WO2025149068A1 - Viral particle, and preparation method therefor and use thereof - Google Patents

Abstract

A viral particle. The surface of the viral particle comprises a T cell activation primary signaling molecule and a T cell activation secondary signaling molecule; the envelope glycoprotein of the viral particle is subjected to a first mutation, so that the receptor binding ability of the viral glycoprotein is weakened or lost with respect to its receptor binding ability before the first mutation; and the envelope glycoprotein of the viral particle can also be subjected to a second mutation, so that the viral particle has enhanced resistance to complement-mediated inactivation or is not subjected to complement-mediated inactivation. The viral particle has improved specificity for targeted activation, stimulation and transduction of non-activated T cells, and is thus more applicable for the preparation of CAR-T cells in subjects.

Description

Virus particle and preparation method and application thereof Technical Field

The present invention relates to the field of gene vectors, and in particular to a virus particle and a preparation method and application thereof.

Background Art

In the field of genetic engineering and cell therapy, lipid nanoparticles (LNPs), virus-like particles (VLPs), adenovirus, adeno-associated virus, retroviral vector (RVV) and lentiviral vector (LVV) are commonly used gene vectors.

Lentiviral vectors and retroviral vectors can integrate exogenous payload genes, such as chimeric antigen receptor (CAR) genes, into the genome of host cells, allowing the CAR genes to be stably expressed in host cells, and are widely used in the preparation of CAR-T cells in vitro and in vivo.

By deleting the virulence genes of HIV (Human Immunodeficiency Virus), such as genes env, vif, vpr, vpu and nef, and performing multiple attenuation, a self-inactivating, replication-defective and biosafe lentiviral vector or retroviral vector is constructed.

However, how to improve the efficiency and specificity of targeted activation and transduction of T cells in vivo and in vitro, the efficiency of membrane expression of CAR molecules in T cells, and the killing efficiency of the prepared CAR-T cells are still technical challenges that need to be solved in the technical field of CAR-T cell therapy.

Summary of the invention

In view of this, in order to solve at least one of the above technical problems, the present invention provides a virus particle in one aspect.

(a) the surface of the virus particle contains T cell activation primary signal molecules and T cell activation secondary signal molecules; and

(b) The surface of the virus particle comprises a glycoprotein, and the glycoprotein undergoes a first mutation, thereby weakening or losing the ability of the glycoprotein to bind to a glycoprotein receptor relative to before the first mutation occurs.

In some embodiments of the present invention, the viral particles target and activate non-activated T cells (Non-Activated T Cells).

In some embodiments of the present invention, the viral particle is a lentiviral vector or a retroviral vector.

Non-activated T cells refer to T cells that are not proliferated, not differentiated, in a resting state, do not recognize antigens, and have not been activated by T cell activation primary signal molecules or T cell activation primary and secondary signal molecules, such as T cells in the G0 phase of the cell cycle, resting (resting/quiescent) T cells, or naive T cells.

T cell activation primary signal molecules bind to T cell surface proteins and participate in T cell receptor (T Cell Receptor, "TCR") mediated T cell activation (TCR-mediated T Cell Activation).

In some embodiments of the present invention, the T cell activation primary signal molecule is involved in converting TCR into active PTK (protein tyrosine kinase), which can phosphorylate a series of substrates to generate a large number of downstream signals. When these signals are properly integrated (together 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).

In some embodiments of the present invention, the T cell activation primary signal molecule binds to a T cell endocytic receptor involved in mediating the generation and/or transmission of the T cell activation primary signal, such as CD3.

In some embodiments of the present invention, the T cell activation primary signal molecule binds to the TCR/CD3 complex.

CD3 and TCR form a TCR/CD3 complex and participate in the activation of helper T cells (CD4 ⁺ T cells) and cytotoxic T cells (CD8 ⁺ T cells).

In some embodiments of the present invention, the T cell activation primary signal molecule binds to the TCR/CD3 complex or the TCR/CD3 complex subunit; the TCR/CD3 complex subunit is selected from at least one of CD3ε, CD3γ, CD3δ, TCRα and TCRβ.

In some embodiments of the present invention, the TCR/CD3 complex subunit is further selected from TCRγ and TCRζ.

In some embodiments of the present invention, the T cell activation primary signal molecule binds to human CD3ε (Unipro ID: P07766).

In some embodiments of the present invention, the T cell activation primary signal molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof comprises an antibody or antigen-binding fragment thereof derived from UCHT1, OKT3, SP34, HuM291 or TR66, or a variant thereof, or a derivative thereof.

In some embodiments of the present invention, the anti-CD3 antibody is a scFv derived from UCHT1 (UCHT1-scFv), and the amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO:9; the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO:81-83, respectively, and the LCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO:84-86, respectively.

T cell activation secondary signal molecules (Secondary Signal), also known as co-stimulatory signal molecules (Co-Stimulatory Signal), bind to other T cell surface receptors to provide additional signals necessary to avoid anergy and effective T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

In some embodiments of the present invention, the T cell activation secondary signal molecule binds to a T cell endocytic receptor involved in mediating the generation and/or transmission of T cell activation secondary signals, such as CD28.

In some embodiments of the present invention, the T cell activation secondary signaling molecule binds to CD28.

In some embodiments of the present invention, the CD28 is human CD28 (Uniprot ID: P10747).

Although other cell surface receptors (co-stimulatory receptors) can also enhance activation signals through TCR, CD28-mediated co-stimulation is stronger than other co-stimulatory receptors (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

In some embodiments of the present invention, the T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof.

In some embodiments of the present invention, the CD28 ligand or its receptor binding fragment includes CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

In some embodiments of the present invention, the receptor binding fragment of the ligand includes at least one of the extracellular domain, functional fragment and derivative of the ligand.

In some embodiments of the present invention, the CD28 ligand or its receptor binding fragment includes CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

In some embodiments of the present invention, the CD80 receptor binding fragment is the CD80 extracellular domain; the CD86 receptor binding fragment is the CD86 extracellular domain.

In some embodiments of the present invention, the anti-CD28 antibody or its antigen-binding fragment is a scFv (15E8-scFv) derived from 15E8, and the amino acid sequence of the 15E8-scFv is shown in SEQ ID NO: 13; the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 87-89, respectively, and the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 90-92, respectively.

In some embodiments of the present invention, the T cell activation secondary signal molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

In some embodiments of the present invention, when the T cell activation secondary signal molecule includes a T cell activation secondary signal molecule that can bind to CD28, it may also include at least one ligand or its receptor binding fragment selected from ICOS (inducible costimulator, "ICOS") ligand (ICOSL) or its receptor binding fragment, 4-1BB ligand (4-1BBL) or its receptor binding fragment and OX40 ligand (OX40L) or its receptor binding fragment.

Unlike CD28, which is stably expressed on both non-activated and activated T cells, ICOS is inducibly expressed on activated T cells (Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, et al. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 1999; 397: 263-6. [PubMed: 9930702])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619). Lack of ICOS will lead to impaired immune responses similar to those in the CD28 knockout model, but less severe than the CD28 knockout model, which means that the two molecules may act in similar pathways (Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000; 13: 95-105. [PubMed: 10933398])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

In addition to the CD28 family of co-stimulatory receptors, TNFR family members OX40 (CD134) and 4-1BB (CD137) provide co-stimulatory signals by binding to their ligands OX40L and 4-1BBL (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

In some embodiments of the present invention, the T cell activation primary signal molecule and/or T cell activation secondary signal molecule is directly or indirectly linked to a transmembrane polypeptide (Polypeptide) and displayed on the surface of the virus particle.

In some embodiments of the present invention, the transmembrane polypeptide is selected from the transmembrane region of the following proteins:

CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD 37. CD40, CD45, CD64, CD80, CD86, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-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;

Preferably, the transmembrane polypeptide is the CD8α transmembrane region.

In some embodiments of the present invention, the T cell activation primary signal molecule and/or T cell activation secondary signal molecule is indirectly connected to the transmembrane polypeptide via a linker domain and displayed on the surface of the viral particle.

