Vector comprising multiple targeting molecules, and method for improving transduction efficiency of vector

Abstract

Provided are a vector comprising multiple targeting molecules, and a method for improving transduction efficiency of a vector. Provided is a vector comprising multiple targeting molecules. The efficiency of transducing non-activated T cells by the vector is improved. Also provided is a method for improving transduction efficiency of a vector. The method can effectively improve the efficiency of transducing non-activated T cells by the vector.

Description

A carrier containing multiple target molecules and a method for improving carrier transduction efficiency. Technical Field

[0001] This invention relates to the field of gene vectors, specifically to a vector containing multiple target molecules and a method for improving vector transduction efficiency. Background Technology

[0002] In the fields of gene engineering and cell therapy, inactive T cells are difficult to infect by vectors containing wild-type VSV-G on their surface, such as lentiviral vectors (LVV) or retroviral vectors (RVV), due to their low expression of LDL-R. Existing techniques typically involve constructing targeting molecules on the surface of these vectors that can bind to surface antigens of inactive T cells to improve the transduction efficiency of these vectors in converting inactive T cells.

[0003] However, the efficiency of transducing inactive T cells using vectors containing a single targeting molecule, such as an anti-CD5 antibody, which are commonly used in existing technologies, still needs to be improved.

[0004] Therefore, the need for a vector that can efficiently transduce inactive T cells and a method to improve the efficiency of vector transduction of inactive T cells has not yet been met. Summary of the Invention

[0005] In view of this, in order to at least solve one of the above-mentioned technical problems, one aspect of the present invention provides a carrier, the surface of which includes a CD3-binding molecule, a T-cell co-stimulatory molecule, and a CD7-binding molecule.

[0006] Preferably, the T cell co-stimulatory molecule includes CD28.

[0007] In some embodiments of the present invention, the carrier is selected from lipid nanoparticles (LNPs), extracellular vesicles, and viral vectors;

[0008] Preferably, the extracellular vesicles include exosomes and microvesicles;

[0009] Preferably, the viral vector includes an enveloped viral vector;

[0010] More preferably, the enveloped viral vector includes LVV and RVV.

[0011] In some embodiments of the present invention, the vector further includes a vector selected from adenovirus and adeno-associated virus.

[0012] In some embodiments of the present invention, the targeting molecule comprises an extracellular binding domain and a transmembrane domain, wherein the extracellular binding domain comprises at least one of an antibody or antigen-binding fragment thereof capable of binding to (a) CD3, (b) T cell co-stimulatory molecules or (c) CD7, and a ligand or receptor-binding fragment thereof;

[0013] Preferably, the T cell co-stimulatory molecule includes CD28.

[0014] In some embodiments of the present invention, the targeting molecule comprises an extracellular binding domain and a transmembrane domain, wherein the extracellular binding domain comprises at least one of an antibody or antigen-binding fragment thereof capable of binding to CD3, CD28 or CD7 and a ligand or receptor-binding fragment thereof.

[0015] In some embodiments of the present invention, the extracellular binding domain of the CD3-binding target molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

[0016] In some embodiments of the present invention, the extracellular binding domain of the CD28-binding target molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

[0017] In some embodiments of the present invention, the extracellular binding domain of the CD28-binding target molecule comprises CD86 or its receptor-binding fragment.

[0018] In some embodiments of the present invention, the extracellular binding domain of the CD28-binding target molecule comprises CD80 or its receptor-binding fragment.

[0019] In some embodiments of the present invention, the extracellular binding domain of the CD7-binding target molecule comprises an anti-CD7 antibody or an antigen-binding fragment thereof.

[0020] In some embodiments of the present invention, the extracellular binding domain binds to human CD3, CD7, or T cell co-stimulatory molecules;

[0021] Preferably, the T cell co-stimulatory molecule includes CD28.

[0022] In some embodiments of the present invention, the extracellular binding domain binds to co-stimulatory molecules of monkey CD3, CD7, or T cells;

[0023] Preferably, the T cell co-stimulatory molecule includes CD28.

[0024] In some embodiments of the present invention, the extracellular binding domain binds to human CD3, CD7, or CD28.

[0025] In some embodiments of the present invention, the extracellular binding domain binds to monkey CD3, CD7, or CD28.

[0026] In some embodiments of the present invention, the extracellular binding domain binds to CD3, CD7, or CD28 of cynomolgus monkeys.

[0027] In some embodiments of the present invention, the extracellular binding domain is directly or indirectly connected to the transmembrane domain and exposed on the surface of the carrier;

[0028] Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins:

[0029] CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70;

[0030] More preferably, the transmembrane domain includes the transmembrane domain of CD8α.

[0031] In some embodiments of the present invention, the targeting molecule further includes a linker domain, and the extracellular binding domain is indirectly connected to the transmembrane region through the linker domain;

[0032] Preferably, the connection structure domain is selected from:

[0033] (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;

[0034] (b) Hinge region, which 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;

[0035] (c) All or a portion of the Fc domains, wherein the Fc domains are selected from at least one of the CH1, CH2, and CH3 domains; and

[0036] (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;

[0037] More preferably, the connection structure domain includes the hinge region of CD8α.

[0038] In some embodiments of the present invention, the carrier is LVV or RVV.

[0039] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV is selected from vesicular stomatitis virus strains glycoprotein and its variants, Nipah virus (NiV) glycoprotein G and its variants, measles virus glycoprotein H and its variants, lentivirus glycoprotein and its variants, rabies virus glycoprotein (RVG) and its variants, gibberish leukemia virus glycoprotein (GaLV) and its variants, biphilic murine leukemia virus glycoprotein (MLV-A) and its variants, feline endogenous virus (RD114) glycoprotein and its variants, avian plague virus (FPV) glycoprotein and its variants, Ebola virus (EboV) glycoprotein and its variants, and lymphocytic choriomeningitis virus (LCMV) glycoprotein and its variants;

[0040] The vesicular stomatitis virus (VSV) glycoproteins and their variants include: VSV Indiana strain glycoprotein and its variants, VSV Cocal strain glycoprotein and its variants, VSV Maraba strain glycoprotein and its variants, VSV Morreton strain glycoprotein and its variants, VSV Alagoas strain glycoprotein and its variants, and VSV New... The glycoproteins of the Jersey strain and its variants, the Carajas strain and its variants, the Chandipura strain and its variants, the Eptesicus strain and its variants, the Isfahan strain and its variants, the Jurona strain and its variants, the Malpais strain and its variants, the Perinet strain and its variants, the Piry strain and its variants, the Radi strain and its variants, the Rhinolopus strain and its variants, and the Yug Bogdanovac strain and its variants are all glycoproteins of the vesicular stomatitis virus.

[0041] Preferably, the viral glycoprotein of the LVV or RVV is a glycoprotein of the Indiana strain of vesicular stomatitis virus or a variant thereof, or a glycoprotein of the Cocal strain of vesicular stomatitis virus or a variant thereof.

[0042] Vesicular stomatitis virus strains, such as the Indiana strain's viral glycoprotein (VSV-G) or the Cocal strain's viral glycoprotein (Cocal-G), have broad infectivity because they can specifically bind to the low-density lipoprotein receptor (LDL-R), which is widely present on the surface of many cells. Therefore, in the field of genetic engineering, VSV-G or Cocal-G is often used to construct viral glycoproteins for lentiviral or retroviral vectors.

[0043] In some embodiments of the present invention, wild-type VSV-G comprises the amino acid sequence shown in SEQ ID NO:1;

[0044] The full-length protein (including the signal peptide) of the wild-type VSV-G contains the amino acid sequence shown in SEQ ID NO:22;

[0045] Among them, the amino acid sequence shown in positions 1-16 of SEQ ID NO:22 is as follows:

[0046] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.

[0047] In some embodiments of the present invention, wild-type Cocal-G comprises the amino acid sequence shown in SEQ ID NO:2;

[0048] The sequence of the full-length protein (including the signal peptide) of the wild-type Cocal-G is shown in SEQ ID NO:29;

[0049] Among them, the sequence shown in positions 1-17 of SEQ ID NO:29 is as follows:

[0050] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild-type Cocal-G.

[0051] In some embodiments of the present invention, the ability of the viral glycoprotein of the LVV or RVV to bind to its receptor is inhibited;

[0052] Preferably, the viral glycoprotein is VSV-G or a variant thereof or Cocal-G or a variant thereof, and the receptor is low-density lipoprotein receptor LDL-R.

[0053] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV contains a first mutation that inhibits the ability of the viral glycoprotein to bind to a receptor.

[0054] In some embodiments of the present invention, the ability of the viral glycoprotein containing the first mutation to bind to its receptor is inhibited but can mediate membrane fusion.

[0055] By inducing the first mutation in VSV-G or Cocal-G, the ability of the mutated VSV-G or Cocal-G to bind LDL-R is reduced, and multiple targeting molecules that can bind CD3, CD28, and CD7 are constructed on the surface of the LVV or RVV. This not only improves the efficiency of the lentiviral vector or retroviral vector in transducing inactive T cells, but also enhances the targeting of the lentiviral vector or retroviral vector in transducing inactive T cells.

[0056] In some embodiments of the present invention, the viral glycoprotein containing the first mutation is VSV-G or a variant thereof, and the receptor is LDL-R;

[0057] Preferably, the first mutation includes mutations at at least one of the following sites:

[0058] (a) Substitutions or deletions of H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, amino acid deletions at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353; and

[0059] (b) After best global alignment with SEQ ID NO:1, it is located at the position equivalent to SEQ ID NO:1 Substitutions or deletions of H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, amino acid deletions at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353.

[0060] More preferably, the first mutation includes a mutation in at least one of the following amino acids:

[0061] (a) Substitution or deletion of K47 located in SEQ ID NO:1, substitution of R354; and

[0062] (b) After best global alignment with SEQ ID NO:1, the substitution or deletion of K47, which is equivalent to SEQ ID NO:1, and the substitution of R354;

[0063] More preferably, the first mutation includes a mutation in at least one of the following amino acids:

[0064] (a) The 47th amino acid in SEQ ID NO:1 is replaced by lysine K with glutamine Q (K47Q) or the 47th amino acid lysine is deleted (K47 deletion), and the 354th amino acid is replaced by arginine R with glutamine Q (R354Q); and

[0065] (b) After best global alignment with SEQ ID NO:1, it is located at K47Q or K47 deletion, R354Q, which is equivalent to SEQ ID NO:1;

[0066] Most preferably, the first mutation includes mutations in the following amino acids:

[0067] (a) Deletion of K47 located at SEQ ID NO:1; or

[0068] (b) After best global alignment with SEQ ID NO:1, the deletion is located at K47, which corresponds to SEQ ID NO:1.

[0069] In some embodiments of the present invention, the viral glycoprotein containing the first mutation is VSV-G or a variant thereof, the receptor is LDL-R, and the first mutation includes mutations at at least one of the following sites:

[0070] (a) I182D or I182E, I331E or I331M located in SEQ ID NO:1;

[0071] (b) After best global alignment with SEQ ID NO:1, it is located at I182D or I182E, I331E or I331M, which are equivalent to SEQ ID NO:1.

[0072] In some embodiments of the present invention, the viral glycoprotein containing the first mutation is Cocal-G or a variant thereof, and the receptor is LDL-R;

[0073] Preferably, the first mutation includes mutations at at least one of the following sites:

[0074] (a) Substitutions or deletions of Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350, T352, E353, R354, amino acid deletions at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353; and

[0075] (b) After best global alignment with SEQ ID NO:2, it is located at the position equivalent to SEQ ID NO:2. Substitutions or deletions of Q8, S9, Q10, K47, K50, A51, D183, A179, T180, V182, T184, Y209, I347, S350, T352, E353, R354, amino acid deletions at positions 1-18, 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, and 345-353 of NO:2.

[0076] More preferably, the first mutation includes a mutation in at least one of the following amino acids:

[0077] (a) Substitution or deletion of K47 in SEQ ID NO:2, substitution of R354; and

[0078] (b) After best global alignment with SEQ ID NO:2, the substitution or deletion of K47, which is equivalent to SEQ ID NO:2, and the substitution of R354;

[0079] More preferably, the first mutation includes a mutation in at least one of the following amino acids:

[0080] (a) K47Q or K47 deletion, R354Q located in SEQ ID NO:2; and

[0081] (b) After best global alignment with SEQ ID NO:2, it is located at K47Q or K47 deletion, R354Q, which is equivalent to SEQ ID NO:2;

[0082] Most preferably, the first mutation includes mutations in the following amino acids:

[0083] (a) Deletion of K47 located at SEQ ID NO:2; or

[0084] (b) After best global alignment with SEQ ID NO:2, the deletion is located at K47, which corresponds to SEQ ID NO:2.

[0085] In some embodiments of the present invention, the viral glycoprotein containing the first mutation is Cocal-G or a variant thereof, the receptor is LDL-R, and the first mutation includes mutations at at least one of the following sites:

[0086] (a) V182D or V182E, I331E or I331M located in SEQ ID NO:2;

[0087] (b) After best global alignment with SEQ ID NO:2, it is located at V182D or V182E, I331E or I331M, which are equivalent to SEQ ID NO:2.

[0088] In some embodiments of the present invention, the number of binding sites available for the viral glycoprotein of the LVV or RVV to bind to its receptor is reduced, thereby inhibiting the ability of the viral glycoprotein to bind to its receptor.

[0089] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV has bound to at least one of its receptor and antibody, thereby reducing the number of binding sites available for the viral glycoprotein to bind to its receptor.

[0090] Preferably, the viral glycoprotein has been bound to at least one of its receptor and antibody in the packaging cells that package the LVV or RVV;

[0091] Preferably, the viral glycoprotein has bound at least one of its receptor and antibody to the cell membrane of the packaging cell that packages the LVV or RVV;

[0092] Preferably, the viral glycoprotein has at least one of its receptor and antibody bound to the viral envelope of the LVV or RVV.

[0093] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV is VSV-G or a variant thereof or Cocal-G or a variant thereof, wherein the VSV-G or a variant thereof or Cocal-G or a variant thereof has bound to at least one of its receptor LDL-R, VSV-G antibody and Cocal-G antibody, thereby reducing the number of binding sites available for the VSV-G or a variant thereof or Cocal-G or a variant thereof to bind to its receptor LDL-R.

[0094] In some embodiments of the present invention, the viral glycoprotein VSV-G or a variant thereof, or Cocal-G or a variant thereof, has been bound to its receptor LDL-R.

[0095] In some embodiments of the present invention, the viral glycoprotein VSV-G or a variant thereof has been bound to a VSV-G antibody.

[0096] In some embodiments of the present invention, the viral glycoprotein Cocal-G or a variant thereof has been bound to a Cocal-G antibody.

[0097] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV contains a second mutation that enhances the ability of the viral glycoprotein to antagonize complement inactivation or prevents complement inactivation.

[0098] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV contains a second mutation that enhances or prevents the viral glycoprotein from being inactivated by complement and can mediate membrane fusion.

[0099] In some embodiments of the present invention, the viral glycoprotein containing the second mutation is VSV-G or a variant thereof; the second mutation includes a mutation in at least one of the following amino acids:

[0100] (a) The 214th amino acid located in SEQ ID NO:1;

[0101] (b) After optimal global alignment with SEQ ID NO:1, the amino acid located at the 214th position, which corresponds to SEQ ID NO:1;

[0102] (c) The 352nd amino acid located in SEQ ID NO:1;

[0103] (d) After optimal global alignment with SEQ ID NO:1, the amino acid located at position 352, which corresponds to SEQ ID NO:1;

[0104] (e) The 50th amino acid located in SEQ ID NO:1;

[0105] (f) After optimal global alignment with SEQ ID NO:1, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:1;

[0106] (g) the amino acid located at position 146 of SEQ ID NO:1; and

[0107] (h) is located at amino acid position 146 of SEQ ID NO:1 after optimal global alignment with SEQ ID NO:1;

[0108] Preferably, the mutation of the amino acid includes the deletion, insertion, or substitution of the amino acid;

[0109] More preferably, the second mutation includes the substitution of at least one of the following amino acids:

[0110] (a) The 214th amino acid located in SEQ ID NO:1;

[0111] (b) After optimal global alignment with SEQ ID NO:1, the amino acid located at the 214th position, which corresponds to SEQ ID NO:1;

[0112] (c) The 352nd amino acid located in SEQ ID NO:1;

[0113] (d) After optimal global alignment with SEQ ID NO:1, the amino acid located at position 352, which corresponds to SEQ ID NO:1;

[0114] (e) The 50th amino acid located in SEQ ID NO:1;

[0115] (f) After optimal global alignment with SEQ ID NO:1, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:1;

[0116] (g) the amino acid located at position 146 of SEQ ID NO:1; and

[0117] (h) is located at amino acid position 146 of SEQ ID NO:1 after optimal global alignment with SEQ ID NO:1;

[0118] More preferably, the second mutation includes at least one mutation at the following sites:

[0119] (a) The amino acid at position 214 of SEQ ID NO:1 is replaced by threonine T with asparagine N (T214N), the amino acid at position 352 is replaced by threonine T with alanine A (T352A), the amino acid at position 50 is replaced by lysine K with threonine T (K50T), and the amino acid at position 146 is replaced by serine S with threonine T (S146T); and

[0120] (b) After best global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located at the equivalent of SEQ ID NO:1.

