mutation selected from one or more of the following site mutations: The amino acid located at position 214 of SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at position 214 of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment; or 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; or The 50th amino acid located in SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at the 50th amino acid of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment; or The amino acid located at the 146th position of SEQ ID NO:1 or SEQ ID NO:2, or the amino acid located at the equivalent of the 146th position of 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.

40. The particles according to claim 39, characterized in that, The VSV-G or Cocal-G, or a variant thereof, or a functional fragment thereof, or a modified version thereof, wherein the second mutation comprises a combination of mutations at the following sites: 1) Substitution of (i) T / K214 and T352 located in SEQ ID NO:1 or SEQ ID NO:2, or (ii) substitution of T / K214, T352, K50 and S146; or 2) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, the substitutions located at (i) T / K214 and T352, or (ii) T / K214, T352, K50 and S146, which are equivalent to 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) (i) T / K214N and T352A located in SEQ ID NO:1 or SEQ ID NO:2, or (ii) T / K214N, T352A, K50T and S146T; or 2) After the best global alignment of SEQ ID NO:1 or SEQ ID NO:2, the (i) T / K214N and T352A or (ii) T / K214N, T352A, K50T and S146T are located at the equivalent of SEQ ID NO:1 or SEQ ID NO:

2.

41. The particles according to any one of claims 1-40, characterized in that, The antigens on the surface of the T cells are selected from one or more of the following: CD2, CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD57, CD71, CD69, CD94, CD95, 4-1BB, CD103, CD122, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), PD-1, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, IL6ST, P2RX7, TIGIT, TIM-3, and LAG-3; Preferably, the antigen on the surface of the T cells is selected from one or more of the following: CD3, CD3γ, CD3δ, CD3ε, CD5, CD7, CD28, CD2, CD127, 4-1BB, OX40, ICOS, TCRγ, TCRδ, TCRα, and TCRβ.

42. The particles according to any one of claims 1-41, characterized in that, The target molecules include activating molecules.

43. The particles according to claim 42, characterized in that, The activated molecule can specifically bind to CD3, CD7, CD5 and / or TCR.

44. The particles according to claim 42 or 43, characterized in that, The activating molecules include anti-CD3 antibodies, anti-CD7 antibodies, anti-CD5 antibodies, anti-TCR antibodies, or their antigen-binding fragments.

45. The particles according to claim 44, characterized in that, The anti-CD3 antibody or its antigen-binding fragment is an anti-CD3 scFv or VHH.

46. ​​The particles according to claim 44 or 45, characterized in that, The anti-CD3 antibody is UCHT1, OKT3, SP34, HuM291, or TR66, or a variant thereof, or a derivative thereof.

47. The particles according to claim 55, 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.

48. The particles according to any one of claims 1-47, characterized in that, The targeting molecule includes at least one co-stimulatory molecule.

49. The particles according to claim 48, characterized in that, The at least one co-stimulatory molecule can specifically bind to one or more of CD28, CD137, OX40, and ICOS.

50. The particles according to claim 49, characterized in that, The co-stimulatory molecules are selected from anti-CD28 antibodies or their antigen-binding fragments, and CD80, CD86, CD137L, OX40L and ICOSL, or their extracellular domains, or their functional fragments; Preferably, the anti-CD28 antibody or its antigen-binding fragment is a 15E8 scFv (15E8-scFv), and 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.

51. The particles according to any one of claims 1-50, characterized in that, The targeting molecule is a transmembrane protein that can be membrane-exposed on the surface of the particle.

52. The particles according to claim 51, characterized in that, The target molecule is a recombinant transmembrane protein.

53. The particles according to claim 52, characterized in that, The recombinant transmembrane protein includes a transmembrane region; preferably, the recombinant transmembrane protein further includes a linker domain.

54. The particles according to any one of claims 48-50, characterized in that, The activating molecule is linked to the co-stimulatory molecule via a first polypeptide linker (PolypeptideLinker1).

55. The particles according to claim 54, characterized in that, The viral glycoprotein, or its variants, or its functional fragments, or its modified forms, are linked to the activating molecule or the co-stimulatory molecule via a second polypeptide linker.

56. The particles according to claim 55, characterized in that, (a) The viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, is linked to the activating molecule via the second polypeptide linker; the activating molecule is linked to the co-stimulatory molecule via the first polypeptide linker; or (b) The viral glycoprotein, or a variant thereof, or a functional fragment thereof, or a modified version thereof, is linked to the co-stimulatory molecule via the second polypeptide linker; the co-stimulatory molecule is linked to the activating molecule via the first polypeptide linker.

57. The particles according to any one of claims 54-56, characterized in that, The first polypeptide linker and the second polypeptide linker are 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) the stalk domain of a type II C-lectin selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (e) a flexible linker peptide; The preferred (G4S)n linker peptide is n = 1 to 4; linker 1: GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 60); and linker 2: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 94).

58. The particles according to claim 66, characterized in that, The first and second polypeptide linkers are (G4S)3 linker peptides, GGGGSGGGGSGGGGS (SEQ ID NO:32).

59. The particles according to any one of claims 1-58, characterized in that, The particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles.

60. The particles according to claim 59, characterized in that, The particles are enveloped viral particles.

61. The particles according to claim 60, characterized in that, The enveloped viral particles are pseudolentiviral vectors (LVV) and / or retroviral vectors (RVV).

62. A composition, characterized in that, The composition comprises a pharmaceutically acceptable carrier or excipient and particles according to any one of claims 1-61.

63. 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 1-61.

64. A method for preparing CAR-T cells by transducing T cells in a subject in need, characterized in that, This includes administering the particles of any one of claims 1-61 to the subject.

65. The method according to claim 64, characterized in that, It also includes administering T cells and / or a cell population containing T cells to the subject.

66. A method for improving, enhancing, or strengthening at least one of the expansion capacity, persistence, and killing capacity of CAR-T cells prepared in a subject in need, while reducing or decreasing the release or secretion of cytokines by CAR-T cells, characterized in that, This includes administering the particles according to any one of claims 1-61 to a subject in need.

67. The method according to claim 66, characterized in that, It also includes administering T cells and / or a cell population containing T cells to the subject.

68. The method according to claim 66 or 67, characterized in that, The cytokines are selected from one or more of IL-2, IFN-γ, TNF-α, and IL-6.

69. The method according to any one of claims 66-68, characterized in that, The cell population is selected from one or more of the subject's own, allogeneic, and iPSC-derived cell populations.

70. The method according to claim 69, characterized in that, The cell population was selected from PBMCs, lymphocytes, and leukocytes.

71. Use of the particles according to any one of claims 1-61 in the preparation of a cancer therapeutic drug.

72. The application according to claim 71, characterized in that, The cancer is selected from one or more of hematologic malignancies and solid tumors.

73. The application according to claim 72, 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), and cancers unsuitable for transplantation. One or more of the following: 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, 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 center T-cell lymphoma (FTCL).

74. The application according to claim 72, characterized in that, 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.

75. A method for treating a subject who has or is suspected of having cancer, characterized in that, The treatment includes administering a therapeutically effective amount of the granules according to any one of claims 1-61 to the subject.

76. The method according to claim 75, characterized in that, The cancer is selected from one or more of hematologic malignancies and solid tumors.

77. The method according to claim 76, 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), and cancers unsuitable for transplantation. One or more of the following: 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, 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 center T-cell lymphoma (FTCL).

78. The method according to claim 76, characterized in that, 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.

79. The method according to any one of claims 75-78, 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 or secretion system, and implanted device.

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