In some embodiments of the present invention, the T cell activation primary signal molecule and/or T cell activation secondary signal molecule are indirectly connected to the transmembrane polypeptide through a connecting domain and displayed on the surface of the virus particle;

Preferably, the linking domain is selected from:

(a) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge region;

(b) a hinge region 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 an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain;

(d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and

(e) flexible linker peptide;

More preferably, the connecting domain is the CD8α hinge region.

In some embodiments of the present invention, the connecting domain is the hinge region of human CD8α.

In some embodiments of the present invention, the human CD8α hinge region has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:10.

In some embodiments of the present invention, the T cell activation primary signal molecule comprises the anti-CD3 antibody or its antigen-binding fragment, the CD8α hinge region and the CD8α transmembrane region from the N-terminus to the C-terminus; preferably, the anti-CD3 antibody is the UCHT1-scFv.

In some embodiments of the present invention, the T cell activation secondary signal molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof, a CD8α hinge region and a CD8α transmembrane region from the N-terminus to the C-terminus; preferably, the anti-CD28 antibody is the 15E8-scFv.

In some embodiments of the present invention, the T cell activation secondary signal molecule is selected from at least one of human CD80 or its receptor binding fragment and human CD86 or its receptor binding fragment;

Preferably, the amino acid sequence of human CD80 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:93;

Preferably, the amino acid sequence of human CD86 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:95.

In some embodiments of the present invention, the receptor binding fragment of human CD80 comprises the extracellular domain and transmembrane region of human CD80; the receptor binding fragment of human CD86 comprises the extracellular domain and transmembrane region of human CD86;

Preferably, the amino acid sequences of the extracellular domain and transmembrane region of human CD80 are at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:94;

Preferably, the amino acid sequences of the extracellular domain and transmembrane region of human CD86 are at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:79.

In some embodiments of the present invention, the transmembrane polypeptide is the glycoprotein, and the glycoprotein is directly or indirectly linked to the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule.

In some embodiments of the present invention, the glycoprotein is indirectly connected to the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule through a first polypeptide linker (The First Polypeptide Linker).

In some embodiments of the present invention, the T cell activation primary signal molecule is directly or indirectly linked to the T cell activation secondary signal molecule.

In some embodiments of the present invention, the T cell activation primary signal molecule is indirectly connected to the T cell activation secondary signal molecule through a second polypeptide linker (The Second Polypeptide Linker).

In some embodiments of the present invention, the polypeptide linker is a flexible linker peptide.

In some embodiments of the present invention, the flexible connecting peptide is selected from (G ₄ S) _n connecting peptide, linker 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 97) and linker 2: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 98); wherein n=1-4.

In some embodiments of the present invention,

(a) the glycoprotein is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( _G4S ) _n connecting peptide, and the anti-CD3 antibody or antigen-binding fragment thereof is indirectly linked to the anti-CD28 antibody or antigen-binding fragment thereof, human CD86 extracellular domain or human CD80 extracellular domain via a ( _G4S ) _n connecting peptide; and/or

(b) the glycoprotein is indirectly linked to the anti-CD28 antibody or antigen-binding fragment thereof, the extracellular domain of human CD86 or the extracellular domain of human CD80 via a (G ₄ S) _n connecting peptide, and the anti-CD28 antibody or antigen-binding fragment thereof, the extracellular domain of human CD86 or the extracellular domain of human CD80 is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a (G ₄ S) _n connecting peptide;

Preferably, n=3;

Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is the UCHT1-scFv;

Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is the 15E8-scFv.

In some embodiments of the present invention, the glycoprotein is indirectly linked to the UCHT1-scFv via a (G ₄ S) ₃ linker peptide, and the UCHT1-scFv is indirectly linked to the 15E8-scFv via a (G ₄ S) ₃ linker peptide.

In some embodiments of the present invention, the glycoprotein is indirectly linked to the 15E8-scFv via a (G ₄ S) ₃ linker peptide, and the 15E8-scFv is indirectly linked to the UCHT1-scFv via a (G ₄ S) ₃ linker peptide.

In some embodiments of the present invention, the glycoprotein is selected from at least one of the envelope glycoproteins of vesicular stomatitis virus strains and their variants, the envelope glycoproteins of baboon endogenous retrovirus BaEV and their variants, the envelope glycoproteins RD114 of feline endogenous retrovirus and their variants, and the envelope glycoproteins GALV of gibbon ape leukemia virus and their variants.

In some embodiments of the present invention, the glycoprotein is selected from at least one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof;

Preferably, the envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain include the following envelope glycoproteins and variants thereof: envelope glycoprotein and variants of Indiana strain of vesicular stomatitis virus, envelope glycoprotein and variants of Cocal strain of vesicular stomatitis virus, envelope glycoprotein and variants of Maraba strain of vesicular stomatitis virus, envelope glycoprotein and variants of Morreton strain of vesicular stomatitis virus, envelope glycoprotein and variants of Alagoas strain of vesicular stomatitis virus, envelope glycoprotein and variants of New Jersey strain of vesicular stomatitis virus, envelope glycoprotein and variants of Carajas strain of vesicular stomatitis virus, envelope glycoprotein and variants of Chandipura strain of vesicular stomatitis virus and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

The envelope glycoproteins of vesicular stomatitis virus strains, such as Indiana strain (VSV-G) and Cocal strain (Cocal-G), can bind to the low-density lipoprotein receptor (LDL-R) widely expressed on the surface of various cells and have a wide range of infectivity.

The extracellular domain of the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus comprises the amino acid sequence shown in SEQ ID NO:1; the extracellular domain of the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus comprises the amino acid sequence shown in SEQ ID NO:2.

In some embodiments of the present invention, the amino acid sequence of the full-length protein of the wild-type VSV-G (including the VSV-G signal peptide) is shown in SEQ ID NO: 22;

Wherein, the amino acid sequence shown in positions 1 to 16 of SEQ ID NO: 22:

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

In some embodiments of the present invention, the amino acid sequence of the full-length protein of the wild-type Cocal-G (including the Cocal-G signal peptide) is shown in SEQ ID NO:99;

Among them, the sequence shown in the 1st to 17th positions of SEQ ID NO:99:

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

Extracellular domain of wild-type VSV-G:

Extracellular domain of wild-type Cocal-G:

Full-length protein of wild-type VSV-G:

Full-length protein of wild-type Cocal-G:

Activated T cells express LDL-R, therefore, artificially synthesized, biosafe lentiviral vectors or retroviral vectors usually use wild-type VSV-G to construct their envelope glycoprotein (VSV-G lentiviral vector or retroviral vector) to transduce activated T cells.

In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus or a variant thereof, and the glycoprotein receptor is the low-density lipoprotein receptor LDL-R;

The extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

However, LDL-R is widely expressed on the surface of various cells, such as activated T cells, hepatocytes, cardiomyocytes and endothelial cells. Therefore, VSV-G lentiviral vectors or retroviral vectors can also infect other cells by binding to LDL-R, and the targeting of infecting T cells is relatively low.

By weakening the ability of VSV-G to bind to LDL-R and simultaneously making its envelope contain primary and secondary signal molecules for T cell activation such as anti-CD3 antibodies and anti-CD28 antibodies, the ability of VSV-G type lentiviral vectors or retroviral vectors to target, activate and transduce T cells can be effectively improved.