[0121] In some embodiments of the present invention, the second mutation comprises a combination of any of the following site mutations:

[0122] (a) Substitution of T214 and T352 located in SEQ ID NO:1;

[0123] (b) After best global alignment with SEQ ID NO:1, the substitutions located at T214 and T352, which are equivalent to SEQ ID NO:1;

[0124] (c) Substitutions of T214, T352, K50 and S146 located at SEQ ID NO:1; and

[0125] (d) Substitutions located at T214, T352, K50 and S146 corresponding to SEQ ID NO:1 after best global alignment with SEQ ID NO:1;

[0126] Preferably, the second mutation comprises a combination of any of the following site mutations:

[0127] (a) T214N and T352A located at SEQ ID NO:1;

[0128] (b) After best global alignment with SEQ ID NO:1, it is located at T214N and T352A, which are equivalent to SEQ ID NO:1;

[0129] (c) T214N, T352A, K50T and S146T located at SEQ ID NO:1; and

[0130] (d) After best global alignment with SEQ ID NO:1, T214N, T352A, K50T and S146T are located at the equivalent of SEQ ID NO:1.

[0131] In some embodiments of the present invention, the viral glycoprotein containing the second mutation is Cocal-G or a variant thereof; the second mutation includes a mutation in at least one of the following amino acids:

[0132] (a) The 214th amino acid located in SEQ ID NO:2;

[0133] (b) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 214th position of SEQ ID NO:2;

[0134] (c) The 352nd amino acid located in SEQ ID NO:2;

[0135] (d) After optimal global alignment with SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:2;

[0136] (e) The 50th amino acid located in SEQ ID NO:2;

[0137] (f) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:2;

[0138] (g) the amino acid located at position 146 of SEQ ID NO:2; and

[0139] (h) After optimal global alignment with SEQ ID NO:2, it is located at amino acid position 146, which corresponds to SEQ ID NO:2;

[0140] Preferably, the mutation of the amino acid includes the deletion, insertion, or substitution of the amino acid;

[0141] More preferably, the second mutation includes the substitution of at least one of the following amino acids:

[0142] (a) The 214th amino acid located in SEQ ID NO:2;

[0143] (b) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 214th position of SEQ ID NO:2;

[0144] (c) The 352nd amino acid located in SEQ ID NO:2;

[0145] (d) After optimal global alignment with SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:2;

[0146] (e) The 50th amino acid located in SEQ ID NO:2;

[0147] (f) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:2;

[0148] (g) the amino acid located at position 146 of SEQ ID NO:2; and

[0149] (h) After optimal global alignment with SEQ ID NO:2, it is located at amino acid position 146, which corresponds to SEQ ID NO:2;

[0150] More preferably, the second mutation includes at least one mutation at the following sites:

[0151] (a) The amino acid at position 214 of SEQ ID NO:2 is replaced by lysine K with asparagine N (K214N), T352A, K50T, or S146T; and

[0152] (b) After best global alignment with SEQ ID NO:2, it is located at K214N, T352A, K50T, S146T, which are equivalent to SEQ ID NO:2.

[0153] In some embodiments of the present invention, the second mutation comprises a combination of any of the following site mutations:

[0154] (a) Substitution of K214 and T352 located in SEQ ID NO:2;

[0155] (b) Substitutions located at K214 and T352 corresponding to SEQ ID NO:2 after best global alignment with SEQ ID NO:2;

[0156] (c) Substitutions of K214, T352, K50, and S146 located at SEQ ID NO:2; and

[0157] (d) Substitutions located at K214, T352, K50 and S146 corresponding to SEQ ID NO:2 after best global alignment with SEQ ID NO:2;

[0158] Preferably, the second mutation comprises a combination of any of the following site mutations:

[0159] (a) K214N and T352A located at SEQ ID NO:2;

[0160] (b) After best global alignment with SEQ ID NO:2, it is located at K214N and T352A, which are equivalent to SEQ ID NO:2;

[0161] (c) K214N, T352A, K50T and S146T located at SEQ ID NO:2; and

[0162] (d) After best global alignment with SEQ ID NO:2, it is located at K214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:2.

[0163] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV contains either the first mutation or the second mutation, such that the ability of the viral glycoprotein to bind to its receptor is inhibited and its ability to antagonize complement inactivation is enhanced or not inactivated by complement.

[0164] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV contains either the first mutation or the second mutation, such that the ability of the viral glycoprotein to bind to its receptor is inhibited, its ability to antagonize complement inactivation is enhanced or it is not inactivated by complement, and it can mediate membrane fusion.

[0165] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1.

[0166] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:2.

[0167] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:3 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3; SEQ ID NO:3 contains a K47 deletion relative to SEQ ID NO:1.

[0168] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:4 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4; SEQ ID NO:4 comprises R354Q relative to SEQ ID NO:1.

[0169] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:5 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5; relative to SEQ ID NO:1, SEQ ID NO:6 comprises K47 deletion, T214N, T352A, K50T, and S146T.

[0170] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:6 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:6; relative to SEQ ID NO:1, SEQ ID NO:6 comprises K47 deletion, T214N, and T352A.

[0171] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:7 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with 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.

[0172] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:21 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:21; relative to SEQ ID NO:1, SEQ ID NO:21 comprises R354Q, T214N, T352A, K50T, and S146T.

[0173] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:23 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:23; SEQ ID NO:23 comprises a K47 deletion relative to SEQ ID NO:2.

[0174] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:24 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:24; SEQ ID NO:24 comprises R354Q relative to SEQ ID NO:2.

[0175] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:25 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:25; relative to SEQ ID NO:2, SEQ ID NO:25 comprises K47 deletion, K214N, T352A, K50T, and S146T.

[0176] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:26 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:26; relative to SEQ ID NO:2, SEQ ID NO:26 comprises K47 deletion, K214N, and T352A.

[0177] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:27 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:27; relative to SEQ ID NO:2, SEQ ID NO:27 comprises R354Q, K214N, T352A, K50T, and S146T.

[0178] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises an amino acid sequence as shown in SEQ ID NO:28 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:28; relative to SEQ ID NO:2, SEQ ID NO:28 comprises R354Q, K214N, and T352A.

[0179] In some embodiments of the present invention, the viral glycoprotein of the LVV or RVV comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

[0180] In some embodiments of the present invention, any of the aforementioned vectors comprises exogenous nucleic acids;

[0181] Preferably, the exogenous nucleic acid encodes at least one of a chimeric antigen receptor (CAR) and a T cell receptor chimeric protein (TCP);

[0182] The CAR includes an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction domain; more preferably, the CAR also includes a hinge region and a co-stimulatory signal transduction domain.

[0183] The TCP comprises: (a) a TCR / CD3 complex subunit related peptide (TSP), the TSP comprising at least one of a TCR / CD3 complex subunit, a TCR / CD3 complex subunit functional fragment, a TCR / CD3 complex subunit variant, and a variant of a TCR / CD3 complex subunit functional fragment; and (b) an extracellular antigen-binding region; more preferably, the TCP further comprises a linker peptide.

[0184] In some embodiments of the present invention, the extracellular antigen-binding regions of the CAR and TCP can bind at least one cancer-associated antigen;

[0185] Preferably, the cancer is selected from: acute myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendiceal cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, and fallopian tube cancer. Cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, common germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin's lymphoma, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, colorectal cancer, intrahepatic cholangiocarcinoma, invasive / invasive breast cancer, islet cell carcinoma, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, stromal tumor Tumors, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal carcinoma, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord-stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, sinus cancer, parathyroid carcinoma, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma At least one of the following: cytokine tumor, pineal region tumor, pineal blastoma, pituitary adenoma, primary central nervous system cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, bone, sarcoma, soft tissue, sarcoma, uterine, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymic cancer, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, renal tubular cancer, undiagnosed cancer, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0186] In some embodiments of the present invention, the extracellular antigen-binding regions of the CAR and TCP can bind TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, and DR5. , TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3 , ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD.2, GD.3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, soybean protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, spermin 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2 ETS fusion gene / ERG, NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut At least one of the following: hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, and GCC (guanylate cyclase).

[0187] Preferably, the extracellular antigen-binding regions of the CAR and TCP can bind to at least one of CD19, CD20, CD33, CD79A, CD79B, HER2, CEA, and BCMA.

[0188] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR and TCP comprises at least one of an antibody or its antigen-binding fragment and a ligand or its receptor-binding fragment.

[0189] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from at least one of the following: immunoglobulin (full-length antibody), hapten, Fab, Fab', F(ab')2, Fv fragment, single-chain variable region fragment (scFv), disulfide bond stable antibody (dsFv), heavy chain variable region (VH) or light chain variable region (VL) of antibody, Fd fragment composed of VH and CH1 domains, linear antibody, heavy chain antibody and nanobody (VHH).

[0190] In some embodiments of the present invention, the CAR and TCP are single-specific, bi-specific, or multi-specific.

[0191] 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.

[0192] In some embodiments of the present invention, the transmembrane region of the CAR includes the transmembrane region of CD8α.

[0193] 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.

[0194] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR includes the intracellular signal transduction domain of CD3ζ.

[0195] In some embodiments of the present invention, the CAR further includes a hinge region, wherein the hinge region of the CAR is sequentially connected to the extracellular antigen-binding region of the CAR and the transmembrane region of the CAR.

[0196] In some embodiments of the present invention, the hinge region of the CAR is selected from the hinge regions of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

[0197] In some embodiments of the present invention, the hinge region of the CAR includes the hinge region of CD8α.

[0198] In some embodiments of the present invention, the CAR further includes a co-stimulatory signal transduction domain.

[0199] In some embodiments of the present invention, the co-stimulatory signal transduction domain of the CAR comprises one or more of the following proteins' co-stimulatory signal transduction domains: 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.

[0200] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR includes the costimulatory signal transduction domains of 4-1BB and / or CD28.

[0201] In some embodiments of the present invention, the CAR and TCP further comprise a signal peptide encoded by a leader sequence;

[0202] Preferably, the signal peptide is selected from CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide;

[0203] More preferably, the signal peptide is the CD8α signal peptide.

[0204] In some embodiments of the present invention, the TCR / CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ζ, and CD3ε.

[0205] In some embodiments of the present invention, the functional fragment of the TCR / CD3 complex subunit includes at least one of the extracellular region, transmembrane region, intracellular region, variable region, and constant region of the TCR / CD3 complex subunit.

[0206] In some embodiments of the present invention, the TSP comprises CD3γ or a functional fragment thereof.

[0207] In some embodiments of the present invention, the TSP includes CD3ε or a functional fragment thereof.

[0208] In some embodiments of the present invention, the TSP comprises TCRα or a functional fragment thereof and TCRβ or a functional fragment thereof.

[0209] In some embodiments of the present invention, the TSP includes a constant region of TCRα and a constant region of TCRβ.

[0210] In some embodiments of the present invention, the constant region of the TCRα contains a mutation, the mutation including a cysteine ​​substitution in the constant region of the TCRα; the mutation may enhance disulfide bond-based interchain interactions.

[0211] In some embodiments of the present invention, the TCRβ constant region contains a mutation, the mutation including a cysteine ​​substitution in the TCRβ constant region; the mutation may enhance disulfide bond-based interchain interactions.

[0212] In some embodiments of the present invention, the TCRα constant region contains a mutation, the mutation comprising replacing at least one uncharged amino acid in the TCRα constant region with a hydrophobic amino acid; the mutation increases the hydrophobicity of the TCRα transmembrane region, counteracting the instability caused by the positive charge carried by the TCRα transmembrane region, so that the TCRα and the dimer formed with TCRβ can be expressed more stably on the T cell membrane, thereby obtaining better function.

[0213] In some embodiments of the present invention, the extracellular antigen-binding region of the TCP is operatively linked to the N-terminus of any of the aforementioned TSPs via a linker peptide.

[0214] In some embodiments of the present invention, the C-terminus of the extracellular antigen-binding region of the TCP is operatively linked to the N-terminus of any of the aforementioned TSPs via a linker peptide.

[0215] In some embodiments of the present invention, the linker peptide includes a flexible linker peptide.

[0216] In some embodiments of the present invention, the flexible linker peptide is selected from (G4S). n Linking peptide and linking peptide 1 containing the amino acid sequence shown in SEQ ID NO:30; wherein n = 1 to 4.

[0217] In some embodiments of the present invention, the flexible linker peptide is linker peptide 1 or (G4S)3 linker peptide.

[0218] Referring to Chinese invention patent application CN2024101450536 and PCT patent application PCT / CN2024 / 107675 (which are incorporated herein by reference in their entirety), the TCP in T cells or on the T cell membrane can (1) incorporate the endogenous TCR / CD3 complex, endogenous TCR / CD3 complex subunits or their functional fragments; and / or (2) interact with the endogenous TCR / CD3 complex, endogenous TCR / CD3 complex subunits or their functional fragments, for example, form a TCR / CD3 complex or its functional fragments with endogenous CD3 subunits or their functional fragments; thereby expressing and anchoring on the T cell membrane; and the TCP is difficult to express and anchor on the cell membrane effectively in cells other than T cells.

[0219] In some embodiments of the present invention, the efficiency of transducing inactive T cells using any of the vectors carrying the exogenous nucleic acid is improved.

[0220] In some embodiments of the present invention, the increased efficiency of any of the vectors carrying the exogenous nucleic acid in transducing inactive T cells is relative to vectors whose surfaces do not contain target molecules that can bind CD3, CD28, or CD7.

[0221] In another aspect, the present invention also provides a method for improving the efficiency of vector transduction of inactive T cells, wherein the vector contains exogenous nucleic acid, and the method includes constructing a target molecule that can bind CD3, a target molecule that can bind T cell co-stimulatory molecules, and a target molecule that can bind CD7 on the surface of the vector.

[0222] Preferably, the T cell co-stimulatory molecule includes CD28.

[0223] In some embodiments of the present invention, the improvement in transduction efficiency is relative to a carrier whose surface does not contain a target molecule that can bind CD3, a target molecule that can bind T cell co-stimulatory molecules, or a target molecule that can bind CD7.

[0224] Preferably, the T cell co-stimulatory molecule includes CD28.

[0225] In some embodiments of the present invention, the improvement in transduction efficiency is relative to a carrier whose surface does not contain a target molecule that can bind CD3, a target molecule that can bind CD28, or a target molecule that can bind CD7.

[0226] In some embodiments of the present invention, the carrier is selected from lipid nanoparticles, extracellular vesicles, and viral vectors;

[0227] Preferably, the extracellular vesicles include exosomes and microvesicles;

[0228] Preferably, the viral vector includes an enveloped viral vector;

[0229] More preferably, the enveloped viral vector includes LVV and RVV.

[0230] In some embodiments of the present invention, the targeting molecule comprises an extracellular binding domain and a transmembrane domain, wherein the extracellular binding domain comprises at least one of an antibody or antigen-binding fragment thereof capable of binding to (a) CD3, (b) T cell co-stimulatory molecules or (c) CD7, and a ligand or receptor-binding fragment thereof;

[0231] Preferably, the T cell co-stimulatory molecule includes CD28.

[0232] In some embodiments of the present invention, the targeting molecule comprises an extracellular binding domain and a transmembrane domain, wherein the extracellular binding domain comprises at least one of an antibody or antigen-binding fragment thereof capable of binding to CD3, CD28 or CD7 and a ligand or receptor-binding fragment thereof.

[0233] In some embodiments of the present invention, the extracellular binding domain of the CD3-binding target molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

[0234] In some embodiments of the present invention, the extracellular binding domain of the CD28-binding target molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

[0235] In some embodiments of the present invention, the extracellular binding domain of the CD28-binding target molecule comprises CD86 or its receptor-binding fragment.

[0236] In some embodiments of the present invention, the extracellular binding domain of the CD28-binding target molecule comprises CD80 or its receptor-binding fragment.

[0237] In some embodiments of the present invention, the extracellular binding domain of the CD7-binding target molecule comprises an anti-CD7 antibody or an antigen-binding fragment thereof.

[0238] In some embodiments of the present invention, the extracellular binding domain binds to human CD3, CD7, or T cell co-stimulatory molecules;

[0239] Preferably, the T cell co-stimulatory molecule includes CD28.

[0240] In some embodiments of the present invention, the extracellular binding domain binds to co-stimulatory molecules of monkey CD3, CD7, or T cells;

[0241] Preferably, the T cell co-stimulatory molecule includes CD28.

[0242] In some embodiments of the present invention, the extracellular binding domain binds to human CD3, CD7, or CD28.

[0243] In some embodiments of the present invention, the extracellular binding domain binds to monkey CD3, CD7, or CD28.

[0244] In some embodiments of the present invention, the extracellular binding domain binds to CD3, CD7, or CD28 of cynomolgus monkeys.

[0245] In some embodiments of the present invention, the extracellular binding domain is directly or indirectly connected to the transmembrane domain and exposed on the surface of the carrier;

[0246] Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins:

[0247] CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70;

[0248] More preferably, the transmembrane domain includes the transmembrane domain of CD8α.

[0249] In some embodiments of the present invention, the targeting molecule further includes a linker domain, and the extracellular binding domain is indirectly connected to the transmembrane region through the linker domain;

[0250] Preferably, the connection structure domain is selected from:

[0251] (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;

[0252] (b) Hinge region, which 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;

[0253] (c) All or a portion of the Fc domains, wherein the Fc domains are selected from at least one of the CH1, CH2, and CH3 domains; and

[0254] (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;

[0255] More preferably, the connection structure domain includes the hinge region of CD8α.

[0256] In some embodiments of the present invention, the carrier is LVV or RVV.

[0257] In some embodiments of the present invention, the exogenous nucleic acid encodes at least one of the aforementioned CAR and TCP.

[0258] In another aspect, the present invention also provides a method for transducing inactive T cells, the method comprising contacting inactive T cells with any of the vectors carrying the exogenous nucleic acid provided by the present invention;

[0259] Preferably, the contact occurs outside the subject, who is an individual given non-activated T cells transduced by the method;

[0260] Preferably, the contact occurs within the body of a subject, who is an individual to whom the carrier is given.

[0261] In some embodiments of the present invention, the contact occurs outside the subject, and the carrier is in contact with non-activated T cells for no more than 2 days, 47 hours, 46 hours, 45 hours, 44 hours, 43 hours, 42 hours, 41 hours, 40 hours, 39 hours, 38 hours, 37 hours, 36 hours, 35 hours, 34 hours, 33 hours, 32 hours, 31 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, or 25 hours. Not exceeding 24 hours, not exceeding 23 hours, not exceeding 22 hours, not exceeding 21 hours, not exceeding 20 hours, not exceeding 19 hours, not exceeding 18 hours, not exceeding 17 hours, not exceeding 16 hours, not exceeding 15 hours, not exceeding 14 hours, not exceeding 13 hours, not exceeding 12 hours, not exceeding 11 hours, not exceeding 10 hours, not exceeding 9 hours, not exceeding 8 hours, not exceeding 7 hours, not exceeding 6 hours, not exceeding 5 hours, not exceeding 4 hours, not exceeding 3 hours, not exceeding 2 hours, not exceeding 1 hour, not exceeding 59 minutes, not exceeding 58 minutes, not exceeding 57 minutes, not exceeding 56 minutes, not exceeding 55 minutes, not exceeding 5 4 minutes, no more than 53 minutes, no more than 52 minutes, no more than 51 minutes, no more than 50 minutes, no more than 49 minutes, no more than 48 minutes, no more than 47 minutes, no more than 46 minutes, no more than 45 minutes, no more than 44 minutes, no more than 43 minutes, no more than 42 minutes, no more than 41 minutes, no more than 40 minutes, no more than 39 minutes, no more than 38 minutes, no more than 37 minutes, no more than 36 minutes, no more than 35 minutes, no more than 34 minutes, no more than 33 minutes, no more than 32 minutes, no more than 31 minutes, no more than 30 minutes, no more than 29 minutes, no more than 28 minutes, no more than 27 minutes, no more than 26 minutes Not exceeding 25 minutes, not exceeding 24 minutes, not exceeding 23 minutes, not exceeding 22 minutes, not exceeding 21 minutes, not exceeding 20 minutes, not exceeding 19 minutes, not exceeding 18 minutes, not exceeding 17 minutes, not exceeding 16 minutes, not exceeding 15 minutes, not exceeding 14 minutes, not exceeding 13 minutes, not exceeding 12 minutes, not exceeding 11 minutes, not exceeding 10 minutes, not exceeding 9 minutes, not exceeding 8 minutes, not exceeding 7 minutes, not exceeding 6 minutes, not exceeding 5 minutes, not exceeding 4 minutes, not exceeding 3 minutes, not exceeding 2 minutes, not exceeding 1 minute, not exceeding 50 seconds, not exceeding 40 seconds, not exceeding 30 seconds, not exceeding 20 seconds, or not exceeding 10 seconds.

[0262] In some embodiments of the present invention, when the contact occurs outside the subject, the inactive T cells are contained in the blood of the subject's own or allogeneic individual.

[0263] In some embodiments of the present invention, when the contact occurs outside the subject, the inactive T cells are contained in the peripheral blood of the subject's own or allogeneic individual.

[0264] In some embodiments of the present invention, when the contact occurs outside the subject, the inactive T cells are contained in the umbilical cord blood of the subject's own or allogeneic individual.

[0265] In some embodiments of the present invention, when the contact occurs outside the subject, the inactive T cells are contained in inactive PBMCs of the subject's own body or an allogeneic individual.

[0266] In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, intratumoral, subcutaneous, continuous release system and implanted device administration;

[0267] In some embodiments of the present invention, the administration is at least one of the following techniques: intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection, and infusion.

[0268] In some embodiments of the present invention, the exogenous nucleic acid encodes at least one of the aforementioned CAR and TCP.

[0269] In another aspect, the present invention also provides an engineered T cell containing exogenous nucleic acid; the engineered T cell is prepared by the method for transducing inactive T cells provided by the present invention.

[0270] In some embodiments of the present invention, the engineered T cells are CAR-T cells.

[0271] In some embodiments of the present invention, the engineered T cells are TCP-T cells.

[0272] In another aspect, the present invention also provides a composition comprising a pharmaceutically acceptable excipient or carrier and any one of the following components:

[0273] The present invention provides any of the aforementioned vectors and any of the aforementioned engineered T cells.

[0274] In some embodiments of the present invention, the composition comprises any of the vectors carrying the exogenous nucleic acid provided by the present invention or any of the engineered T cells.

[0275] In another aspect, the present invention also provides the use of any of the carriers or compositions provided by the present invention in the preparation of medicaments for treating and / or preventing diseases;

[0276] Preferably, the disease is selected from at least one of hematologic malignancies and solid tumors.

[0277] In another aspect, the present invention also provides a method for treating a subject with cancer and killing the subject's cancer cells, the method comprising administering to the subject any of the vector carrying exogenous nucleic acid provided by the present invention, any of the engineered T cells, or any composition comprising any of the vector carrying exogenous nucleic acid provided by the present invention or any of the engineered T cells.

[0278] Preferably, the cancer is selected from at least one of hematologic malignancies and solid tumors.

[0279] In some embodiments of the present invention, the disease is selected from at least one type of blood cancer;

[0280] Preferably, the hematologic malignancy is selected from at least one of acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), multiple myeloma (MM), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), and follicular lymphoma (FL).

[0281] In some embodiments of the present invention, the disease is selected from at least one type of solid cancer;

[0282] Preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, and ovarian cancer.

[0283] In some embodiments of the present invention, the hematologic malignancy includes relapsed or refractory hematologic malignancies.

[0284] The beneficial effects of this invention include:

[0285] The surface of any of the vectors provided by the present invention contains a target molecule that can bind CD3, a target molecule that can bind T cell co-stimulatory molecules such as CD28, and a target molecule that can bind CD7, thereby significantly improving the efficiency of transducing inactive T cells; any of the methods provided by the present invention for improving the efficiency of vector transduction of inactive T cells can significantly improve the efficiency of vector transduction of inactive T cells.

[0286] In this article:

[0287] “T cell activation molecules”: As used herein, “T cell activation molecules” include T cell surface antigens or markers involved in TCR-mediated T cell activation. T cell activation molecules can convert the TCR / CD3 complex into an active PTK (protein tyrosine kinase), which phosphorylates a series of substrates, thereby generating a large number of downstream signals. When these signals are properly integrated (along with signals from other co-receptors), they lead to T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27:591-619).

[0288] Inactive T cells express endocytic receptors CD3 and CD7. Therefore, carriers containing CD3-binding target molecules (such as anti-CD3 antibodies or their antigen-binding fragments) and CD7-binding target molecules (such as anti-CD7 antibodies or their antigen-binding fragments) can enter inactive T cells through endocytosis induced by binding to CD3 and CD7. Furthermore, after binding to target molecules involved in T cell activation signaling pathways, such as anti-CD3 antibodies or their antigen-binding fragments, activated T cells will also express T cell co-stimulatory molecules involved in T cell activation co-stimulatory (secondary) signaling pathways, including but not limited to T cell surface antigens, CD28, 4-1BB (CD137), and OX40 (CD134).

[0289] "T cell costimulatory molecules": These include T cell markers involved in the costimulatory (secondary) signaling pathway for T cell activation. T cell costimulatory molecules bind to their ligands (including but not limited to CD80 and CD86) or their receptor-binding fragments and / or antibodies (anti-CD28 antibodies) or their antigen-binding fragments, providing additional signals necessary to avoid anergy and for effective T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27:591-619).

[0290] Various activators that can bind to T cell activation molecules or co-stimulatory molecules are known in the art, including but not limited to anti-CD3 antibodies or their antigen-binding fragments, CD28 ligands (CD28L, including CD80 and CD86, etc.) or their receptor-binding fragments, anti-CD28 antibodies or their antigen-binding fragments, and 4-1BB ligands (4-1BBL or CD137L) or their receptor-binding fragments. In conventional techniques for preparing CAR-T cells in vitro, additional exogenous activators, such as anti-CD3 antibody magnetic beads and / or anti-CD28 antibody magnetic beads, are typically required to stimulate and activate inactive T cells.

[0291] Therefore, constructing multiple targeting molecules on the surface of the vector that target CD3, T cell co-stimulatory molecules (such as CD28) and CD7 can effectively improve the efficiency of the vector in transducing inactive T cells.

[0292] Endocytosis: Endocytosis is a process by which substances enter a cell. In endocytosis, the substance to be taken in is surrounded by a region of the plasma membrane, which then buds inside the cell to form a vesicle containing the taken-in substance. Endocytosis can be classified into four types: receptor-mediated endocytosis (also known as clathrin-mediated endocytosis), caveolae, pinocytosis, and phagocytosis (Marsh M, Endocytosis. Oxford University Press. p.vii., 2001).

[0293] Receptor-mediated endocytosis, or clathrin-mediated endocytosis, is mediated by the production of small (100 nm in diameter) vesicles with a morphologically distinctive shell composed of the cytoplasmic protein clathrin (McMahon HT, Boucurt E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nature Reviews. Molecular Cell Biology. 2011).

[0294] Clathrin-coated vesicles (CCVs) are present in almost all cells and form plasma membrane domains called clathrin-coated pits. Clathrin-coated pits can bind extracellular macromolecules to various receptors responsible for receptor-mediated ligand endocytosis, such as low-density lipoprotein, transferrin, growth factors, antibodies, and many other substances (Marsh M, McMahon HT (July 1999). The structural era of endocytosis. Science. 285(5425):215-220.).

[0295] The complement system, composed of a series of proteins, is part of the innate immune system. Complement (C) is present in the serum, tissue fluid, and cell membrane surface of normal humans and animals. Upon activation, it possesses enzymatic activity and undergoes complex cascade reactions. The complement system is initiated through a series of enzyme interactions, ultimately reaching the target...

[0296] Microorganisms form pore-like membrane attack complexes, causing them to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, clearing immune complexes through cytolysis, opsonization, phagocytosis, and mediating inflammatory responses, thus exhibiting corresponding biological functions. Complement is widely involved in the body's defense responses against microbial infections and immune regulation, and also mediates immunopathological damage responses, making it an important effector system and effector mechanism system in the body.

[0297] “Inhibition”: When referring to the ability of a viral glycoprotein to bind to its receptor, the term “inhibition” includes the complete elimination of the binding of the viral glycoprotein to its receptor, as well as a significant reduction in the binding. In a specific embodiment, “significant reduction” means the ability of a viral glycoprotein to bind to its receptor relative to the absence of a reduction in available binding sites and / or the absence of any of the first mutations; “significant 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%.

[0298] "T cells": T cells are one of the important types of white blood cells in the human immune system and play a crucial role in the acquired immune response. One of the main functions of T cells is immune-mediated cell death, a function primarily accomplished by two T cell subtypes: CD8+ and CD9+. + T cells (Cytotoxic T cells) and CD4 + T cells (Helper T cells).

[0299] In some embodiments of the present invention, the T cells are CD4 cells. + / CD8 - CD4 - / CD8 + CD4 + / CD8 + CD4 - / CD8 - T cells or combinations thereof. In some embodiments of the present invention, CD4 + T cells produce IL-2, TFN, TNF, or combinations thereof after expressing TCP and / or CAR and binding to target tumor cells. In some embodiments of the present invention, CD8... + T cells lyse antigen-specific target cells after expressing TCP and / or CAR and binding to target cells.

[0300] "Non-activated T cells" refer to T cells that are not proliferating, undifferentiated, resting, do not recognize antigens, and have not been activated by T cell activation and / or stimulated by co-stimulatory molecules in the T cell activation signaling pathway and / or co-stimulatory signaling pathway. Examples include T cells in the G0 phase of the cell cycle, resting / quiiescent T cells, or immature T cells. T cells.

[0301] "T-cell receptor chimeric protein" (TCP) is a recombinant protein comprising various polypeptides derived from the TCR / CD3 complex, such as TCR subunits or functional fragments thereof, and an extracellular antigen-binding region capable of specifically binding to at least one antigen. The TCP typically binds to antigens on the surface of target cells via its included extracellular antigen-binding region. In some embodiments of the present invention, the extracellular antigen-binding region can be linked to the TCR subunit or its functional fragment via a linker peptide; exemplaryly, the extracellular antigen-binding region can be operatively linked to the TCR subunit or its functional fragment via a flexible linker peptide. In some embodiments of the present invention, the TCP further comprises a signal peptide encoded by a leader sequence.

[0302] "Chimeric Antigen Receptor" (CAR) refers to an artificial cell surface receptor modified to be expressed on immune effector cells, including T cells, and specifically bind to antigens. It comprises at least (1) an extracellular antigen-binding region, such as scFv or VHH; (2) a transmembrane region anchoring the CAR to the cell membrane; and (3) an intracellular signal transduction domain. The extracellular structure of the CAR may further include a hinge region; the intracellular structure of the CAR may further include a co-stimulatory signal transduction domain. The CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner using its extracellular antigen-binding region. In some embodiments of the present invention, the CAR further comprises a signal peptide encoded by a leader sequence.

[0303] "Flexible linkers" are typically used when linked domains require a certain degree of movement or interaction (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10):1357-69.). Flexible linkers are usually composed of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990; 211:943–958.). These small amino acids provide flexibility and also allow for the movement of linked functional domains. For commonly used flexible linker peptides, see Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10):1357-69, which is incorporated herein by reference in its entirety.

[0304] "Antibody" refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or the variable regions of the immunoglobulin light chain, enabling it to specifically bind to an antigen. In this article, "antibody" encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0305] The term "antibody" in this article includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin class consisting of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain from its N-terminus to its C-terminus. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain refers to a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) from its N-terminus to its C-terminus. The heavy chains are linked to each other and to each other with light chains by disulfide bonds, forming a "Y"-shaped structure.