In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a) substitution or deletion of the amino acid at position 8, substitution or deletion of the amino acid at position 9, substitution or deletion of the amino acid at position 10, substitution or deletion of the amino acid at position 47, substitution or deletion of the amino acid at position 50, substitution or deletion of the amino acid at position 51, substitution or deletion of the amino acid at position 183, substitution or deletion of the amino acid at position 179, substitution or deletion of the amino acid at position 180, substitution or deletion of the amino acid at position 182, substitution or deletion of the amino acid at position 184, substitution or deletion of the amino acid at position 209 of SEQ ID NO:1 or SEQ ID NO:2. 347, substitution or deletion of amino acid 350, substitution or deletion of amino acid 352, substitution or deletion of amino acid 353, substitution of amino acid 354, 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;

(b) after optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184 The present invention relates to a substitution or deletion of the amino acid at position 350, a substitution or deletion of the amino acid at position 352, a substitution or deletion of the amino acid at position 353, a substitution or deletion of the amino acid at position 354, a deletion of the amino acids at positions 1-18, a deletion of the amino acids at positions 19-36, a deletion of the amino acids at positions 37-51, a deletion of the amino acids at positions 314-384, a deletion of the amino acids at positions 321-374, a deletion of the amino acids at positions 331-364, a deletion of the amino acids at positions 344-354, and a deletion of the amino acids at positions 345-353.

In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a) deletion of amino acids 331 to 364, deletion of amino acids 344 to 354, substitution of K47, deletion of K47, or substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2;

(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletion at positions 331 to 364, amino acid deletion at positions 344 to 354, replacement of K47, deletion of K47, and replacement of R354 are located at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a) The amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 in SEQ ID NO: 1 or SEQ ID NO: 2;

(b) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 are located corresponding to SEQ ID NO:1 or SEQ ID NO:2.

In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises the following amino acids:

(a) K47 deletion located at SEQ ID NO:1 or SEQ ID NO:2;

(b) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the K47 deletion is located at the position equivalent to SEQ ID NO:1 or SEQ ID NO:2.

In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a) substitution of K47, deletion of K47, substitution of I182, substitution of R354, and substitution of Y209 in SEQ ID NO: 1;

(b) After optimal global alignment with SEQ ID NO: 1, the substitutions at K47, K47 deletion, I182 substitution, R354 substitution, and Y209 substitution are located at positions equivalent to those of SEQ ID NO: 1;

(c) substitution of K47, deletion of K47, substitution of V182, substitution of R354, and substitution of Y209 at SEQ ID NO:2;

(d) After optimal global alignment with SEQ ID NO: 2, the substitution of K47, K47 deletion, V182 substitution, R354 substitution, and Y209 substitution are located at positions equivalent to SEQ ID NO: 2;

Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a) K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q located in SEQ ID NO:1;

(b) After optimal global alignment with SEQ ID NO:1, K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q are located at the positions equivalent to SEQ ID NO:1;

(c) K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q located in SEQ ID NO:2;

(d) After optimal global alignment with SEQ ID NO:2, it is located at K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q equivalent to SEQ ID NO:2.

In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 23 or SEQ ID NO: 24.

In some embodiments of the present invention, SEQ ID NO:3 comprises a K47 deletion relative to SEQ ID NO:1.

In some embodiments of the present invention, relative to SEQ ID NO:1, SEQ ID NO:4 contains R354Q.

In some embodiments of the present invention, SEQ ID NO:23 comprises a K47 deletion relative to SEQ ID NO:2.

In some embodiments of the present invention, relative to SEQ ID NO:2, SEQ ID NO:24 comprises R354Q.

In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 23 or SEQ ID NO: 24.

In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:23 or SEQ ID NO:24, or has at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:23 or SEQ ID NO:24.

In some embodiments of the present invention, any of the aforementioned glycoproteins having the first mutation retains the ability to mediate membrane fusion and endosomal/lysosomal escape.

In some embodiments of the present invention, the glycoprotein further undergoes a second mutation, which enhances the ability of the glycoprotein to antagonize inactivation by complement or prevents inactivation by complement relative to before the second mutation.

In some embodiments of the present invention, the glycoprotein having the second mutation is any of the aforementioned glycoproteins.

The complement system is composed of a series of proteins and is part of the innate immune system. Complement (complement, C) exists in the serum, tissue fluid and cell membrane surface of normal humans and animals. After activation, it has enzymatic activity and can undergo complex cascade reactions. The complement system is activated by a series of enzymes (enzymes) cutting each other, and eventually forms a membrane attack complex similar to a hole on the target microorganism, causing the microorganism to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, and clear immune complexes through lysis, conditioning, phagocytosis and mediating inflammatory reactions, showing corresponding biological functions. Complement is widely involved in the body's defense response against microbial infection and immune regulation, and also mediates immunopathological damage reactions. It is an effector system and effector method system with important biological functions in the body.

The regulatory complement components exist in soluble or membrane-bound forms, including properdin (P factor), C1 inhibitor (C1INH), factor I, factor H, C4 binding protein (C4BP), S protein, SP40/40, membrane cofactor protein (MCP), decay accelerating factor (DAF), homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL).

After entering the serum, VSV-G lentiviral vectors or retroviral vectors may be recognized and inactivated by complement, making it difficult for them to efficiently reach the target cells and exert their effects; therefore, when used in vivo to prepare CAR-T cells, the efficiency of VSV-G lentiviral vectors or retroviral vectors in transducing non-activated T cells is low.

By causing VSV-G or Cocal-G to undergo the second mutation, the ability of VSV-G or Cocal-G to antagonize complement inactivation is improved, thereby making the mutant VSV-G lentiviral vector or retroviral vector more suitable for use in the preparation of CAR-T cells in vivo.

In some embodiments of the present invention, the glycoprotein that undergoes the second mutation is an envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus or a variant thereof, and the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, or has at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

In some embodiments of the present invention, the second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a) amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 214th amino acid corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(c) amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

(d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 352nd amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

(f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at the 50th amino acid equivalent to SEQ ID NO: 1 or SEQ ID NO: 2;

(g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

(h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at the 146th amino acid corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

Preferably, the amino acid mutation is selected from at least one of amino acid deletion, insertion and substitution;

More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids:

(a) amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 214th amino acid corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(c) amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

(d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 352nd amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

(f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at the 50th amino acid equivalent to SEQ ID NO: 1 or SEQ ID NO: 2;

(g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

(h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 146th amino acid equivalent to SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 1, comprising at least one of the following site mutations:

(a) substitution of T214, T352, K50 and S146 in SEQ ID NO: 1;

(b) After optimal global alignment with SEQ ID NO: 1, the substitutions at T214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 1;

Preferably, the second mutation includes that the amino acid sequence comprises at least one of the following site mutations:

(a) T214N, T352A, K50T, S146T located in SEQ ID NO: 1;

(b) After optimal global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO:1.

In some embodiments of the present invention, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

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

(b) after optimal global alignment with SEQ ID NO:1, substitutions at positions corresponding to (1) T214 and T352 of SEQ ID NO:1; or (2) substitutions at T214, T352, K50, and S146;

Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

(a) (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T located at SEQ ID NO:1;

(b) After optimal global alignment with SEQ ID NO:1, located at (1) T214N and T352A equivalent to SEQ ID NO:1; or (2) T214N, T352A, K50T and S146T.

In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:2, comprising at least one of the following site mutations:

(a) substitution of K214, T352, K50 and S146 in SEQ ID NO: 2;

(b) After optimal global alignment with SEQ ID NO: 2, the substitutions at K214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 2;

Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence:

(a) K214N, T352A, K50T, S146T located in SEQ ID NO: 2;

(b) After optimal global alignment with SEQ ID NO:2, K214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO:2.

In some embodiments of the present invention, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

(a) replacement of (1) K214 and T352; or (2) replacement of K214, T352, K50 and S146 at SEQ ID NO:2;

(b) after optimal global alignment with SEQ ID NO:2, the position is equivalent to (1) replacement of K214 and T352 of SEQ ID NO:2; or (2) replacement of K214, T352, K50 and S146;

Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

(a) (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T located at SEQ ID NO:2;

(b) After optimal global alignment with SEQ ID NO:2, located at (1) K214N and T352A; or (2) K214N, T352A, K50T and S146T equivalent to SEQ ID NO:2.

In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

In some embodiments of the present invention, relative to SEQ ID NO:1, SEQ ID NO:5 comprises K47 deletion, T214N, T352A, K50T and S146T.

In some embodiments of the present invention, relative to SEQ ID NO:1, SEQ ID NO:6 comprises K47 deletion, T214N and T352A.