[0306] The term "antibody" in this article also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by camels (Camelus dromedarius), Bactrian camels (Camelus Bactrianus), llamas (Lama Glama), guanicoes (Lama Guanicoe), and alpacas (Vicugna Pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0307] The terms "VHH domain," "nanoantibody," and "single-domain antibody" (sdAb) used in this article have the same meaning and are used interchangeably. They refer to the cloning of the variable region of a heavy chain antibody to construct a single-domain antibody consisting of only one heavy chain variable region. This is the smallest antigen-binding fragment with complete function. Typically, a naturally occurring heavy chain antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0308] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. Non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, alpacas, ostriches, cynomolgus monkeys (e.g., cynomolgus monkeys and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0309] In this article, "antigen-binding fragment" refers to a part or a variant of a complete antibody that does not possess the full structure of the complete antibody, but only contains a part or a variant of the complete antibody, and the part or variant has the ability to bind antigens.

[0310] For example, in this document, "antibody or its antigen-binding fragment" includes, but is not limited to: immunoglobulin (full-length antibody), hapten, Fab, Fab', F(ab')2, Fv fragment, single-chain variable region fragment (scFv), disulfide bond stable antibody (dsFv), heavy chain variable region (VH) or light chain variable region (VL) of antibody, Fd fragment composed of VH and CH1 domains, linear antibody, heavy chain antibody and nanobody (VHH).

[0311] In some embodiments of the present invention, there is no particular limitation on the order in which scFv contains VH or VL from the N-terminus to the C-terminus, such as VH-Linker-VL or VL-Linker-VH from the N-terminus to the C-terminus; the linker peptide may be selected from flexible linker peptides.

[0312] "Ligand": In receptor-ligand binding, the ligand is typically a molecule that binds to a site on the receptor to generate a signal. This binding usually leads to a conformational change in a complex structure, thereby inducing related physiological activities.

[0313] "Receptor-binding fragment" refers to a fragment that does not possess the complete structure of the complete ligand, but only contains a portion or a variant of the complete ligand, which has the ability to bind to a receptor. For example, "receptor-binding fragment" herein includes, but is not limited to, the extracellular domain and variable region of the ligand.

[0314] "Variant": A variant is a mutant that has at least 50% identity with the amino acid sequence of a non-mutant (wild type). "At least 50% identity" means that the variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the amino acid sequence of a non-mutant (wild type).

[0315] "Shuttle gene": also known as "transgenic gene", includes exogenous target genes / nucleic acids carried by vectors such as LVV or RVV.

[0316] “Nucleic acid” refers to any compound and / or substance, such as a polynucleotide, that comprises polymers containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5' to 3'. In this document, the term “nucleic acid” encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. “Nucleic acid” can be linear or circular. Furthermore, “nucleic acid” includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Moreover, the “nucleic acid” described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbones or chemically modified residues.

[0317] "Exogenous" refers to any molecule originating from outside the organism, including nucleic acids, proteins, polypeptides, or small molecule compounds. In contrast, the term "endogenous" refers to any molecule originating from within the organism (i.e., naturally produced by the organism).

[0318] "Viral envelope": This refers to the outermost layer of many viruses (HURLBERT, RONALD E., Fundamentals of Microbiology, 102. Chapter #11: Viruses. Archived from the original on 2008-11-10.). As viruses shuttle through host cells, the viral envelope protects their genetic material throughout their life cycle. Not all viruses have a viral envelope. Many human pathogenic viruses are encased in a lipid bilayer, and they transduce target cells by fusing the viral envelope with the cell membrane.

[0319] "Retrovirus": A retrovirus is a double-stranded RNA enveloped virus, characterized by its ability to "reverse transcribe" its genome from RNA to DNA. Viral particles are measured to be 100-120 nm in diameter and contain a dimer genome of the same positive RNA strand complexed with nucleocapsid proteins. The genome is encapsulated in a protein capsid that also contains enzymes—reverse transcriptase, integrase, and protease—required for viral infection. Matrix proteins form a layer outside the capsid core, interacting with the lipid bilayer derived from the host cell membrane surrounding the viral core particle. Anchored to this bilayer are viral envelope glycoproteins responsible for recognizing specific receptors on the host cell and initiating the infection process. These envelope (glyco)proteins consist of two subunits: one anchoring the protein to the transmembrane (TM) lipid membrane, and the other binding to the surface (SU) of the cell receptor.

[0320] Retroviruses contain reverse transcriptase and integrase. Upon entering a target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecules into DNA molecules. 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 sequence).

[0321] Based on their genomic structure, retroviruses are classified into simple retroviruses, such as MLV and murine leukemia virus, and complex retroviruses, such as HIV and EIAV. Retroviruses contain four genes: gag (group-specific antigen), pro (protease), pol (polymerase), and env (env). The gag sequence encodes three main structural proteins: matrix protein, nucleocapsid protein, and capsid protein. The pro sequence encodes the protease responsible for cleaving Gag and Gag-Pol during viral particle assembly, budding, and maturation. The pol sequence encodes reverse transcriptase and integrase; the former catalyzes the reverse transcription of the viral genome from RNA to DNA during infection, while the latter is responsible for integrating proviral DNA into the host cell genome. The env sequence encodes the SU and TM subunits of the envelope glycoprotein. In addition, retroviral genomes exhibit non-coding cis-acting sequences, such as: two LTRs (long terminal repeats) containing elements necessary for driving gene expression, reverse transcription, and integration into the host cell chromosome; a sequence called the packaging signal (ψ) required for the specific packaging of viral RNA into newly formed virions; and a polypurine tract (PPT) that serves as the site for initiating positive-strand DNA synthesis during reverse transcription. Besides gag, pro, pol, and env, complex retroviruses such as lentiviruses have accessory genes including vif, vpr, vpu, nef, tat, and rev, which regulate viral gene expression in infected cells, assembly of infected viral particles, and regulation of viral replication.

[0322] Lentivirals are complex retroviruses that contain not only the common retroviral genes gag, pol, and env, but also other genes with regulatory or structural functions. This high complexity allows viruses to regulate their life cycle, as they do during latent infection. Lentivirals belong to the genus of retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV types I and II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simultaneous immunodeficiency virus (SIV).

[0323] "Lentiviral vector (LVV) and retroviral vector (RVV)": As used herein, the term "lentiviral vector (LVV) or retroviral vector (RVV)" is intended to refer to self-inactivated viral particles that include a viral envelope, possess one or more characteristics of lentiviruses or retroviruses, are capable of invading target cells and delivering the gene of interest (GOI), and are not capable of self-replication.

[0324] LVV and RVV can stably integrate exogenous load genes, such as CAR genes, into the chromosomes of target cells, allowing target cells to express the delivered transgenes long-term, providing a significant advantage for gene therapy. Furthermore, they do not transfer viral genes, thus avoiding the problem of transduced cells that can be destroyed by cytotoxic T cells. They also possess relatively large cloning capacity, sufficient to meet most anticipated clinical applications.

[0325] "Packaging system": as used herein, refers to a vector system containing one or more nucleic acid vectors that, when introduced into packaging cells / packaging cell lines for packaging LVV or RVV, contain nucleic acids necessary for the production, assembly, and / or packaging of lentiviral or retroviral vectors in the packaging cells / packaging cell lines.

[0326] In some embodiments of the present invention, the packaging system comprises (a) nucleic acids encoding viral proteins necessary for the production, assembly, and / or packaging of LVV or RVV in packaging cell lines and (b) signals providing the signals required for key functions such as viral replication, packaging, reverse transcription, and integration.

[0327] Commonly used lentiviral vector packaging systems include the so-called third-generation LVV packaging system. The third-generation LVV packaging system includes four plasmids: typically, a transfer plasmid and three packaging plasmids: a transfer plasmid / master plasmid containing transgenic / shuttle genes encoding the target gene / GOI, such as the CAR gene; a GagPol plasmid; a Rev plasmid; and an envelope plasmid (containing viral glycoprotein genes such as VSV-G or its variants, or Cocal-G or its variants).

[0328] Typically, a "shuttle / transfer vector" contains the lentiviral backbone genes, transgenes, and signals necessary for key functions such as viral replication, packaging, reverse transcription, and integration. Transfer vectors usually contain one or more transgenes flanked by long terminal repeats (LTRs), which facilitate the integration of transgenes, such as CAR genes, contained in the transfer vector into the host genome. LTRs are responsible for the reverse transcription and integration of the viral genome. Through these sequences, lentiviruses can integrate transgenes into the host cell's genome. For safety reasons, transfer vectors are often engineered to prevent the resulting viral vector from self-replicating; for example, the transfer vector may lack the genetic elements necessary for the production of infectious LVV in the host cell. Furthermore, transfer plasmids can be engineered to lack the 3'LTR, thereby achieving "self-inactivating" of the virus. Compared to traditional second-generation pseudotyped LVV packaging systems (typically containing a single packaging plasmid and a separate envelope plasmid encoding nucleic acids for Gag, Pol, Rev, and Tat), the TAT gene is eliminated from third-generation pseudotyped LVV packaging systems by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) to the transfer plasmid. Transfer plasmids typically contain a Ψ sequence (Psi sequence, also known as the Ψ packaging signal) downstream of the 5'LTR, responsible for packaging the transgenic RNA into the viral vector. The Ψ sequence ensures that only the transgenic RNA is packaged into the viral vector. Optionally, the transfer plasmid may also include an internal ribosome entry site. Sites (“IRES”) are used to allow simultaneous translation of two or more open reading frames (ORFs) on a single mRNA, thereby enabling multigene expression. Some transfer plasmids, such as the master plasmid / transfer plasmid used in some embodiments of the present invention, may also contain selection marker genes, such as antibiotic resistance genes (e.g., PuroR, encoding puromycin resistance) or fluorescent protein genes (e.g., GFP), for screening or tracking transduced cells.

[0329] Transfer plasmids for LVV packaging systems 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 No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, transfer plasmids contain basic nucleic acid sequences configured to carry for selecting vector-containing cells, for incorporating foreign nucleic acids into lentiviral particles, and for transferring nucleic acids to target cells.

[0330] Lentiviral backbone genes typically refer to the most basic and essential cis-acting elements that make up the transfer plasmid. These sequences do not encode viral proteins but provide the signals needed for key functions such as viral replication, packaging, reverse transcription, and integration. Specifically, lentiviral backbone genes typically include: long terminal repeats (LTRs): located at both ends of the genome, containing promoters, enhancers, and terminators that regulate viral gene transcription and integration; packaging signals (Ψ): determining which RNA molecules can be recognized and encapsulated into viral particles; central polypurine region (cPPT) and central termination signal (CTS): contributing to improved reverse transcription efficiency and nuclear transport; and Rev response elements (RREs): binding to Rev proteins and regulating the transport of viral RNA from the nucleus to the cytoplasm.

[0331] When constructing transfer plasmids, to ensure safety and efficiency, only the essential backbone sequences are typically retained in the transfer plasmid, while sequences encoding viral structural proteins and enzymes (such as gag, pol, env, etc.) are removed and provided by the packaging system in helper plasmids. This design not only ensures the functionality of the vector but also reduces the risk of generating replicating viruses.

[0332] In some embodiments of the invention, the "backbone gene" is intended to include a nucleic acid encoding a lentiviral or retroviral cis-nucleotide sequence required for genome packaging. The backbone gene may also encode other cis-nucleotide sequences beneficial for gene delivery, including, for example, cis-sequences required for reverse transcription, proviral integration, or genome transcription. Therefore, the exact composition of the backbone gene will depend on the genetic material desired to be introduced into the target cell. Thus, the backbone gene may encode, for example, additional polypeptides or functions besides those required for packaging, reverse transcription, integration, or transcription. Such functions typically include cis-elements encoding those required for the expression of the target nucleic acid / shuttle gene.

[0333] Transfer plasmids for lentiviral and retroviral vectors 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.

[0334] For a comparison and discussion of the packaging systems of lentiviral vectors and retroviral vectors, and the transfer plasmids they contain, please see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, KK, Vorburger, SA, Swisser, SG (eds). Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0335] The packaging system of third-generation lentiviral vectors typically includes three packaging plasmids: GagPol plasmid, Rev plasmid, and envelope plasmid. The envelope plasmid usually carries a viral envelope glycoprotein gene; for example, wild-type VSV-G or Cocal-G are commonly used viral glycoproteins. The viral glycoprotein gene is operatively linked to a promoter, typically a CMV promoter, to initiate transcription of the viral glycoprotein gene. In some embodiments of the present invention, the envelope plasmid contains an encoding of any of the aforementioned targeting molecules provided by the present invention.

[0336] The third-generation lentiviral vector packaging system also includes two packaging plasmids: one containing genes encoding the Gag and Pol proteins (GagPol packaging plasmid), and the other containing a gene encoding the Rev protein (Rev plasmid) as a further safety feature, an improvement over the single packaging plasmid of the so-called second-generation packaging system. The Gag gene encodes the Gag polyprotein precursor, which contains lentiviral structural proteins including the matrix, capsid, and nucleocapsid; the Pol gene encodes the Pol polyprotein precursor, which provides the lentiviral enzyme functions necessary for replication, containing a protease, reverse transcriptase, and integrase; the Rev gene encodes the Rev protein, which binds Rev response elements (RREs) to allow the nuclear export of unspliced ​​and single-spliced ​​HIV RNA during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral vector preparation. The Rev protein binds to the Rev response element (RRE) sequence on the viral RNA, facilitating the transport of incompletely cleaved viral RNA from the nucleus to the cytoplasm through interaction with the host cell's nuclear export mechanism. This unspliced ​​RNA can then be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral vectors.

[0337] For example, the packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0338] Compared to LVV packaging systems, RVV packaging systems typically do not contain Rev plasmids. 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. RVV packaging systems typically contain one transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgenic sequence contained in the transfer plasmid is flanked by long terminal repeats (LTRs), which facilitate the integration of the transfer plasmid sequence into the host genome. Generally, during viral transduction, sequences between and including LTRs are integrated into the host genome. The genomes of MMLV or Murine Stem Cell Virus (MSCV), containing their respective LTRs, are often used to construct the transfer plasmid in the RVV packaging system. The GagPol packaging plasmid contains the Gag and Pol genes; the envelope plasmid typically contains a polynucleotide encoding a viral glycoprotein, such as VSV-G or Cocal-G. In some embodiments of the present invention, the envelope plasmid may also contain a polynucleotide encoding any of the aforementioned target molecules.

[0339] In some embodiments, production cells are transfected with transfer plasmids, GagPol plasmids, envelope plasmids, and Rev plasmids in defined ratios. 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 lentiviral or retroviral vector. In some embodiments, the mass of each of the transfer plasmids and GagPol plasmids packaging the lentiviral vector is higher than the mass of each of the envelope plasmids and Rev plasmids. In some embodiments, the defined ratio of transfer plasmids, GagPol plasmids, Rev plasmids, and envelope plasmids is from about 1:1:1:1 to about 10:5:4:4. In some embodiments, the defined ratio of transfer plasmids, GagPol plasmids, envelope plasmids, and Rev plasmids is about 9:4:2:2. In some embodiments of the invention, the envelope plasmid may contain nucleic acids encoding one or more target molecules.

[0340] In some embodiments, the envelope plasmid contains a targeting molecule encoding the surface of any of the aforementioned LVV or RVV. In some embodiments, the tandem expression cassette contained in the envelope plasmid contains a polynucleotide encoding a first signal peptide, a polynucleotide encoding the target molecule, a polynucleotide encoding one of the internal ribosome entry site (IRES), a furin cleavage site, or viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof. In some embodiments, the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof is located at the 5' end of the polynucleotide encoding the target molecule. In other embodiments, the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof is located at the 3' end of the polynucleotide encoding the target molecule. The polynucleotide encoding the target molecule and the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof are separated in the tandem cassette by the polynucleotide encoding IRES, the furin cleavage site, or viral 2A peptide, which allows co-expression of both proteins by a single mRNA. In some embodiments, the viral 2A peptide is porcine cyclovir-1 (P2A), *Thosea asigna* virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. In some embodiments, the viral 2A peptide includes derivatives thereof, such as derivatives of the T2A peptide, T2A cleavage sites with furin cleavage sites and GSG linkers, and FT2A peptides. In some embodiments, at least two different promoters independently drive the expression of polynucleotides encoding any of the aforementioned viral glycoproteins or variants thereof and polynucleotides encoding the target molecule, respectively.