In some embodiments of the present invention, relative to SEQ ID NO:1, SEQ ID NO:7 comprises R354Q, T214N and T352A.

In some embodiments of the present invention, relative to SEQ ID NO:1, SEQ ID NO:21 comprises R354Q, T214N, T352A, K50T and S146T.

In some embodiments of the present invention, relative to SEQ ID NO:2, SEQ ID NO:25 comprises K47 deletion, K214N, T352A, K50T and S146T.

In some embodiments of the present invention, relative to SEQ ID NO:2, SEQ ID NO:26 comprises K47 deletion, K214N and T352A.

In some embodiments of the present invention, relative to SEQ ID NO:2, SEQ ID NO:27 comprises R354Q, K214N, T352A, K50T and S146T.

In some embodiments of the present invention, relative to SEQ ID NO:2, SEQ ID NO:28 comprises R354Q, K214N and T352A.

In some embodiments of the present invention, any of the aforementioned glycoproteins having the second mutation retains the ability to mediate membrane fusion and endosomal/lysosomal escape.

In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof;

The extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1;

The glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the glycoprotein to bind to LDL-R is weakened or lost relative to before the first mutation; the glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation, or is not inactivated by complement.

In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus or a variant thereof;

The extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:2;

The glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the glycoprotein to bind to LDL-R is weakened or lost relative to before the first mutation; the glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation, or is not inactivated by complement.

In some embodiments of the present invention, any of the aforementioned glycoproteins having the first mutation and the second mutation retains the ability to mediate membrane fusion and endosomal/lysosomal escape.

In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

In some embodiments of the present invention, any of the aforementioned viral particles contains an exogenous payload gene.

In some embodiments of the present invention, the exogenous load gene encodes a therapeutic protein or polypeptide, i.e., "Therapeutic Proteins or Polypeptide"; the therapeutic protein or polypeptide is grouped according to its molecular type, and is selected from at least one of antibody-based drugs, chimeric antigen receptors, T cell receptors, cytokine receptors, cytokines, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytic agents.

In some embodiments of the present invention, the exogenous cargo gene encodes a chimeric antigen receptor (CAR).

In some embodiments of the present invention, the chimeric antigen receptor comprises an extracellular antigen binding region, a transmembrane region and an intracellular signaling domain.

In some embodiments of the present invention, the extracellular antigen binding region of the CAR binds to an antigen associated with the disease.

In some embodiments of the present invention, the antigen is selected from:

TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD3 8. CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD1 38. DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin (MSLN), 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, EphA 2. 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, bean curd protein, HPV E6/E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, P At least one of CTA-1/Galectin 8, MelanA/MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2 ETS fusion gene/ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, and NKG2D.

In some embodiments of the present invention, the antigen is GC-C (guanylate cyclase C).

In some embodiments of the present invention, the antigen is CD19.

In some embodiments of the present invention, the antigen is selected from one or more of CD19, CD20, BCMA, CD33, HER2 and CEA.

In some embodiments of the present invention, the extracellular antigen binding region of the CAR comprises an antibody or an antigen binding fragment thereof and/or a ligand or a receptor binding fragment thereof, wherein the antibody or antigen binding fragment thereof is selected from at least one of an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab') ₂ , an Fv fragment, a single-chain variable region fragment (scFv), a disulfide bond-stabilized antibody (dsFv), an antibody heavy chain variable region (VH) or a light chain variable region (VL), an Fd fragment consisting of a VH and a CH1 domain, a linear antibody, and a single-domain antibody (nanoantibody).

In some embodiments of the present invention, the extracellular antigen binding region of the CAR includes scFv.

In some embodiments of the present invention, the extracellular antigen binding region of the CAR is monospecific, bispecific or multispecific.

In some embodiments of the present invention, the extracellular antigen binding region of the CAR is an antibody or an antigen binding fragment thereof and/or a ligand or a receptor binding fragment thereof derived from mouse, rat, monkey, human or humanized.

In some embodiments of the present invention, the transmembrane region of the CAR is derived from the transmembrane region of at least one of the following proteins:

CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-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.

In some embodiments of the present invention, the transmembrane region of the CAR is derived from the transmembrane region of CD8α.

In some embodiments of the present invention, the transmembrane region of the CAR is derived from the transmembrane region of human CD8α.

In some embodiments of the present invention, the amino acid sequence of the transmembrane region of human CD8α is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11.

In some embodiments of the present invention, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of at least one of the following proteins:

CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FcεRlγ, FcεRβ, FcγRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP12 and other intracellular signaling domains of proteins whose intracellular signaling domains contain at least one ITAM.

In some embodiments of the present invention, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of CD3ζ.

In some embodiments of the present invention, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of human CD3ζ.

In some embodiments of the present invention, the amino acid sequence of the intracellular signaling domain of human CD3ζ is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18.

In some embodiments of the present invention, any of the aforementioned chimeric antigen receptors further comprises a hinge region; the hinge region sequentially connects the extracellular antigen binding region and the transmembrane region.

In some embodiments of the present invention, the hinge region of the CAR is derived from the hinge region of at least one of the following proteins:

CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.

In some embodiments of the present invention, the hinge region of the CAR is derived from the hinge region of CD8α.

In some embodiments of the present invention, the hinge region of the CAR is derived from the hinge region of human CD8α.

In some embodiments of the present invention, the amino acid sequence of the hinge region of human CD8α is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10.

In some embodiments of the present invention, any of the aforementioned chimeric antigen receptors further comprises a co-stimulatory signaling domain.

In some embodiments of the present invention, the costimulatory signaling domain of the CAR is derived from the costimulatory signaling domain of at least one of the following proteins:

CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

In some embodiments of the present invention, the costimulatory signaling domain of the CAR is derived from the costimulatory signaling domain of 4-1BB.

In some embodiments of the present invention, the costimulatory signaling domain of the CAR is derived from the costimulatory signaling domain of human 4-1BB.

In some embodiments of the present invention, the costimulatory signaling domain of the CAR includes the costimulatory signaling domain of human 4-1BB and the costimulatory signaling domain of human CD28.

In some embodiments of the present invention, the amino acid sequence of the co-stimulatory signaling domain of human 4-1BB is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:17.

In some embodiments of the present invention, any of the aforementioned CARs further comprises a signal peptide; the signal peptide is not particularly limited as long as it can mediate the expression of CAR outside the cell membrane.

In some embodiments of the present invention, the signal peptide of the CAR is selected from CD8α signal peptide, CD28 signal peptide, and IgG1 signal peptide.

In some embodiments of the present invention, the signal peptide of the CAR is a human CD8α signal peptide.

In some embodiments of the present invention, the amino acid sequence of the human CD8α signal peptide is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8.

In some embodiments of the present invention, the efficiency of transducing non-activated T cells by the viral particles is increased compared to a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules.

In some embodiments of the present invention, compared with a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules, the efficiency of expression of the exogenous load gene in T cells is improved after the virus particles contact non-activated T cells.

In some embodiments of the present invention, relative to a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules, after the virus particles contact non-activated T cells, the efficiency of the chimeric antigen receptor in T cell membrane expression is improved.

In some embodiments of the present invention, compared with a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules, the killing efficiency of the prepared CAR-T cells is enhanced after the virus particles contact non-activated T cells.

In some embodiments of the present invention, the control carrier comprises at least one T cell targeting molecule on its surface, and the T cell targeting molecule binds to a T cell endocytic receptor.

In some embodiments of the present invention, the T cell endocytosis receptor is selected from at least one of CD5 and CD7.

In some embodiments of the present invention, the T cell targeting molecule binds to CD7.

In some embodiments of the present invention, the T cell targeting molecule comprises at least one selected from an anti-CD7 antibody or an antigen-binding fragment thereof and a CD7 ligand or a receptor-binding fragment thereof;

Optionally, the anti-CD7 antibody or its antigen-binding fragment is a scFv (TH69-scFv) derived from the monoclonal antibody TH-69; the amino acid sequence of the TH69-scFv is as shown in SEQ ID NO:37; the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO:38-40, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO:41-43, respectively.