[0341] The use of lentiviral vector systems relies on "packaging cells." Generally, packaging cells are cells that, when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cell, produce a lentiviral vector that lacks self-replication capability and can infect / transduce target cells. Exemplarily, plasmids can be introduced into packaging cells using transduction / transfection methods including chemically mediated, physically mediated, or biologically mediated methods. For example, chemically mediated transduction / transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-glucan, or PEI (polyethylenimine transduction reagent), while physically mediated transduction methods include transduction methods such as electroporation.

[0342] The packaging cells are genetically engineered to improve the immune properties of the lentiviral and retroviral vectors disclosed in this invention and / or promote the transduction of target cells by the lentiviral and retroviral vectors in other ways; the other ways include, but are not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0343] In some embodiments of the present invention, the packaging cells include, but are not limited to, at least one of the following cells: CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, and 211 cells;

[0344] Preferably, the packaging cells are HEK-293T cells.

[0345] "Viral glycoproteins" are glycoproteins that coat the outer layer of a virus. They play an important role in viral adsorption and penetration into host cells, pathogenicity, downregulation of host surface protein expression, and increased viral packaging and budding processes.

[0346] “Subject”: As used herein, “subject” or “individual” is used synonymously, including but not limited to mammals, such as humans or non-human mammals, such as livestock, agricultural animals or wild animals, and birds and aquatic animals.

[0347] "Patient" is a subject who suffers from a disease, condition or illness, is at risk of developing a disease, condition or illness, or otherwise requires any of the lentiviral or retroviral vectors, CAR-T cells, TCP-T cells, compositions, methods or applications provided herein.

[0348] "Administer" refers to "administer" (including administration). The carrier disclosed in this invention can be administered via any route, including but not limited to at least one of the following: oral, nasal, intravenous, intraperitoneal, intracerebral (within brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, intratumoral, subcutaneous, continuous release system, and implantable device. In some embodiments of this invention, the administration includes at least one of the following: intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection, and infusion techniques.

[0349] "And / or": should be understood as referring to one or two alternatives.

[0350] “About” / “Approximately”: As used herein, the term “about” refers to a common range of error for a corresponding value that is readily known to those skilled in the art, exemplarily including, but not limited to, a variation of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length. References herein to “about” a value or parameter include (and describe) embodiments of said value or parameter itself; for example, a description of “about X” includes a description of “X”.

[0351] Numerical ranges: Unless otherwise stated, any concentration range, percentage range, ratio range or integer range in this document shall be understood to include any integer value within the range, and, where appropriate, its fraction (such as one-tenth and one-hundredth of an integer).

[0352] "Comprising": In this document, unless the context otherwise requires, the word "comprising" will be understood to mean including the specified steps, elements, or groups of steps or elements, but not excluding any other steps, elements, or groups of steps or elements. In some embodiments of the invention, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0353] "Embodiments": Throughout this specification, references to "some embodiments," "some embodiments," "implementation," "related embodiments," "a particular embodiment," or "other embodiments," or combinations thereof, refer to specific features, structures, or characteristics described in connection with embodiments that are included in at least one embodiment of the invention. Therefore, the appearance of the foregoing phrases in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0354] “Treatment”: As used in this article, “treatment” includes any beneficial or desired effect associated with treatment. “Treatment” does not necessarily mean the complete eradication or cure of a disease or condition, or its associated symptoms.

[0355] "Prevention": As used herein, "prevention" and similar terms, such as "avoidance," indicate methods used to prevent, suppress, or reduce the likelihood of the occurrence or recurrence of a condition. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition before its onset or recurrence.

[0356] "Stable integration": also known as "stable transduction" or "stable gene expression", refers to the integration of exogenous nucleic acids into the host cell genome after they are introduced into the host cell, and their stable expression in the host cell over a long period of time.

[0357] "Specific binding": As used herein, the term "specific binding" refers to binding that occurs between paired molecular species (e.g., receptor and ligand, antibody and antigen). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, specific binding between one or more species is direct. In some embodiments of the invention, the affinity of specific binding is 1, 1.5, 2, 5, 10, 20, 50, 100, or 1000 times or more of background binding (non-specific binding).

[0358] "Sequence identity": Generally speaking, "sequence identity" or "sequence homology" refers to the precise correspondence between nucleotides or amino acids of two nucleic acid sequences or protein / peptide sequences. Typically, techniques used to determine sequence identity involve identifying the nucleotide sequence of the nucleic acid and / or the amino acid sequence it encodes, and comparing these sequences to control nucleotide or amino acid sequences. Two or more sequences (nucleic acids or amino acids) can be compared by determining their "percentage of identity." Whether it's a nucleic acid or amino acid sequence, the percentage of identity between two sequences is the number of precise matches between the two aligned sequences divided by the length of the shorter sequence, and then multiplied by 100. For example, the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percentage of identity. The BLAST procedure is based on the following alignment methods: Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). In short, the BLAST procedure defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of shorter symbols in both sequences. The procedure can be used to determine the percentage of identity across the entire length of the compared protein / peptide.

[0359] “Signal peptide”: Signal peptide, sometimes also called signal sequence, target signal, localization signal, localization sequence, transport peptide is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), including a leader sequence encoding a leader signal peptide; signal peptides are present at the N-terminus of most newly synthesized proteins leading to the secretory pathway (occasionally non-classically present at the C-terminus or inside) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Journal of Cell Biology. 97(6):422-441. (2018)) (Blobel G, Dobberstein B, et al., Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, The Journal of Cell Biology, 67(3):835-51. (1975). The role of signal peptides includes facilitating the transfer of proteins within cells, typically to the cell membrane.

[0360] "MOI" stands for "Multiplicity of Infection (MOI)," which refers to the number of viral particles added to each cell during viral infection or transduction. For example, when one million viral particles are added to one million cells, MOI = 1.

[0361] "Operably linked": Nucleic acids are "operably linked" when they are functionally related to each other. For example, operably linked nucleic acid sequences can be adjacent to each other; and, for example, are in the same reading frame when binding to two protein-coding regions is required. For example, if the DNA of a leader sequence or secretory leader sequence is expressed as a preprotein involved in polypeptide secretion, then that DNA is operably linked to the DNA of that polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then that promoter or enhancer is operably linked to that sequence; or, if a ribosome binding site is located to facilitate translation, then that ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked nucleic acids are adjacent, and in the case of a secretory leader sequence, adjacent and in the reading frame. However, enhancers do not necessarily need to be adjacent. Linkage is achieved by linking at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0362] "2A peptide": The term "2A peptide" refers to a self-cleaving peptide configured to generate two or more proteins from a single open reading frame, including FT2A peptide, F2A peptide, E2A peptide, T2A peptide, and P2A peptide. 2A peptides are 18 to 22 residue-long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" can refer to peptides with different amino acid sequences. In this disclosure, it should be understood that in the case where vectors such as lentiviral or retroviral vectors contain two or more 2A peptides, the 2A peptides may be the same or different from each other. Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) ColdSpring Harb. Protoc. 2012:199-204.

[0363] "Self": As used in this article, the term "self" means any material derived from the same individual that is subsequently reintroduced into that individual.

[0364] "Allogeneic" as used in this article refers to grafts that are derived from different individuals of the same species.

[0365] “Transduction”: As used in this article, the terms “transfect” or “transduce” refer to the process of introducing exogenous nucleic acids into host cells, packaging cells, or other cells. “Transfected” or “transduced” cells are cells that have been transduced or transduced with exogenous nucleic acids. These cells include primary cells and their progeny.

[0366] “Therapeutic effective amount”: As used herein, “therapeutic effective amount” is the amount of a composition or its active substance administered to an individual that is sufficient to provide a beneficial effect or otherwise reduce harmful, non-beneficial events. “Therapeutic effective dose” here means, as a dose that produces one or more desired or anticipated (e.g., beneficial) effects as a result of its administration, given once or more times over a specified period of time. The exact dose will depend on the therapeutic purpose and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0367] "Pharmaceutically acceptable excipients or carriers": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration used.

[0368] All publications, documents, and patents mentioned herein are hereby incorporated in their entirety by reference, as are each publication, document, or patent not specifically and individually indicated to be incorporated herein by reference in its entirety. In case of conflict, this application (including any definitions herein) shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or recommendation of any kind.

[0369] The section headings used in this article are for organizational purposes only and are not intended to limit the topics described. Attached Figure Description

[0370] Figure 1: In Example 1, LVV-G1-GFP, LVV-G1A3-GFP, LVV-G1A3 / 28-GFP, LVV-G1A7-GFP, and LVV-G1A3 / 28 / 7-GFP transduced non-activated PBMCs of Donor 1 and Donor 2, respectively. The CD3+ levels in each group of PBMCs were detected. + Flow cytometry results of GFP positivity rate in cells;

[0371] Figure 2: Flow cytometry results of the CAR-19 positivity rate in each group of PBMCs transduced by LVV-G1-CAR19, LVV-G1A3-CAR19, LVV-G1A3 / 28-CAR19, LVV-G1A7-CAR19 and LVV-G1A3 / 28 / 7-CAR19 in Example 2, respectively.

[0372] Figure 3: Flow cytometry results of the CAR-19 positivity rate in each group of PBMCs transduced by LVV-G2-CAR19, LVV-G2A3-CAR19, LVV-G2A7-CAR19, LVV-G2A3 / 28-CAR19 and LVV-G2A3 / 28 / 7-CAR19 in Example 3, respectively.

[0373] Figure 4: A bar chart showing the detection and calculation of the titers of LVV-G2A3-CAR19, LVV-G2A3 / 28-CAR19, LVV-G2A7-CAR19 and LVV-G2A3 / 28 / 7-CAR19 in Example 3;

[0374] Figure 5: A bar chart showing the detection and calculation of the titers of LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, LVV-G2mA7-CAR19 and LVV-G2mA3 / 28 / 7-CAR19 in Example 4;

[0375] Figure 6: Flow cytometry results of the CAR-19 positivity rate in each group of PBMCs transduced with LVV-G2-CAR19, LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, LVV-G2mA7-CAR19 and LVV-G2mA3 / 28 / 7-CAR19 in Example 4, respectively.

[0376] Figure 7: The plasmid map of the main plasmid CAR19 in Example 2;

[0377] Figure 8: The plasmid map of the envelope plasmid G1Atri in Example 1. Detailed Implementation

[0378] The present invention and its technical effects will be clearly and completely described below with reference to embodiments, so as to fully understand the technical solution, the technical problem solved, and the beneficial effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments; other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0379] Experimental methods not specifically described in the following examples were performed according to conventional methods and conditions known in the art, or according to the product instructions. Reagents and raw materials not specifically described in this invention are all commercially available.

[0380] Example 1

[0381] The packaging virus envelope contains LVV-G1A3 / 28 / 7-GFP (LVV-G1A3 / 28 / 7-GFP) that expresses anti-CD3, anti-CD28, and anti-CD7 antibodies against human CD3, CD28, and CD7.

[0382] 1. Construct membrane expression structures for anti-CD3 antibody × anti-CD28 antibody × anti-CD7 antibody

[0383] In this embodiment, the polynucleotide encoding the membrane-expressed anti-CD3 antibody × anti-CD28 antibody × anti-CD7 antibody (membrane-expressed triantibody) sequentially encodes from the 5' end to the 3' end as follows: human CD8α signal peptide, anti-CD3 antibody (scFv derived from UCHT1, scFv-UCHT1), human CD8α hinge region, human CD8α transmembrane region, FT2A peptide, human CD8α signal peptide, anti-CD28 antibody (scFv derived from 15E8, scFv-15E8), human CD8α hinge region, human CD8α transmembrane region, FT2A peptide, human CD8α signal peptide, anti-CD7 antibody (scFv derived from TH69, scFv-TH69), human CD8α hinge region, human CD8α transmembrane region;

[0384] (1) The amino acid sequence of the human CD8α signal peptide is shown in SEQ ID NO:8;

[0385] (2) The amino acid sequence of scFv-UCHT1 is shown in SEQ ID NO:9, the amino acid sequence of the VH region of scFv-UCHT1 is shown in SEQ ID NO:35, the amino acid sequence of the VL region of scFv-UCHT1 is shown in SEQ ID NO:36, the amino acid sequences of the HCDR1-3 regions of scFv-UCHT1 are shown in SEQ ID NO:37-39, the amino acid sequences of the LCDR1-3 regions of scFv-UCHT1 are shown in SEQ ID NO:40-42, the VH region of scFv-UCHT1 is connected to the VL region of scFv-UCHT1 through the (G4S)3 linker peptide, and the amino acid sequence of the (G4S)3 linker peptide is shown in SEQ ID NO:16;

[0386] (3) The amino acid sequence of the hinge region of the human CD8α is shown in SEQ ID NO:10;

[0387] (4) The amino acid sequence of the transmembrane region of the human CD8α is shown in SEQ ID NO:11;

[0388] (5) The amino acid sequence of the FT2A peptide is shown in SEQ ID NO:12;

[0389] (6) The amino acid sequence of the scFv-15E8 is shown in SEQ ID NO:13, the amino acid sequence of the VH region of the scFv-15E8 is shown in SEQ ID NO:43, the amino acid sequence of the VL region of the scFv-15E8 is shown in SEQ ID NO:44, the amino acid sequences of the HCDR1-3 regions of the scFv-15E8 are shown in SEQ ID NO:45-47, and the amino acid sequences of the LCDR1-3 regions of the scFv-15E8 are shown in SEQ ID NO:48-50.

[0390] (7) The amino acid sequence of the scFv-TH69 is shown in SEQ ID NO:51, the amino acid sequence of the VH region of the scFv-TH69 is shown in SEQ ID NO:19, the amino acid sequence of the VL region of the scFv-TH69 is shown in SEQ ID NO:20, the VH region of the scFv-TH69 is connected to the VL region of the scFv-TH69 through the (G4S)3 linker peptide, the amino acid sequences of the HCDR1-3 regions of the scFv-TH69 are shown in SEQ ID NO:52-54, and the amino acid sequences of the LCDR1-3 regions of the scFv-TH69 are shown in SEQ ID NO:55-57.

[0391] 2. Packaging LVV-G1A3 / 28 / 7-GFP

[0392] A. Prepare the following four plasmids: an envelope plasmid (encapsulation plasmid G1Atri) containing a polynucleotide encoding wild-type VSV-G (expressed by the CMV promoter) and a polynucleotide encoding the membrane-expressed triple antibody (expressed by the PGK promoter); a pMDLg / pRRE packaging plasmid; a pRSV-REV packaging plasmid; and a pGClenti-GFP plasmid (master plasmid GFP) carrying a polynucleotide (GFP gene) encoding GFP (Green Fluorescent Protein); the envelope plasmid G1Atri was synthesized using conventional molecular cloning methods; the plasmid map of the envelope plasmid G1Atri is shown in Figure 8.

[0393] The wild-type VSV-G contains the amino acid sequence shown in SEQ ID NO:1; the amino acid sequence of the full-length protein of the wild-type VSV-G containing the VSV-G signal peptide is shown in SEQ ID NO:22.

[0394] B. Packaging LVV-G1A3 / 28 / 7-GFP:

[0395] (1) The four plasmids were mixed and transfected into HEK-293T packaging cells using PEI reagent. The specific steps are as follows:

[0396] 9 μg of the main plasmid GFP, 4 μg of the pMDLg / pRRE packaging plasmid, 2 μg of the pRSV-REV packaging plasmid, and 2 μg of the envelope plasmid G1Atri 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 culture medium of HEK-293T cells. The culture 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 LVV-G1A3 / 28 / 7-GFP was resuspended in 200 μL of F12 medium and stored at -80 °C.

[0397] Opti-MEM alpha serum-reduced culture medium, brand: GIBCO, catalog number: #SP0272;

[0398] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog No.: #C12430500BT; FBS: Brand: EXCELL, Catalog No.: #FSP500;

[0399] F12 medium: Brand: GIBCO, Product No.: #C11330500BT;

[0400] Needle filter: Brand: SORFA, Item No.: #622120.

[0401] 3. Packaging control group LVV

[0402] Following the method described for packaging LVV-G1A3 / 28 / 7-GFP, multiple control group LVVs were packaged, with the viral envelope either not containing any targeting molecule or containing anti-CD3 antibody, anti-CD7 antibody, or anti-CD3 antibody × anti-CD28 antibody (double antibody).

[0403] A. Packaging control group LVV-G1A3-GFP

[0404] In this example, a membrane-expressing anti-CD3 antibody is constructed. The polynucleotide encoding the membrane-expressing anti-CD3 antibody sequentially encodes the following from the 5' end to the 3' end: the human CD8α signal peptide, the scFv-UCHT1, the human CD8α hinge region, and the human CD8α transmembrane region.