The "control vector" includes, but is not limited to, a control group vector having the same structure as the virus particle except that the surface of the control group vector does not contain the T cell activation primary signal molecule and the T cell activation secondary signal molecule.

In another aspect, the present invention provides an engineered T cell, wherein the engineered T cell expresses a chimeric antigen receptor, and the engineered T cell is prepared by contacting a T cell with any of the aforementioned virus particles provided by the present invention carrying a polynucleotide encoding a chimeric antigen receptor.

In some embodiments of the present invention, the chimeric antigen receptor includes but is not limited to any of the aforementioned chimeric antigen receptors.

In some embodiments of the present invention, the T cells include non-activated T cells and activated T cells.

In some embodiments of the present invention, the contacting occurs in vivo and/or in vitro in a subject, and the subject is an individual who is administered the engineered T cells and/or any of the aforementioned viral particles carrying a polynucleotide encoding a chimeric antigen receptor.

In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device administration.

In some embodiments of the present invention, the T cells are non-activated T cells, and the efficiency of the engineered T cells expressing CAR is improved;

The improvement in expression efficiency is relative to the engineered T cells prepared by contacting a control vector not containing the T cell activation primary signal molecule and the T cell activation secondary signal molecule on its surface with the non-activated T cells.

In some embodiments of the present invention, the T cells are non-activated T cells, and the killing efficiency of the engineered T cells is improved;

The improvement in killing efficiency is relative to the engineered T cells prepared by contacting a control vector whose surface does not contain the T cell activation primary signal molecule and the T cell activation secondary signal molecule with the non-activated T cells.

In some embodiments of the present invention, the control vector is any of the aforementioned control vectors.

In another aspect, the present invention provides a composition comprising a pharmaceutically acceptable excipient or carrier and any one of the following components: any one of the aforementioned virus particles provided by the present invention and engineered T cells.

In another aspect, the present invention provides use of any of the aforementioned viral particles, engineered T cells or compositions in the preparation of a drug for preventing and/or treating a disease.

In some embodiments of the present invention, the disease is an autoimmune disease or cancer, including solid cancer and blood cancer.

In some embodiments of the present invention, the cancer is a blood cancer.

In some embodiments of the present invention, the cancer is B-lymphocyte cancer, B-lymphocyte leukemia, Hodgkin's lymphoma or multiple myeloma.

In some embodiments of the present invention, the blood cancer is selected from non-Hodgkin lymphoma (NHL), acute B-cell lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FLCL), and The patient may be one or more of: lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma/leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), primary plasma cell leukemia (pPCL), peripheral T-cell lymphoma (PTCL-NHL), NK/T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, T-large granular lymphocytic leukemia (T-LGL) and germ center T-cell lymphoma (FTCL).

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

In some embodiments of the present invention, the cancer is selected from one or more of CD19 ⁺ cancer, CD20 ⁺ cancer, BCMA ⁺ cancer, CD33 ⁺ cancer, HER2 ⁺ cancer and CEA ⁺ cancer.

In another aspect, the present invention provides a method for treating cancer in a subject or killing cancer cells in a subject, comprising administering to the subject any one of the aforementioned viral particles, engineered T cells or compositions provided by the present invention; the viral particles or the composition contain an exogenous load gene.

In some embodiments of the present invention, the exogenous load gene is any one of the exogenous load genes mentioned above.

In some embodiments of the present invention, the exogenous cargo gene encodes a chimeric antigen receptor.

In some embodiments of the present invention, the chimeric antigen receptor includes but is not limited to any of the aforementioned chimeric antigen receptors.

In some embodiments of the present invention, the administration is at least one of the aforementioned administrations.

In another aspect, the present invention also provides a cell, wherein the cell is configured to produce any of the aforementioned virus particles provided by the present invention.

In some embodiments of the present invention, the cell line is selected from at least one of NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK cells, BHK cells and MDCKⅡ cells;

Preferably, the cells are HEK-293T cells.

In some embodiments of the present invention, the cells are selected from HEK cells.

In some embodiments of the present invention, the cell is a HEK-293T cell.

In some embodiments of the present invention, the subject is a subject in need thereof.

In another aspect, the present invention also provides a method for preparing virus particles, the method comprising culturing any of the aforementioned cells provided by the present invention until sufficient to produce the virus particles.

In another aspect, the present invention also provides a method for transducing T cells, the method comprising using any of the aforementioned virus particles provided by the present invention to carry an exogenous load gene and contact the T cells.

In some embodiments of the present invention, the exogenous load gene is any one of the exogenous load genes mentioned above.

In some embodiments of the present invention, the exogenous cargo gene encodes a chimeric antigen receptor.

In some embodiments of the present invention, the chimeric antigen receptor includes but is not limited to any of the aforementioned chimeric antigen receptors.

In some embodiments of the present invention, the T cells include activated T cells and non-activated T cells.

In some embodiments of the present invention, the contact occurs in vivo and/or in vitro in a subject; the subject is an individual who is given T cells transduced by the transduction method and/or any of the aforementioned viral particles carrying exogenous load genes.

In some embodiments of the present invention, the contacting occurs in a subject, and the subject is an individual who is administered any of the aforementioned viral particles carrying the exogenous load gene.

In some embodiments of the present invention, the viral particle is any of the aforementioned viral particles carrying the CAR gene.

In some embodiments of the present invention, the administration is at least one of the aforementioned administrations.

The beneficial effects of the present invention include:

The surface of any of the aforementioned virus particles provided by the present invention contains T cell activation primary signal molecules such as anti-CD3 antibodies and T cell activation secondary signal molecules such as anti-CD28 antibodies, which can effectively target and activate non-activated T cells; and the glycoprotein on the surface of the virus particle undergoes a first mutation, so that its ability to bind to the glycoprotein receptor is weakened or lost, further significantly improving the ability of the virus particle to target and activate and transduce non-activated T cells; the glycoprotein of the virus particle can also undergo a second mutation, so that the ability of the glycoprotein to antagonize complement inactivation is enhanced, making it more suitable for use in the preparation of CAR-T cells in vivo; at the same time, compared with the control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules, after the virus particle delivers the CAR gene to the genome of the T cell, the efficiency of the CAR molecule in the membrane expression of the T cell is higher, and the killing efficiency of the prepared CAR-T cells is better.

In this article:

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

In some embodiments of the present invention, the T cells are CD4 ⁺ /CD8 ^- , CD4 ^- /CD8 ⁺ , CD4 ⁺ /CD8 ⁺ , CD4 ^- /CD8 ^- T cells or a combination thereof. In some embodiments of the present invention, CD4 ⁺ T cells produce IL-2, TFN, TNF or a combination thereof after expressing CAR and binding to target cells such as CD19 ⁺ tumor cells. In some embodiments of the present invention, CD8 ⁺ T cells lyse antigen-specific target cells after expressing CAR and binding to target cells.

Non-activated T cells refer to T cells that are not proliferated, differentiated, in a resting state, do not recognize antigens, and have not been activated by primary signaling molecules such as TCR-CD3 complex binding molecules such as anti-CD3 antibodies and secondary signaling molecules/co-stimulatory molecules such as anti-CD28 antibodies, CD80 and CD86, such as T cells in the G0 phase of the cell cycle, resting/quiescent T cells, or naive T cells. T cells. Resting T cells, also called quiescent T cells or naive T cells, refer to T cells that are not mitotically active or have not been exposed to cognate antigens presented on antigen presenting cells, such as macrophages or dendritic cells.

"Antibody": refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of the immunoglobulin heavy chain and/or sufficient sequence from the variable region of the immunoglobulin light chain to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures as long as they exhibit the desired antigen binding activity.

The "antibody" herein includes a typical "four-chain antibody", which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from its N-terminus to its C-terminus; and, when the full-length antibody is an IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain refers to a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction from its N-terminus to its C-terminus; the heavy chains and the light chains are connected by disulfide bonds to form a "Y"-shaped structure.