[0405] Following the method described above for packaging LVV-G1A3 / 28 / 7-GFP, the control group LVV-G1A3-GFP was packaged. The specific method is as follows:

[0406] Using PEI reagent, an envelope plasmid (enveloping plasmid G1A3) containing a polynucleotide encoding the wild-type VSV-G and a polynucleotide encoding the membrane-expressing anti-CD3 antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the main plasmid GFP were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing anti-CD3 antibody and the wild-type VSV-G and carried the GFP gene in the control group LVV-G1A3-GFP.

[0407] B. Packaging control group LVV-G1A7-GFP

[0408] In this example, a membrane-expressing anti-CD7 antibody is constructed. The polynucleotide encoding the membrane-expressing anti-CD7 antibody sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the VH region of the scFv-TH69, the (G4S)3 linker peptide, the VL region of the scFv-TH69, the human CD8α hinge region, and the human CD8α transmembrane region.

[0409] Following the method described for packaging LVV-G1A3 / 28 / 7-GFP, the control group LVV-G1A7-GFP was packaged. The specific method is as follows:

[0410] Using PEI reagent, an envelope plasmid (enveloping plasmid G1A7) containing a polynucleotide encoding the wild-type VSV-G and a polynucleotide encoding the membrane-expressing anti-CD7 antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the main plasmid GFP were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing anti-CD7 antibody and the wild-type VSV-G and carried the GFP gene in the control group LVV-G1A7-GFP.

[0411] C. Packaging control group LVV-G1A3 / 28-GFP

[0412] In this example, a membrane-expressing anti-CD3 antibody × anti-CD28 antibody (biantibody) is constructed. The polynucleotide encoding the membrane-expressing biantibody sequentially encodes the following from the 5' end to the 3' end: the human CD8α signal peptide, the scFv-UCHT1, the human CD8α hinge region, the human CD8α transmembrane region, the FT2A peptide, the human CD8α signal peptide, the scFv-15E8, the human CD8α hinge region, and the human CD8α transmembrane region.

[0413] Following the method described above for packaging LVV-G1A3 / 28 / 7-GFP, the control group LVV-G1A3 / 28-GFP was packaged. The specific method is as follows:

[0414] Using PEI reagent, an envelope plasmid (enveloping plasmid G1A3 / 28), pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the main plasmid GFP, containing polynucleotides encoding the wild-type VSV-G and polynucleotides encoding the membrane-expressing bispecific antibody, were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing bispecific antibody and the wild-type VSV-G and carried the GFP gene in the control group LVV-G1A3 / 28-GFP.

[0415] D. Packaging control group LVV-G1-GFP

[0416] Following the method described for packaging LVV-G1A3 / 28 / 7-GFP, the control group LVV-G1-GFP was packaged. The specific method is as follows:

[0417] Using PEI reagent, pMD2.G envelope plasmid (containing a polynucleotide encoding the wild-type VSV-G), pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the main plasmid GFP were transfected into HEK-293T packaging cells. The control group LVV-G1-GFP contained the wild-type VSV-G envelope and carried the GFP gene.

[0418] 4. Transduced inactive human PBMCs (MOI=2)

[0419] Five groups of 1×10⁵ PBMCs were resuscitated from cryopreserved inactive PBMCs of Donor 1 (healthy individuals) and Donor 2 (healthy individuals), respectively. 5 Individual inactive PBMCs (the method of thawing and freezing PBMCs is well known to those skilled in the art); each group of inactive human PBMCs was resuspended in 200 μL of PBMCs culture medium (including XVT medium, IL-7 at a final concentration of 20 ng / mL and IL-15 at a final concentration of 20 ng / mL).

[0420] On Day 0, at MOI=2, the control group LVV-G1-GFP, LVV-G1A3-GFP, LVV-G1A3 / 28-GFP, LVV-G1A7-GFP, and the LVV-G1A3 / 28 / 7-GFP were added to the PBMC cell culture medium of Donor 1 and Donor 2, respectively. After mixing and infecting at room temperature for 10 minutes, 10 mL of DPBS buffer was added, mixed, and then centrifuged at 500g for 3 minutes. The supernatant was discarded, and each group of PBMCs was resuspended in 1 mL of the PBMC culture medium and incubated in a 5% CO2, 37℃ incubator. On Day 5, flow cytometry was used to detect CD3+ in each group of PBMCs. + The expression of GFP in cells is shown in Figures 1A-E.

[0421] As shown in Figure 1, the control group LVV-G1-GFP, LVV-G1A3-GFP, LVV-G1A3 / 28-GFP, LVV-G1A7-GFP and the LVV-G1A3 / 28 / 7-GFP transduced into non-activated PBMCs of Donor 1 and Donor 2, respectively, showed CD3+. + The positive rates of GFP expression in cells were 0.19% and 0.17%, 27.39% and 39.84%, 43.94% and 55.61%, 16.10% and 4.99%, and 69.14% and 59.61%, respectively.

[0422] The transduction efficiency of the control group LVV-G1A3-GFP, LVV-G1A3 / 28-GFP, LVV-G1A7-GFP, and LVV-G1A3 / 28 / 7-GFP in inactive PBMCs was significantly better than that of the control group LVV-G1-GFP, whose viral envelope did not contain any target molecules. This indicates that because inactive PBMCs express low levels of LDL-R, and the viral envelope only contains wild-type VSV-G, the control group LVV-G1-GFP, which does not contain any target molecules, is difficult to effectively transduce inactive T cells in inactive PBMCs. In contrast, the control group LVV-G1A3-GFP, LVV-G1A3 / 28-GFP, LVV-G1A7-GFP, and LVV-G1A3 / 28 / 7-GFP can all bind to the endocytic receptors CD3, CD28, and CD7 on the surface of inactive T cells through endocytosis and effectively transduce inactive T cells.

[0423] Furthermore, the efficiency of LVV-G1A3 / 28 / 7-GFP transducing Donor 1 and Donor 2 with the viral envelope containing the membrane expressing triple antibodies was significantly better than that of the control group LVV-G1A3-GFP, LVV-G1A7-GFP and LVV-G1A3 / 28-GFP with the viral envelope containing anti-CD3 antibody, anti-CD7 antibody or anti-CD3 antibody × anti-CD28 antibody;

[0424] This demonstrates that using anti-CD3, anti-CD7, and anti-CD28 antibodies simultaneously as targeting molecules can significantly improve the transduction efficiency of vectors, such as lentiviral vectors, in transducing T cells in non-activated PBMCs.

[0425] XVT culture medium: Trade name PRIME-XV T cell CDM, brand: IRVINE (FUJIFILM), catalog number: #91154;

[0426] IL-7: Product Name: IL-7 Protein, Human, Recombinant; Brand: Yi Qiao Shen Zhou; Product Code: #11821-HNAE;

[0427] IL-15: Product Name: IL-15 Protein, Human, Recombinant (His Tag), Brand: Yi Qiao Shen Zhou, Product Number: 10360-H07E.

[0428] Example 2

[0429] 1. Design of chimeric antigen receptors

[0430] In this embodiment, a chimeric antigen receptor (CAR-19) targeting human CD19 is constructed. The polynucleotide encoding the CAR-19 sequentially encodes the following from the 5' end to the 3' end: the human CD8α signal peptide, the extracellular antigen-binding region targeting human CD19, the human CD8α hinge region, the human CD8α transmembrane region, the human 4-1BB co-stimulatory signal transduction domain, and the human CD3ζ intracellular signal transduction domain. The extracellular antigen-binding region targeting CD19 includes scFv (scFv-FMC63) derived from the anti-human CD19 antibody FMC-63, and the VH region of scFv-FMC63 is connected to the VL region through the (G4S)3 linker peptide.

[0431] (1) The amino acid sequence of the VH region of the scFv-FMC63 is shown in SEQ ID NO:14, the amino acid sequence of the VL region of the scFv-FMC63 is shown in SEQ ID NO:15, the amino acid sequences of the HCDR1-3 regions of the scFv-FMC63 are shown in SEQ ID NO:58-60, and the amino acid sequences of the LCDR1-3 regions of the scFv-FMC63 are shown in SEQ ID NO:61-63.

[0432] (2) The amino acid sequence of the human 4-1BB costimulatory domain is shown in SEQ ID NO:17;

[0433] (3) The amino acid sequence of the intracellular signal transduction domain of the human CD3ζ is shown in SEQ ID NO:18.

[0434] Human CD19: Uniprot ID: P15391; CD19 is an effective target for the treatment of B-cell malignancies, especially B-cell lymphoma and acute lymphoblastic leukemia.

[0435] 2. Packaging LVV-G1A3 / 28 / 7-CAR19

[0436] Following the method described for packaging LVV-G1A3 / 28 / 7-GFP, LVV-G1A3 / 28 / 7-CAR19 is packaged as follows:

[0437] Using PEI reagent, the envelope plasmid G1Atri, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the master plasmid (master plasmid CAR19) containing the polynucleotide encoding CAR-19 were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing triple antibody, the wild-type VSV-G, and LVV-G1A3 / 28 / 7-CAR19 containing the polynucleotide encoding CAR-19 (CAR-19 gene). The plasmid map of the master plasmid CAR19 is shown in Figure 7.

[0438] 3. Packaging of each group's control group LVV

[0439] Referring to the method for packaging LVV-G1A3 / 28 / 7-CAR19, the viral envelope containing membrane-expressed anti-CD3 antibody, anti-CD7 antibody, or anti-CD3 antibody × anti-CD28 antibody, as well as multiple control group LVVs without any targeting molecule, were packaged. The specific method is as follows:

[0440] Using PEI reagent, the envelope plasmids G1A3, G1A7, G1A3 / 28 or pMD2.G envelope plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the main plasmid CAR19 were transfected into HEK-293T cells, respectively, to package viral envelopes that do not contain any target molecules or contain the membrane expressing anti-CD3 antibody, anti-CD7 antibody or anti-CD3 antibody × anti-CD28 antibody, as well as control group LVV-G1-CAR19, LVV-G1A3-CAR19, LVV-G1A7-CAR19 or LVV-G1A3 / 28-CAR19 with viral envelopes containing the wild-type VSV-G and carrying the CAR-19 gene.

[0441] 4. Transducer inactive PBMCs

[0442] Following the method described above for transducing Donor 1 and Donor 2 human inactive PBMCs with LVV-G1A3 / 28 / 7-GFP, on Day 0, at MOI=5, the inactive PBMCs of Donor 1 and Donor 2 were transduced using the control group LVV-G1-CAR19, LVV-G1A3-CAR19, LVV-G1A3 / 28-CAR19, LVV-G1A7-CAR19 and the LVV-G1A3 / 28 / 7-CAR19, respectively; on Day 5, the expression of CAR-19 in each group of PBMCs was detected by flow cytometry, and the results are shown in Figures 2A-E.

[0443] As shown in Figure 2, the control group LVV-G1-CAR19, LVV-G1A3-CAR19, LVV-G1A3 / 28-CAR19, LVV-G1A7-CAR19 and LVV-G1A3 / 28 / 7-CAR19 transduced Donor 1 and Donor 2, respectively. The positive rates of CAR-19 expression in non-activated PBMCs were 3.20% and 1.20%, 52.11% and 39.10%, 59.47% and 47.92%, 69.52% and 61.02%, and 81.19% and 77.82%, respectively. The transduction efficiency of non-activated PBMCs by the control group LVV-G1-CAR19, LVV-G1A3-CAR19, LVV-G1A3 / 28-CAR19, LVV-G1A7-CAR19 and LVV-G1A3 / 28 / 7-CAR19 was significantly better than that of the control group LVV-G1-CAR19, which did not contain any target molecules in its viral envelope.

[0444] Furthermore, the efficiency of PBMCs transducing Donor 1 and Donor 2 with the viral envelope containing the membrane expressing the triple antibodies was significantly better than that of the control group LVVs with the viral envelope containing anti-CD3 antibody, anti-CD7 antibody, or anti-CD3 antibody × anti-CD28 antibody.

[0445] This demonstrates that using anti-CD3, anti-CD7, and anti-CD28 antibodies simultaneously as targeting molecules can significantly improve the efficiency of vectors, such as lentiviral vectors, in transducing T cells in inactive PBMCs to deliver and express CAR genes on membranes.

[0446] The flow cytometry antibody used in the detection:

[0447] Flow cytometry antibody for detecting CAR molecules: Trade name: PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1(Y45)(Site-specific conjugation)(0.03% Proclin) DMF Filed, Brand: Acro, Catalog Number: #FM3-PY54A2-200 tests.

[0448] Example 3

[0449] The packaging viral envelope contains a VSV-G variant whose ability to bind to LDL-R is inhibited, and also contains LVV-G2A3 / 28 / 7-CAR19 targeting three molecules.

[0450] 1. Packaging LVV-G2A3 / 28 / 7-CAR19

[0451] The LVV-G2A3 / 28 / 7-CAR19 is packaged using the same method as the LVV-G1A3 / 28 / 7-GFP. The specific method is as follows:

[0452] Using PEI reagent, an envelope plasmid (enveloping plasmid G2Atri), pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the master plasmid CAR19, containing polynucleotides encoding mutant VSV-G and polynucleotides encoding the membrane-expressing triple antibody, were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing triple antibody, the mutant VSV-G, and LVV-G2A3 / 28 / 7-CAR19 encoding the CAR-19 gene.

[0453] The mutant VSV-G contains the amino acid sequence shown in SEQ ID NO:3; relative to SEQ ID NO:1, SEQ ID NO:3 contains a K47 deletion, which inhibits the ability of the mutant VSV-G to bind its receptor LDL-R relative to wild-type VSV-G, while retaining the ability of the mutant VSV-G to mediate membrane fusion and endosome / lysosome escape.

[0454] 2. Packaging multiple control groups LVV

[0455] Following the method for packaging LVV-G2A3 / 28 / 7-CAR19, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the main plasmid CAR19, along with an envelope plasmid containing any of the following polynucleotides, were transfected into HEK-293T cells using PEI reagent to package control groups LVV-G2-CAR19, LVV-G2A3-CAR19, LVV-G2A7-CAR19, and LVV-G2A3 / 28-CAR19.

[0456] (a) A polynucleotide encoding the mutant VSV-G;

[0457] (b) The polynucleotide encoding the mutant VSV-G and the polynucleotide encoding the membrane-expressing anti-CD3 antibody;

[0458] (c) the polynucleotide encoding the mutant VSV-G and the polynucleotide encoding the membrane-expressing anti-CD7 antibody; or

[0459] (d) The polynucleotide encoding the mutant VSV-G and the polynucleotide encoding the membrane-expressed bispecific antibody.

[0460] 3. Detect and calculate the viral titer for each group.

[0461] The viral titers (transducing units, TU) of the control group LVV-G2A3-CAR19, LVV-G2A3 / 28-CAR19, LVV-G2A7-CAR19 and LVV-G2A3 / 28 / 7-CAR19 were detected and calculated.

[0462] Day 0: Jurkat cells were counted, and 1×10⁶ cells were added to each well of a 96-well plate. 5 Jurkat cells (in 1640 medium containing 10% FBS), 12 wells in total;

[0463] Subsequently, crude venom solutions of LVV-G2A3-CAR19, LVV-G2A3 / 28-CAR19, LVV-G2A7-CAR19, and LVV-G2A3 / 28 / 7-CAR19 were added to each well in gradients of 200 μL, 100 μL, and 20 μL, respectively. After 48 hours, the expression of CAR-19 in each well was detected by flow cytometry, and the titers of LVV-G2A3-CAR19, LVV-G2A3 / 28-CAR19, LVV-G2A7-CAR19, and LVV-G2A3 / 28 / 7-CAR19 were calculated by linear regression. The titer of LVV-G2A3-CAR19 was set to 1. The results are shown in Figure 4.

[0464] As shown in Figure 4, under the same packaging conditions, the titer of LVV-G2A3 / 28 / 7-CAR19 containing the membrane expressing the triple antibody is significantly better than that of packaged LVV-G2A3-CAR19, LVV-G2A3 / 28-CAR19, and LVV-G2A7-CAR19.

[0465] 4. Transducer inactive PBMCs

[0466] Following the method described above for transducing Donor 1 and Donor 2 human inactive PBMCs with LVV-G1A3 / 28 / 7-GFP, on Day 0, at MOI=5, the inactive PBMCs of Donor 1 and Donor 2 were transduced using the control group LVV-G2-CAR19, LVV-G2A3-CAR19, LVV-G2A7-CAR19, LVV-G2A3 / 28-CAR19 and the LVV-G2A3 / 28 / 7-CAR19, respectively; on Day 5, the expression of CAR-19 in each group of PBMCs was detected by flow cytometry, and the results are shown in Figures 3A-E.