In the context of antibodies, the term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains (VH and VL regions, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th edition, W.H.Freeman and Co., p. 91 (2007)). A single VH region or VL region may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be separated using a VH region or VL region from an antibody that binds to that specific antigen to screen a library of complementary VL regions or VH regions, respectively. See, e.g., Portolano et al., J.Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and/or binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

In some embodiments of the present invention, the CDR regions are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme or the Contact numbering scheme.

In some embodiments of the invention, the CDR regions are defined according to the Kabat numbering scheme.

“Antibody” in this article also includes single-chain variable region fragment (Single-Chain Fragment Variable, “scFv”).

"Antibodies" in this article also include antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus Dromedarius), Bactrian camels (Camelus Bactrianus), llamas (Lama Glama), guanacos (Lama Guanicoe) and alpacas (Vicugna Pacos), as well as immunoglobulin new antigen receptors (Ig New Antigen Receptor, IgNAR) found in cartilaginous fish such as sharks.

In this article, the terms "VHH domain" and "single domain antibody" (sdAb) have the same meaning and can be used interchangeably. They refer to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, a heavy chain antibody that naturally lacks the light chain and 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.

"Antibodies" herein also include monoclonal antibodies or antigen-binding portions thereof. Monoclonal antibodies or antigen-binding portions thereof may be non-human, chimeric, humanized or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., ed., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, monkeys (e.g., cynomolgus monkeys and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

Herein, "antigen-binding fragment" refers to a fragment that does not have the entire structure of an intact antibody and only contains a portion or a partial variant of the intact antibody, wherein the portion or partial variant has the ability to bind to an antigen.

Exemplarily, herein, “antibody or antigen-binding fragment thereof” includes, but is not limited to, immunoglobulin (full-length antibody), half antibody, Fab, Fab', F(ab') ₂ , Fv fragment, single-chain variable fragment (scFv), disulfide bond-stabilized antibody (dsFv), heavy chain variable region (VH) or light chain variable region (VL) of antibody, Fd fragment consisting of VH and CH1 domains, linear antibody, heavy chain antibody and nanobody (VHH).

In some embodiments of the present invention, there is no particular limitation on the order in which the VH region or VL region of the scFv is included 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 connecting peptide can be selected from a flexible connecting peptide.

"Ligand": In receptor-ligand binding, a ligand is generally a molecule that binds to a site on a receptor to generate a signal, such binding typically resulting in a conformational change in the complex structure, thereby inducing the relevant physiological activity.

"Receptor binding fragment" refers to a fragment that does not have the entire structure of a complete ligand, but only contains a portion or a partial variant of the complete ligand, and the portion or partial variant has the ability to bind to the receptor. Exemplarily, the "binding fragment of a ligand" herein includes but is not limited to the extracellular domain, functional fragment, epitope, binding region and variable region of the ligand.

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

"Chimeric Antigen Receptor": Chimeric Antigen Receptor (CAR) refers to an artificial cell surface receptor that has been modified to be expressed on immune effector cells such as lymphocytes and specifically binds to antigens, which at least includes (1) an extracellular antigen binding region, such as scFv or VHH; (2) a transmembrane region that anchors the CAR molecule into the immune effector cell, and (3) an intracellular signal transduction domain; the extracellular structure of CAR may further include a hinge region, and the intracellular structure may further include a co-stimulatory signal transduction domain. CAR molecules can use the extracellular antigen binding region to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner. In some embodiments of the present invention, each polypeptide structure contained in the CAR is of human origin.

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

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

"Weakened": When referring to the "weakened" ability of the glycoprotein variant to specifically bind to its receptor, the term "weakened" includes completely eliminating the ability of the glycoprotein variant to specifically bind to its receptor, as well as significantly weakening the binding ability. In a specific embodiment, "significantly weakened" means relative to the wild-type viral glycoprotein; "weakened" is selected from the group consisting of weakening by 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%.

"Nucleic acid": refers to any compound and/or substance including a polymer containing nucleotides, such as a polynucleotide. Herein, "nucleic acid", "polynucleotide" and "gene" are used synonymously. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, 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 generally expressed as 5' to 3'. Herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. "Nucleic acid" can be linear or cyclic. In addition, "nucleic acid" includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Furthermore, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases modified with derivatized sugars, phosphate backbone linkages, or chemically modified residues.

"Nucleic acid vector" means a vector that carries, contains or expresses any nucleic acid. The nucleic acid vector may have specific functions such as expression, packaging, pseudotyping or transduction. If the nucleic acid vector is suitable for use as a cloning vector or shuttle vector, it may also have a manipulation function. The structure of the vector may include any desired form that is feasible to manufacture and suitable for a particular purpose. Such forms include, for example, circular forms such as plasmids and phagemids, as well as linear or branched forms. Nucleic acid vectors may be composed of, for example, DNA or RNA, and contain some or all nucleotide derivatives, analogs and mimetics. Such nucleic acid vectors may be obtained from natural sources, recombinantly produced or chemically synthesized.

"Transgene": Transgene, also known as payload gene (Payload gene). As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., CAR or engineered TCR, etc.), the expression of which is desired in host cells/target cells and has been transferred into cells by genetic engineering technology. Transgenes can encode therapeutically significant proteins as well as proteins that serve as reporters, tags, markers, suicide proteins, etc. Transgenes can come from natural sources, modifications of natural genes, or recombinant or synthetic molecules. In certain embodiments, the transgene is a component of a vector, such as a particle (including NIL).

"Expression cassette": As used herein, the term "expression cassette" refers to a unique component of a vector nucleic acid comprising at least one transgene and regulatory sequences (e.g., promoter, 3'UTR) that control its expression in a host cell. A tandem expression cassette refers to a component of a vector nucleic acid comprising at least two transgenes under the control of a set of identical regulatory sequences for tandem expression of the at least two transgenes. In certain embodiments, the tandem expression cassette comprises at least two transgenes under the control of the same promoter. In certain embodiments, the first transgene and the second transgene are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to allow co-expression of two proteins from a single mRNA.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds.

"Encoding" refers to the inherent properties of a specific polynucleotide sequence (such as DNA, cDNA and mRNA sequences) used as a template for synthesizing other polymers and macromolecules in biological processes, and the template has a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to the polynucleotide produces a protein in a cell or other biological system, the polynucleotide encodes the protein. Both the coding strand and the non-coding strand can be referred to as encoding proteins or other products of the polynucleotide. Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

"Self-cleaving peptide" or "self-cleaving peptide" or "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, etc. 2A peptides are 18 to 22 residues long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" may refer to peptides with different amino acid sequences. In the present disclosure, it should be understood that in the case where the particle (including NIL) contains 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) Cold Spring Harb. Protoc. 2012: 199-204.

"Exogenous" refers to any molecule that originates from outside an organism, including nucleic acids, proteins, peptides, or small molecules. In contrast, the term "endogenous" refers to any molecule that originates from within an organism (i.e., produced naturally by the organism).

"Promoter": As used herein, the term "promoter" is defined as a DNA sequence recognized by the cellular synthetic machinery or introduced synthetic machinery required for initiating specific transcription of a polynucleotide sequence. As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence required for expressing a gene product operably linked to a promoter/regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may contain an enhancer sequence and other regulatory elements required for expressing a gene product. The promoter/regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes production of a gene product in a cell under most or all physiological conditions of the cell.

An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

"Viral envelope": 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.). The viral envelope protects the genetic material during the virus's life cycle as it travels through host cells. Not all viruses have a viral envelope. Many human pathogenic viruses are encapsulated in a lipid bilayer, and they infect target cells by fusing the viral envelope with the cell membrane.

"Retrovirus" and "Retroviral Vector": Retrovirus and Retroviral Vector. A retrovirus is a virus that can integrate a DNA copy of its RNA genome into the DNA of the host cell it infects, thereby changing the genome of the host cell. An overview of available packaging systems is provided in Cold Spring Harbour Laboratory Press, 1997, by JM Coffin, SM Hughes et al., p. 447, which is incorporated herein by reference in its entirety.