[0467] As shown in Figure 3, the positive rates of LVV-G2-CAR19, LVV-G2A3-CAR19, LVV-G2A7-CAR19, LVV-G2A3 / 28-CAR19 and LVV-G2A3 / 28 / 7-CAR19 in transducing non-activated PBMCs of Donor 1 and Donor 2 to express CAR-19 were 3.63% and 4.66%, 57.82% and 45.05%, 74.58% and 65.28%, 62.89% and 53.15%, and 84.53% and 81.64%, respectively.

[0468] The control group LVV-G2A3-CAR19, LVV-G2A3 / 28-CAR19, and LVV-G2A7-CAR19 with viral envelope containing any of the aforementioned target molecules and the mutant VSV-G, as well as the control group LVV-G2-CAR19 with viral envelope containing the membrane-expressed triple antibody and the mutant VSV-G, showed significantly better transduction efficiency for non-activated PBMCs than the control group LVV-G2-CAR19 with viral envelope containing the mutant VSV-G but not any of the target molecules.

[0469] Furthermore, the efficiency of LVV-G2A3 / 28 / 7-CAR19, whose viral envelope contains the membrane expressing the triple antibody and the mutant VSV-G, in transducing non-activated PBMCs is significantly better than that of control group LVV-G2A3-CAR19, LVV-G2A3 / 28-CAR19, and LVV-G2A7-CAR19, whose viral envelope contains the mutant VSV-G and anti-CD3 antibody, anti-CD7 antibody, or anti-CD3 antibody × anti-CD28 antibody.

[0470] This demonstrates that in cases where the viral envelope contains a VSV-G variant whose ability to bind LDL-R is inhibited, the simultaneous use of anti-CD3, anti-CD7, and anti-CD28 antibodies as targeting molecules can significantly improve the efficiency of vectors, such as lentiviral vectors, in transducing inactive T cells and delivering the CAR gene.

[0471] Example 4

[0472] The packaging virus envelope contains anti-monkey (cynomolgus monkey) CD3, CD28 and CD7 membranes expressing anti-monkey anti-CD3 antibody, anti-CD28 antibody and anti-CD7 antibody LVV-G2mA3 / 28 / 7-GFP.

[0473] 1. Construct a membrane expression system for anti-monkey anti-CD3 antibody × anti-CD28 antibody × anti-CD7 antibody.

[0474] In this embodiment, the polynucleotide encoding the membrane-expressed anti-monkey anti-CD3 antibody × anti-CD28 antibody × anti-CD7 antibody (membrane-expressed anti-monkey triantibody) sequentially encodes from the 5' end to the 3' end as follows: the human CD8α signal peptide, scFv derived from anti-monkey anti-CD3 antibody SP34 (scFv-SP34), the human CD8α hinge region, the human CD8α transmembrane region, the FT2A peptide, the human CD8α signal peptide, the anti-CD28 antibody scFv-15E8 (anti-CD28 antibody 15E8 can effectively bind to human CD28 and monkey CD28), the human CD8α hinge region, the human CD8α transmembrane region, the FT2A peptide, the human CD8α signal peptide, scFv derived from anti-monkey anti-CD7 antibody G09 (scFv-G09), the human CD8α hinge region, and the human CD8α transmembrane region;

[0475] (1) The amino acid sequence of the scFv-SP34 is shown in SEQ ID NO:64, the amino acid sequence of the VH region of the scFv-SP34 is shown in SEQ ID NO:31, the amino acid sequence of the VL region of the scFv-SP34 is shown in SEQ ID NO:32, the VH region of the scFv-SP34 is connected to the VL region of the scFv-SP34 through a (G4S)3 linker peptide, the amino acid sequences of the HCDR1-3 regions of the scFv-SP34 are shown in SEQ ID NO:65-67, and the amino acid sequences of the LCDR1-3 regions of the scFv-SP34 are shown in SEQ ID NO:68-70, respectively;

[0476] (2) The amino acid sequence of scFv-G09 is shown in SEQ ID NO:71, the amino acid sequence of the VH region of scFv-G09 is shown in SEQ ID NO:33, the amino acid sequence of the VL region of scFv-G09 is shown in SEQ ID NO:34, the VH region of scFv-G09 is connected to the VL region of scFv-SP34 through a (G4S)3 linker peptide, the amino acid sequences of the HCDR1-3 regions of scFv-G09 are shown in SEQ ID NO:72-74, and the amino acid sequences of the LCDR1-3 regions of scFv-G09 are shown in SEQ ID NO:75-77.

[0477] 2. Packaging LVV-G2mA3 / 28 / 7-CAR19

[0478] Following the method described for packaging LVV-G1A3 / 28 / 7-GFP, LVV-G2mA3 / 28 / 7-CAR19 is packaged as follows:

[0479] Using PEI reagent, an envelope plasmid (enveloping plasmid G2mAtri), pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and the master plasmid CAR19, containing polynucleotides encoding the mutant VSV-G and polynucleotides encoding the membrane-expressing anti-monkey triple antibody, were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing anti-monkey triple antibody, the mutant VSV-G, and the CAR-19 gene, LVV-G2mA3 / 28 / 7-CAR19.

[0480] 3. Packaging multiple control groups LVV

[0481] Referring to the method for packaging LVV-G1A3 / 28 / 7-CAR19, control groups of LVV were packaged respectively, including those whose viral envelope did not contain any targeting molecule, those containing the anti-monkey anti-CD3 antibody, anti-CD7 antibody or anti-CD3 antibody × anti-CD28 antibody, and the mutant VSV-G.

[0482] A. Packaging control group LVV-G2mA3-CAR19

[0483] In this example, a membrane-expressing anti-monkey anti-CD3 antibody was constructed. The polynucleotide encoding the membrane-expressing anti-monkey anti-CD3 antibody sequentially encodes the following from the 5' end to the 3' end: the human CD8α signal peptide, the scFv-SP34, the human CD8α hinge region, and the human CD8α transmembrane region.

[0484] Following the method described for packaging LVV-G1A3 / 28 / 7-CAR19, the control group LVV-G2mA3-CAR19 was packaged, specifically as follows:

[0485] Using PEI reagent, an envelope plasmid containing polynucleotides encoding the mutant VSV-G and polynucleotides encoding the membrane-expressing anti-monkey anti-CD3 antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the main plasmid CAR19 were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing anti-monkey anti-CD3 antibody and the mutant VSV-G, and carried the CAR-19 gene in the control group LVV-G2mA3-CAR19.

[0486] B. Packaging control group LVV-G2mA7-CAR19

[0487] In this example, a membrane-expressing anti-monkey anti-CD7 antibody was constructed. The polynucleotide encoding the membrane-expressing anti-monkey anti-CD7 antibody sequentially encodes the following from the 5' end to the 3' end: the human CD8α signal peptide, the scFv-G09, the human CD8α hinge region, and the human CD8α transmembrane region.

[0488] Following the method described for packaging LVV-G1A3 / 28 / 7-CAR19, the control group LVV-G2mA7-CAR19 was packaged, specifically as follows:

[0489] Using PEI reagent, an envelope plasmid containing polynucleotides encoding the mutant VSV-G and polynucleotides encoding the membrane-expressing anti-monkey anti-CD7 antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the main plasmid CAR19 were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing anti-monkey anti-CD7 antibody and the mutant VSV-G and carried the CAR-19 gene in the control group LVV-G2mA7-CAR19.

[0490] C. Packaging control group LVV-G2mA3 / 28-CAR19

[0491] In this example, a membrane-expressing anti-monkey anti-CD3 antibody × anti-CD28 antibody (anti-monkey bispecific antibody) was constructed. The polynucleotide encoding the membrane-expressing anti-monkey bispecific antibody sequentially encodes the following from the 5' end to the 3' end: the human CD8α signal peptide, the scFv-SP34, the human CD8α hinge region, the human CD8α transmembrane region, the FT2A peptide, the human CD8α signal peptide, the scFv-15E8, the human CD8α hinge region, and the human CD8α transmembrane region.

[0492] Following the method described for packaging LVV-G1A3 / 28 / 7-CAR19, the control group LVV-G2mA3 / 28-CAR19 was packaged, specifically as follows:

[0493] Using PEI reagent, an envelope plasmid containing polynucleotides encoding the mutant VSV-G and polynucleotides encoding the membrane-expressing anti-monkey bispecific antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the main plasmid CAR19 were transfected into HEK-293T packaging cells. The packaging virus envelope contained the membrane-expressing anti-monkey bispecific antibody and the mutant VSV-G and carried the CAR-19 gene in the control group LVV-G2mA3 / 28-CAR19.

[0494] D. Packaging LVV-G2-CAR19

[0495] The same batch of packaged viral envelope contains the mutant VSV-G but does not contain any target molecule, and the control group LVV-G2-CAR19 carries the CAR-19 gene.

[0496] 4. Detect and calculate the viral titer for each group.

[0497] The viral titers of the control group LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, LVV-G2mA7-CAR19 and LVV-G2mA3 / 28 / 7-CAR19 were detected and calculated.

[0498] Day 0: Jurkat cells were counted, and 1×10⁶ cells were added to each well of a 96-well plate. 5 Jurkat cells (in 1640 medium containing 10% FBS), 12 wells in total;

[0499] Subsequently, crude venom solutions of LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, LVV-G2mA7-CAR19, and LVV-G2mA3 / 28 / 7-CAR19 were added to each well in gradients of 200 μL, 100 μL, and 20 μL, respectively. After 48 hours, the expression of CAR-19 in each well was detected by flow cytometry.

[0500] The titers of LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, LVV-G2mA7-CAR19 and LVV-G2mA3 / 28 / 7-CAR19 were calculated by linear regression; the titer of LVV-G2mA3-CAR19 was set to 1, and the results are shown in Figure 5.

[0501] As shown in Figure 5, under the same packaging conditions, the titer of LVV-G2mA3 / 28 / 7-CAR19 containing the membrane expressing anti-monkey triple antibodies is significantly better than that of LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, and LVV-G2mA7-CAR19.

[0502] 5. Transduced monkey non-activated PBMCs

[0503] Following the method described above for transducing Donor 1 and Donor 2 human inactive PBMCs with LVV-G1A3 / 28 / 7-CAR19, on Day 0, at MOI=5, inactive PBMCs of cynomolgus monkeys (purchased from Huazhen Biotechnology) were transduced using LVV-G2-CAR19, LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, LVV-G2mA7-CAR19, and LVV-G2mA3 / 28 / 7-CAR19, respectively. On Day 5, the expression of CAR-19 in the monkey PBMCs of each group was detected by flow cytometry, and the results are shown in Figures 6A-E.

[0504] As shown in Figure 6, the positive rates of LVV-G2-CAR19, LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, LVV-G2mA7-CAR19, and LVV-G2mA3 / 28 / 7-CAR19 in transducing CAR-19 expression in non-activated PBMCs of monkeys were approximately 0.98%, 19.68%, 28.52%, 29.62%, and 43.99%, respectively. The viral envelope contained any of the aforementioned anti-monkey targeting molecules and the mutation... The control groups LVV-G2mA3-CAR19, LVV-G2mA3 / 28-CAR19, and LVV-G2mA7-CAR19 containing the mutant VSV-G, as well as the control group LVV-G2-CAR19 containing the mutant VSV-G and expressing anti-monkey triple antibodies and the mutant VSV-G, were significantly more efficient than the control group LVV-G2-CAR19 containing the mutant VSV-G and not containing any anti-monkey targeting molecules.

[0505] Furthermore, the efficiency of LVV-G2mA3 / 28 / 7-CAR19 transduced monkeys containing the viral envelope expressing the anti-monkey triple antibody and the mutant VSV-G is significantly better than that of LVV-G2mA3-CAR19, LVV-G2mA7-CAR19, and LVV-G2mA3 / 28-CAR19 containing the mutant VSV-G and the anti-monkey anti-CD3 antibody, anti-monkey anti-CD7 antibody, or anti-monkey bispecific antibody.

[0506] This demonstrates that, in cases where the viral envelope contains a VSV-G variant whose ability to bind LDL-R is inhibited, the simultaneous use of anti-monkey anti-CD3, anti-monkey anti-CD7, and anti-monkey anti-CD28 antibodies as targeting molecules can significantly improve the efficiency of vectors, such as lentiviral vectors, in transducing inactive monkey PBMCs and delivering the CAR gene.

Claims

1. A vector, characterized in that, the carrier surface comprises a targeting moiety that can bind CD3, a targeting moiety that can bind a T cell costimulatory molecule, and a targeting moiety that can bind CD7; Preferably, the T cell costimulatory molecule comprises CD28.

2. The carrier of claim 1, wherein, the carrier is selected from the group consisting of Lipid Nanoparticles ("LNPs"), Extracellular Vesicles, and viral vectors; Preferably, the viral vector comprises an enveloped viral vector; More preferably, the enveloped viral vector comprises a lentiviral vector (LVV) and a retroviral vector (RVV).

3. The carrier of claim 1 or 2, wherein, the targeting moiety comprises an extracellular binding domain and a transmembrane domain, the extracellular binding domain comprising at least one of an antibody or an antigen-binding fragment thereof and a ligand or a receptor-binding fragment thereof that can bind (a) CD3, (b) a T cell costimulatory molecule, or (c) CD7; Preferably, the T cell costimulatory molecule comprises CD28.

4. The carrier of claim 3, wherein, the extracellular binding domain is directly or indirectly linked to the transmembrane domain, exposed on the surface of the carrier; Preferably, the transmembrane domain is selected from the group consisting of transmembrane domains of the following proteins: CD28, CD2, CD4, CD8a, CD5, CD3e, CD3d, CD3z, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRa, FcRp, FcRy, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRp, TIM3, TRIM, LPA5, and Zap70; More preferably, the transmembrane domain comprises a transmembrane domain of CD8a.

5. The carrier of claim 4, wherein, the targeting moiety further comprises a linker domain, the extracellular binding domain being indirectly linked to the transmembrane domain via the linker domain; Preferably, the linker domain is selected from the group consisting of: (a) an immunoglobulin hinge region selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region selected from the wild-type or modified hinge region of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (c) all or a portion of an Fc domain selected from at least one of a CHI domain, a CH2 domain, and a CH3 domain; and (d) a stalk region of a type II C-lectin selected from the stalk region of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the linking domain comprises a hinge region of CD8a.

6. The vector of any one of claims 1-5, wherein, The vector is an LVV or an RVV.

7. The carrier of claim 6, wherein, The viral glycoprotein of the LVV or RVV is selected from the group consisting of a Vesiculovirus strain glycoprotein and variants thereof, a NiV glycoprotein G and variants thereof, a Morbillivirus glycoprotein H and variants thereof, a Lentivirus glycoprotein and variants thereof, a Rabies virus glycoprotein (RVG) and variants thereof, a GaLV glycoprotein and variants thereof, a MLV-A glycoprotein and variants thereof, a RD114 glycoprotein and variants thereof, a Fowlpox virus (FPV) glycoprotein and variants thereof, an Ebola virus (EboV) glycoprotein and variants thereof, and a Lymphocytic choriomeningitis virus (LCMV) glycoprotein and variants thereof; The Vesiculovirus strain glycoprotein and variants thereof include a Vesiculovirus Indiana strain glycoprotein and variants thereof, a Vesiculovirus Cocal strain glycoprotein and variants thereof, a Vesiculovirus Maraba strain glycoprotein and variants thereof, a Vesiculovirus Morreton strain glycoprotein and variants thereof, a Vesiculovirus Alagoas strain glycoprotein and variants thereof, a Vesiculovirus New Jersey strain glycoprotein and variants thereof, a Vesiculovirus Carajas strain glycoprotein and variants thereof, a Vesiculovirus Chandipura strain glycoprotein and variants thereof, a Vesiculovirus Eptesicus strain glycoprotein and variants thereof, a Vesiculovirus Isfahan strain glycoprotein and variants thereof, a Vesiculovirus Jurona strain glycoprotein and variants thereof, a Vesiculovirus Malpais strain glycoprotein and variants thereof, a Vesiculovirus Perinet strain glycoprotein and variants thereof, a Vesiculovirus Piry strain glycoprotein and variants thereof, a Vesiculovirus Radi strain glycoprotein and variants thereof, a Vesiculovirus Rhinolopus strain glycoprotein and variants thereof, and a Vesiculovirus Yug Bogdanovac strain glycoprotein and variants thereof; Preferably, the viral glycoprotein of the LVV or RVV is the vesicular stomatitis virus Indiana strain glycoprotein (VSV-G) or a variant thereof or the vesicular stomatitis virus Cocal strain glycoprotein (Cocal-G) or a variant thereof.

8. The carrier of claim 6 or 7, wherein, the ability of the viral glycoprotein of the LVV or RVV to bind its receptor is inhibited; Preferably, the viral glycoprotein is VSV-G or a variant thereof or Cocal-G or a variant thereof and the receptor is the Low Density Lipoprotein Receptor ("LDL-R").

9. The carrier of claim 8, wherein, the viral glycoprotein comprises a first mutation such that the ability of the viral glycoprotein to bind a receptor is inhibited.

10. The carrier of claim 9, wherein, the viral glycoprotein is VSV-G or a variant thereof and the receptor is the LDL-R; Preferably, the first mutation comprises a mutation of at least one of the following: (a) a substitution or deletion at H8, a substitution or deletion at N9, a substitution or deletion at Q10, a substitution or deletion at K47, a substitution or deletion at K50, a substitution or deletion at A51, a substitution or deletion at S183, a substitution or deletion at S179, a substitution or deletion at N180, a substitution or deletion at 1182, a substitution or deletion at M184, a substitution or deletion at Y209, a substitution or deletion at 1347, a substitution or deletion at T350, a substitution or deletion at T352, a substitution or deletion at E353, a substitution at R354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1; and (b) a substitution or deletion at H8, a substitution or deletion at N9, a substitution or deletion at Q10, a substitution or deletion at K47, a substitution or deletion at K50, a substitution or deletion at A51, a substitution or deletion at S183, a substitution or deletion at S179, a substitution or deletion at N180, a substitution or deletion at 1182, a substitution or deletion at M184, a substitution or deletion at Y209, a substitution or deletion at 1347, a substitution or deletion at T350, a substitution or deletion at T352, a substitution or deletion at E353, a substitution at R354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1; More preferably, the first mutation comprises a mutation of at least one of the following amino acids: (a) a substitution or deletion at K47, a substitution at R354 of SEQ ID NO: 1; and (b) a substitution or deletion at H8, a substitution or deletion at N9, a substitution or deletion at Q10, a substitution or deletion at K47, a substitution or deletion at K50, a substitution or deletion at A51, a substitution or deletion at S183, a substitution or deletion at S179, a substitution or deletion at N180, a substitution or deletion at 1182, a substitution or deletion at M184, a substitution or deletion at Y209, a substitution or deletion at 1347, a substitution or deletion at T350, a substitution or deletion at T352, a substitution or deletion at E353, a substitution at R354, a deletion of amino acids 1-18, a deletion of amino acids 19-36, a deletion of amino acids 37-51, a deletion of amino acids 314-384, a deletion of amino acids 321-374, a deletion of amino acids 331-364, a deletion of amino acids 344-354, a deletion of amino acids 345-353 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1; and (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; 11. The carrier of claim 9, wherein, (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal global alignment with SEQ ID NO: 1 ; (b) substitution or deletion at a position corresponding to K47 of SEQ ID NO: 1, R354 of SEQ ID NO: 1, upon optimal (b) substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of 1347, substitution or deletion of S350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 2 following the best global alignment with SEQ ID NO: 2; More preferably, the first mutation comprises a mutation of at least one of the following amino acids: (a) substitution or deletion of K47, substitution of R354 of SEQ ID NO: 2; and (b) substitution or deletion of K47, substitution of R354 of SEQ ID NO: 2 following the best global alignment with SEQ ID NO: 2; More preferably, the first mutation comprises a mutation of at least one of the following amino acids: (a) K47Q or deletion of K47, R354Q of SEQ ID NO: 2; and (b) K47Q or deletion of K47, R354Q of SEQ ID NO: 2 following the best global alignment with SEQ ID NO: 2; Most preferably, the first mutation comprises a mutation of: (a) deletion of K47 of SEQ ID NO: 2; or (b) deletion of K47 of SEQ ID NO: 2 following the best global alignment with SEQ ID NO:

2.

12. The carrier of claim 8, wherein, The binding site of the viral glycoprotein available for binding to its receptor is reduced, such that the ability of the viral glycoprotein to bind to its receptor is inhibited.

13. The carrier of claim 12, wherein, The binding site of the viral glycoprotein available for binding to its receptor is reduced, such that the ability of the viral glycoprotein to bind to its receptor is inhibited. Preferably, the viral glycoprotein has bound at least one of its receptor and an antibody on the cell membrane of a packaging cell that packages the LVV or RVV. Preferably, the viral glycoprotein has bound at least one of its receptor and an antibody on the cell membrane of a packaging cell that packages the LVV or RVV. Preferably, the viral glycoprotein has bound at least one of its receptor and an antibody on the viral envelope of the LVV or RVV.

14. The carrier of claim 13, wherein, The viral glycoprotein is VSV-G or a variant thereof or Cocal-G or a variant thereof, which has bound at least one of its receptors LDL-R, VSV-G antibody and Cocal-G antibody, rendering the VSV-G or variant thereof or Cocal-G or variant thereof less available for binding to the binding site of its receptor LDL-R.

15. The vector of any one of claims 6-14, wherein, The viral glycoprotein of the LVV or RVV comprises a second mutation rendering the viral glycoprotein more or not inactivated by complement.

16. The carrier of claim 15, wherein, The viral glycoprotein is VSV-G or a variant thereof, the second mutation comprising a mutation of at least one of the following amino acids: (a) the amino acid at position 214 of SEQ ID NO: 1 ; (b) the amino acid corresponding to the amino acid at position 214 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (c) the amino acid at position 352 of SEQ ID NO: 1 ; (d) the amino acid corresponding to the amino acid at position 352 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (e) the amino acid at position 50 of SEQ ID NO: 1 ; (f) the amino acid corresponding to the amino acid at position 50 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (g) the amino acid at position 146 of SEQ ID NO: 1 ; and (h) the amino acid corresponding to the amino acid at position 146 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; Preferably, the mutation of the amino acid comprises a deletion, insertion or substitution of the amino acid; More preferably, the second mutation comprises a substitution of at least one of the following amino acids: (a) the amino acid at position 214 of SEQ ID NO: 1 ; (b) the amino acid corresponding to the amino acid at position 214 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (c) the amino acid at position 352 of SEQ ID NO: 1 ; (d) the amino acid corresponding to the amino acid at position 352 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (e) the amino acid at position 50 of SEQ ID NO: 1 ; (f) the amino acid corresponding to the amino acid at position 50 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (g) the amino acid at position 146 of SEQ ID NO: 1 ; and (h) the amino acid corresponding to the amino acid at position 146 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; Still more preferably, the second mutation comprises a substitution of at least one of the following amino acids: (a) the amino acid at position 214 of SEQ ID NO: 1 ; (b) the amino acid corresponding to the amino acid at position 214 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (c) the amino acid at position 352 of SEQ ID NO: 1 ; (d) the amino acid corresponding to the amino acid at position 352 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (e) the amino acid at position 50 of SEQ ID NO: 1 ; (f) the amino acid corresponding to the amino acid at position 50 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (g) the amino acid at position 146 of SEQ ID NO: 1 ; and (h) the amino acid corresponding to the amino acid at position 146 of SEQ ID NO: 1 after optimal global alignment of SEQ ID NO: 1 ; (a) The amino acid at position 214 of SEQ ID NO:1 is replaced by threonine T with asparagine N (T214N), the amino acid at position 352 is replaced by threonine T with alanine A (T352A), the amino acid at position 50 is replaced by lysine K with threonine T (K50T), and the amino acid at position 146 is replaced by serine S with threonine T (S146T); and (b) After best global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located at the equivalent of SEQ ID NO:

1.

17. The carrier of claim 16, wherein, The second mutation includes any combination of mutations at the following sites: (a) Substitution of T214 and T352 located in SEQ ID NO:1; (b) After best global alignment with SEQ ID NO:1, the substitutions located at T214 and T352, which are equivalent to SEQ ID NO:1; (c) Substitutions of T214, T352, K50 and S146 located at SEQ ID NO:1; and (d) Substitutions located at T214, T352, K50 and S146 corresponding to SEQ ID NO:1 after best global alignment with SEQ ID NO:1; Preferably, the second mutation comprises a combination of any of the following site mutations: (a) T214N and T352A located at SEQ ID NO:1; (b) After best global alignment with SEQ ID NO:1, it is located at T214N and T352A, which are equivalent to SEQ ID NO:1; (c) T214N, T352A, K50T and S146T located at SEQ ID NO:1; and (d) After best global alignment with SEQ ID NO:1, T214N, T352A, K50T and S146T are located at the equivalent of SEQ ID NO:

1.

18. The carrier of claim 15, wherein, The viral glycoprotein is Cocal-G or a variant thereof, and the second mutation includes a mutation in at least one of the following amino acids: (a) The 214th amino acid located in SEQ ID NO:2; (b) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 214th position of SEQ ID NO:2; (c) The 352nd amino acid located in SEQ ID NO:2; (d) After optimal global alignment with SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:2; (e) The 50th amino acid located in SEQ ID NO:2; (f) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:2; (g) the amino acid located at position 146 of SEQ ID NO:2; and (h) After optimal global alignment with SEQ ID NO:2, it is located at amino acid position 146, which corresponds to SEQ ID NO:2; Preferably, the mutation of the amino acid includes the deletion, insertion, or substitution of the amino acid; More preferably, the second mutation includes the substitution of at least one of the following amino acids: (a) The 214th amino acid located in SEQ ID NO:2; (b) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 214th position of SEQ ID NO:2; (c) The 352nd amino acid located in SEQ ID NO:2; (d) After optimal global alignment with SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:2; (e) The 50th amino acid located in SEQ ID NO:2; (f) After optimal global alignment with SEQ ID NO:2, the amino acid located at the 50th amino acid position corresponding to SEQ ID NO:2; (g) the amino acid located at position 146 of SEQ ID NO:2; and (h) After optimal global alignment with SEQ ID NO:2, it is located at amino acid position 146, which corresponds to SEQ ID NO:2; More preferably, the second mutation includes at least one mutation at the following sites: (a) The amino acid at position 214 of SEQ ID NO:2 is replaced by lysine K with asparagine N (K214N), T352A, K50T, or S146T; and (b) After best global alignment with SEQ ID NO:2, it is located at K214N, T352A, K50T, S146T, which are equivalent to SEQ ID NO:

2.

19. The carrier of claim 18, wherein, The second mutation includes any combination of mutations at the following sites: (a) Substitution of K214 and T352 located in SEQ ID NO:2; (b) Substitutions located at K214 and T352 corresponding to SEQ ID NO:2 after best global alignment with SEQ ID NO:2; (c) Substitutions of K214, T352, K50, and S146 located at SEQ ID NO:2; and (d) Substitutions located at K214, T352, K50 and S146 corresponding to SEQ ID NO:2 after best global alignment with SEQ ID NO:2; Preferably, the second mutation comprises a combination of any of the following site mutations: (a) K214N and T352A located at SEQ ID NO:2; (b) After best global alignment with SEQ ID NO:2, it is located at K214N and T352A, which are equivalent to SEQ ID NO:2; (c) K214N, T352A, K50T and S146T located at SEQ ID NO:2; and (d) After best global alignment with SEQ ID NO:2, it is located at K214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:

2.

20. The vector of any one of claims 1-19, wherein, The vector contains exogenous nucleic acid; Preferably, the exogenous nucleic acid encodes at least one of a chimeric antigen receptor (CAR) and a T cell receptor chimeric protein (TCP). The CAR includes an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction domain; more preferably, the CAR also includes a hinge region and a co-stimulatory signal transduction domain. The TCP comprises: (a) a TCR / CD3 complex subunit related peptide ("TSP"), the TSP comprising at least one of a TCR / CD3 complex subunit, a TCR / CD3 complex subunit functional fragment, a TCR / CD3 complex subunit variant, and a variant of a TCR / CD3 complex subunit functional fragment; and (b) an extracellular antigen-binding region; more preferably, the TCP further comprises a linker peptide.

21. The carrier of claim 20, wherein, The vector improves the efficiency of transducing inactive T cells.

22. A method of increasing the efficiency of a vector in transducing non-activated T cells, the vector comprising an exogenous nucleic acid, characterized in that, The method includes constructing a CD3-binding target molecule, a T-cell co-stimulatory molecule-binding target molecule, and a CD7-binding target molecule on the surface of the carrier. Preferably, the T cell co-stimulatory molecule includes CD28.

23. The method of claim 22, wherein, The carrier is selected from lipid nanoparticles, extracellular vesicles, and viral vectors; Preferably, the viral vector includes an enveloped viral vector; More preferably, the enveloped viral vector includes LVV and RVV.

24. The method of claim 22 or 23, wherein, The targeting molecule comprises an extracellular binding domain and a transmembrane domain, wherein the extracellular binding domain comprises at least one of an antibody or antigen-binding fragment thereof capable of binding to (a) CD3, (b) T cell co-stimulatory molecules or (c) CD7, and a ligand or receptor-binding fragment thereof. Preferably, the T cell co-stimulatory molecule includes CD28.

25. The method of claim 24, wherein, The extracellular binding domain is directly or indirectly connected to the transmembrane domain and exposed on the surface of the carrier; Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70; More preferably, the transmembrane domain includes the transmembrane domain of CD8α.

26. The method of claim 25, wherein, The targeting molecule further includes a linker domain, and the extracellular binding domain is indirectly connected to the transmembrane region through the linker domain; Preferably, the connection structure domain is selected from: (a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions; (b) Hinge region, which 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 a portion of the Fc domains, wherein the Fc domains are selected from at least one of the CH1, CH2, and CH3 domains; and (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; More preferably, the connection structure domain includes the hinge region of CD8α.

27. The method of any one of claims 22-26, wherein, The carrier is LVV or RVV.

28. The method of any one of claims 22-27, wherein, The exogenous nucleic acid encodes at least one of CAR and TCP; The CAR includes an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction domain; preferably, the CAR also includes a hinge region and a co-stimulatory signal transduction domain. The TCP comprises: (a) a TCR / CD3 complex subunit-associated polypeptide ("TSP"), the TSP comprising at least one of a TCR / CD3 complex subunit, a TCR / CD3 complex subunit functional fragment, a TCR / CD3 complex subunit variant, and a variant of a TCR / CD3 complex subunit functional fragment; and (b) an extracellular antigen-binding region; preferably, the TCP further comprises a linker peptide.

29. A method of transducing non-activated T cells, comprising, The method includes contacting inactive T cells with the carrier of claim 20; Preferably, the contact occurs outside the subject, who is an individual given non-activated T cells transduced by the method; Preferably, the contact occurs within the body of a subject, who is an individual to whom the carrier is given.

30. An engineered T cell comprising an exogenous nucleic acid, characterized in that, The engineered T cells are prepared by the method of claim 29.

31. A composition comprising, The composition comprises a pharmaceutically acceptable excipient or carrier and any one of the following components: The vector according to any one of claims 1-21 and the engineered T cell according to claim 30.

32. The composition of claim 31, wherein, The composition comprises the vector of claim 20 or the engineered T cells of claim 30.

33. The use of the carrier according to any one of claims 1-21 or the composition according to any one of claims 31-32 in the preparation of a medicament for treating and / or preventing diseases; Preferably, the disease is selected from at least one of hematologic malignancies and solid tumors.

34. The use according to claim 33, wherein The disease is selected from at least one type of blood cancer; Preferably, the hematologic malignancy is selected from at least one of acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), multiple myeloma (MM), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), and follicular lymphoma (FL).

35. The use of claim 33, wherein, The disease is selected from at least one type of solid cancer; Preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, and ovarian cancer.

36. A method of treating a subject for a cancer and killing cancer cells of a subject, comprising, The method includes administering to the subject the vector of claim 20, the engineered T cells of claim 30, or the composition of claim 32; Preferably, the cancer is selected from at least one of hematologic malignancies and solid tumors.

37. The method of claim 36, wherein, The cancer is selected from at least one type of blood cancer; Preferably, the hematologic malignancy is selected from at least one of acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, and follicular lymphoma.

38. The method of claim 36, wherein, The cancer is selected from at least one type of solid cancer; Preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, and ovarian cancer.

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