"Lentivirus": Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes Gag, Pol and env, other genes with regulatory or structural functions. The higher complexity allows the virus to regulate its life cycle, as it does during latent infection. Lentiviruses belong to a genus of retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type I and HIV type II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).

"Lentiviral vector": Lentiviral Vector, is a vector derived from a lentivirus and contains one or more lentiviral packaging proteins and/or lentiviral proteins necessary for the expression of one or more genes carried by the vector. It is produced by multiple attenuation of the virulence genes of lentiviruses such as HIV through gene editing, genetic engineering and other technical means, for example, by deleting the genes env, vif, vpr, vpu and nef, so that the lentiviral vector has biosafety.

As used herein, the term "lentiviral vector" is intended to mean a lentiviral particle that includes a viral envelope, has at least one characteristic of a lentivirus and is capable of invading a target cell and does not have the ability to replicate itself.

Lentiviral vectors or retroviral vectors are generally packaged in packaging cells by a lentiviral vector packaging system or a retroviral vector packaging system. Exemplary, the process and method of packaging lentiviral vectors refer to Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. (2016), 3, 16017, which is incorporated herein by reference in its entirety.

Commonly used pseudotyped lentiviral vectors include the so-called third-generation lentiviral vector packaging system. The third-generation lentiviral vector packaging system includes four plasmids, generally speaking, generally including a transfer plasmid and three packaging plasmids: a transfer plasmid containing a gene of interest ("GOI"), such as a transgenic, 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).

The "transfer plasmid" (Transfer Vector) contains the lentiviral vector backbone genome and transgene. The transfer plasmid usually has one or more transgenes flanked by long terminal repeat (LTRs) sequences, which facilitate the integration of the transgene contained in the transfer plasmid into the host genome. LTRs are responsible for the reverse transcription and integration process of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell. For safety reasons, the transfer plasmid is usually designed so that the resulting viral vector cannot replicate itself. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious lentiviral particles in host cells. In addition, the transfer plasmid can be designed to lack the 3'LTR, thereby making the virus "self-inactivating (Self-Inactivating). Compared with the traditional second-generation pseudo-lentiviral vector packaging system (usually a single packaging plasmid containing nucleic acids encoding Gag, Pol, Rev and Tat and a separate envelope plasmid), the TAT gene is eliminated from the third-generation pseudo-lentiviral vector packaging system by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) on the transfer plasmid. The transfer plasmid usually contains a Ψ sequence (Psi sequence, also known as Ψ packaging signal) located downstream of the 5'LTR, which is responsible for packaging the transgenic RNA (Pack The Ψ sequence ensures that only RNA containing the transgene is packaged into the viral particles. The transfer plasmid may also optionally contain an internal ribosome entry site (Internal Ribosome Entry Site, "IRES") to allow simultaneous translation of two or more open reading frames (ORFs) on one mRNA, thereby achieving multi-gene expression. Some transfer plasmids, such as the lentiviral master plasmid/transfer plasmid used in some embodiments of the present invention, may also contain a selection marker gene, such as an antibiotic resistance gene (such as PuroR, encoding puromycin resistance) or a fluorescent protein gene (such as GFP) for screening or tracking transduced cells.

For details about the transfer plasmid in the lentiviral vector packaging system, please refer to DuLl, et al., J. Virol. 72:8463-71 (1998); Miyoshi, et al., J. Virol. 72:8150-57 (1998).

The third-generation lentiviral vector system also generally includes three packaging plasmids: GagPol plasmid, Rev plasmid and envelope plasmid. The envelope plasmid generally carries viral glycoprotein genes, and wild-type VSV-G or Cocal-G is one of the commonly used viral glycoproteins; the viral glycoprotein gene is operably linked to a promoter, which is generally a CMV promoter, to initiate transcription of the viral glycoprotein gene. The third-generation lentiviral vector system also includes two packaging plasmids, one containing genes encoding Gag protein and Pol protein (GagPol packaging plasmid), and the other containing genes encoding Rev protein (Rev plasmid) as a further safety feature, which is an improvement over the single packaging plasmid of the so-called second-generation packaging system. The Gag gene encodes the Gag polyprotein precursor containing lentiviral structural proteins, which includes the matrix, capsid and nucleocapsid; the Pol gene encodes the Pol polyprotein precursor that provides the lentiviral enzyme functions necessary for replication, which includes protease, reverse transcriptase and integrase; the Rev gene encodes the Rev protein, which binds to the Rev response element (RRE) to allow nuclear export of unspliced and singly spliced HIV RNA during viral replication. Gag and Pol polyprotein precursors are cleaved during viral particle preparation. The Rev protein binds to the Rev response element (RRE) sequence on the viral RNA and promotes the transport of incompletely spliced viral RNA from the nucleus to the cytoplasm by interacting with the host cell's nuclear export machinery. These unspliced RNAs can be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral particles.

Exemplarily, the packaging plasmid includes but is not limited to pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

Lentiviral vectors and lentiviral vector backbone genomes are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey, et al., (1998) J. Virol. 72:9873-9880; DuLl, et al., (1998) J. Virol. 72:8463-8471, U.S. Pat. No. 6,013,516 and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety.

Compared with pseudotyped lentiviral vector packaging systems, pseudotyped retroviral vector packaging systems usually do not contain Rev plasmids. This is because the genomic RNA derived from retroviruses such as Moloney Murine Leukemia Virus (MMLV) can be naturally transported from the nucleus to the cytoplasm for translation and assembly, so there is no need to rely on specific nuclear export mechanisms such as Rev protein. Pseudotyped retroviral vector packaging systems usually contain a transfer plasmid and two packaging plasmids: envelope plasmid and GagPol packaging plasmid. The transgene sequence contained in the transfer plasmid is sandwiched on both sides by long terminal repeat sequences (LTRs), which promote the integration of the transfer plasmid sequence into the host genome. Usually, during viral transduction, the sequences between and including the LTRs will be integrated into the host genome. The backbone genome of MMLV or Murine Stem Cell Virus ("MSCV") containing their respective LTRs is generally used to construct transfer plasmids in pseudoretroviral vector packaging systems. The GagPol packaging plasmid contains the Gag gene and the Pol gene; the envelope plasmid generally 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 nucleic acid encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule.

In some embodiments, the production cells are transfected with transfer plasmids, GagPol plasmids, envelope plasmids, and Rev plasmids at a defined ratio. In some embodiments, the ratio of each plasmid is determined by mass, and there is no particular limitation as long as it can package a non-integrated lentiviral vector with biological activity. In some embodiments, the mass of each of the transfer plasmid and the GagPol plasmid is higher than the mass of each of the envelope plasmid and the Rev plasmid. In some embodiments, the defined ratio of the transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid is about 1:1:1:1 to about 9:4:2:2; in some embodiments of the present invention, the envelope plasmid may contain a nucleic acid encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule.

In some embodiments, the envelope plasmid comprises a tandem expression cassette encoding VSV-G or its variants or Cocal-G or its variants and the T cell activation primary signal molecule and/or T cell activation secondary signal molecule as disclosed herein. In a specific embodiment, the tandem expression cassette contained in the envelope plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule, a polynucleotide encoding an internal ribosome entry site (IRES), a furin protease cleavage site or a viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding VSV-G or its variants or Cocal-G or its variants. In certain embodiments, the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 5' end of the polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule. In other embodiments, the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 3' end of the polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule. The polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule and the polynucleotide encoding VSV-G or its variant or Cocal-G or its variant are separated in a tandem box by a polynucleotide encoding an IRES, a furin cleavage site or a viral 2A peptide, which allows co-expression of the two proteins by a single mRNA. In certain embodiments, the viral 2A peptide is porcine Teschovirus-1 (P2A), Thosea asigna virus (T2A), Equine rhinovirus (E2A), foot-and-mouth disease virus (F2A) or variants thereof.

The use of lentiviral/retroviral vector or particle packaging systems relies on a "packaging cell line". In general, a packaging cell line is a cell line whose cells are capable of producing a lentiviral or retroviral vector that is not self-replicating and can infect/transduce target cells when a transfer plasmid, one or more packaging plasmids, are introduced into the cells. An overview of available packaging lines is provided in Cold Spring Harbour Laboratory Press, 1997, by JM Coffin, SM Hughes et al., p. 447, which is incorporated herein by reference in its entirety.

Exemplarily, various plasmids can be introduced into the packaging cell line using transfection methods including chemical-mediated transfection methods, physical-mediated transfection methods or biological-mediated transfection methods. For example, chemical-mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physical-mediated transfection methods include transfection methods such as electroporation.

Production/host/packaging cells that can be used to prepare the particles disclosed herein include human embryonic kidney (HEK) 293 cells and their derivatives. The production cells can be adherent cell lines such as HEK293T production cells, or suspension cell lines such as HEK293T/17SF production cells.

Exemplarily, the packaging cell/host cell is selected from CHO cells, BHK cells, MDCK cells, C3H-10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells and 211 cells;

Preferably, the packaging cell/host cell is a HEK-293T cell.

"Retrovirus" and "Retroviral Vector": Retrovirus and Retroviral Vector. "Retrovirus" refers to an RNA virus with a single-stranded positive-sense RNA molecule. Retroviruses contain reverse transcriptase and integrase. After entering the target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the DNA molecule is integrated into the host cell genome using integrase. After integration into the host cell genome, the sequence from the retrovirus is called a provirus (e.g., a proviral sequence or a proviral sequence). Retroviral vectors generally refer to pseudotyped retroviral vectors derived from retroviruses, illustratively from γ-retroviruses. Unlike lentiviral vectors that can transduce dividing and non-dividing cells, retroviral vectors can only transduce dividing cells, and the exogenous transgenes they can carry are generally relatively small. For a comparison and discussion of lentiviral vectors and retroviral vectors, see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, K.K., Vorburger, S.A., Swisher, S.G. (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

Lentiviral and retroviral vectors can stably integrate exogenous cargo genes such as shuttle genes into the chromosomes of target cells, allowing the target cells to express the delivered shuttle genes for a long time, providing great advantages for gene therapy. In addition, they do not transfer viral genes, thus avoiding the problem of producing transduced cells that can be destroyed by cytotoxic T cells. And they have relatively large cloning capacity, which is sufficient for most expected clinical applications.

"Envelope glycoprotein" refers to the glycoprotein coated on the outer layer of the virus, which plays an important role in the adsorption of the virus and penetration into host cells, pathogenicity, downregulation of host surface protein expression, and increase in viral packaging and budding.

"Variant": A variant refers to a mutant having at least about 50% identity to the amino acid sequence of a non-mutant (wild type), and "at least about 50% identity" refers to about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of a non-mutant (wild type); alternatively, a variant refers to a nucleic acid sequence encoding a variant. A mutant having at least 50% identity with a nucleic acid sequence encoding a non-mutant (wild type), "at least 50% identity" means that the nucleic acid sequence encoding the variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with a nucleic acid sequence encoding a non-mutant (wild type). In some embodiments of the present invention, variants include mutants comprising conservative substitutions relative to non-mutants. "Conservative substitutions" are considered in the art to replace an amino acid with another amino acid having similar properties. For example, conservative substitutions are well known in the art (see, e.g., WO 97/09433, p. 10, published March 13, 1997; Lehninger, Biochemistry, 2nd ed.; Worth Publishers, Inc. NY: NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

"Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives and/or adjuvants. Excipients are preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration employed. Such excipients include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. The optimal pharmaceutical composition may be determined depending on the intended route of administration, mode of delivery, and desired dosage.

"Subject": As used herein, "subject", "patient" and "individual" are used synonymously, including but not limited to mammals, such as humans or non-human mammals, such as livestock, agricultural animals or wild animals, as well as birds and aquatic animals. As used herein, "subject" includes subjects suffering from a disease, disorder or condition, or at risk of developing a disease, disorder or condition, or otherwise in need of any of the viral particles, CAR-T cells, compositions or treatment methods provided herein.

A "disease" is a state of health in a subject in which the subject is unable to maintain homeostasis, and in which the subject's health continues to deteriorate if the disease is not improved. In contrast, a "disorder" or "adverse condition" of a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be if the disorder or adverse condition were not present. Without treatment, the disorder or adverse condition does not necessarily result in a further deterioration in the subject's state of health.

"Cancer": As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Abnormal cells may form solid tumors or constitute blood malignancies. Cancer cells may spread locally or spread to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, blood cancers, such as B-lymphocyte malignancies; solid cancers, such as breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphatic lung cancer, etc.

"Treatment" refers to the use of the treatment methods described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth) in a subject. The treatment method that effectively treats a patient may vary according to a variety of factors (e.g., the patient's disease state, age, weight, and the ability of the therapy to stimulate an anti-cancer response in the subject).

As used herein, "treatment" includes any beneficial or desired effects associated with treatment."Treatment" does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms.

The therapeutically effective amount will depend, for example, on the extent and goals of the treatment. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the delivered drug, molecule, cell, indication, route of administration, and patient condition (body weight, body surface or organ size) and/or condition (age and general health). In certain embodiments, the clinician may titrate the dose and change the route of administration to obtain the best therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the engineered T cells or the viral particles in the formulation used. The clinician typically administers the pharmaceutical composition until the dose that achieves the desired effect is reached. The pharmaceutical composition can therefore be administered as a single dose, or administered over time as two or more doses (which may or may not contain the same amount of the desired molecule), or administered by continuous infusion via an implantable device or catheter.

"Administration": The administration routes of the pharmaceutical composition are conventional in the art, such as oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection. It can also be administered by a sustained release system or by an implant device.

“And/or”: should be understood to mean one or two alternatives.

"About"/"approximately": As used herein, the term "about" refers to the common error range of the corresponding value that is readily known to those skilled in the art, including, by way of example, but not limited to, referring to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. References herein to "about" a value or parameter include (and describe) embodiments for the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

"Comprising": Herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, or elements, or groups of steps or elements, but not the exclusion of any other steps, or elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including", "having", "containing" and "comprising" are used synonymously.

"Embodiments": References throughout this specification to "some embodiments," "some embodiments," "embodiments," "particular embodiments," "related embodiments," "an embodiment," "another embodiment," or "other embodiments" or combinations thereof mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the various occurrences of the aforementioned phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

"Prevention": As used herein, "prevention" and similar words, such as "preventing", etc., indicate methods for preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms and/or burden of a disease or condition prior to onset or recurrence.

"Stable integration": also known as "stable transfection, refers to the integration of exogenous polynucleotides into the host cell genome after introduction into the host cell, and long-term stable expression in the host cell (Stable Gene Expression); the opposite is transient transfection and transient expression (Transient Expression).

"Specific binding": As used herein, the term "specific binding" refers to the binding that occurs between paired molecular species (e.g., a receptor and a ligand). 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, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times the background binding (non-specific binding), about 5 times the background binding, about 10 times the background binding, about 20 times the background binding, about 50 times the background binding, about 100 times the background binding, or about 1000 times the background binding or more.

"Sequence identity": In general, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. In general, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity." Whether it is a nucleic acid or amino acid sequence, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, 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 percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (usually nucleotides or amino acids) divided by the total number of shorter symbols in the two sequences. The program can be used to determine the percent identity over the entire length of the protein being compared.

"Signal peptide": Signal Peptide, sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically present at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Jour nal of Cell Biology.97(6):422-441.(2018))(Blobel G,Dobberstein B,et al.,Transfer of proteins across membranes.I.Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma,The Journal of Cell Biology,67(3):835-51.(1975)). Signal peptides are short peptides present at the N-terminus of newly synthesized proteins. Signal peptides are specific to the plasma membrane or secretory pathway. The signal sequence usually contains a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a signal sequence cleavage site. In eukaryotes, signal sequences cause newly synthesized proteins to translocate to the endoplasmic reticulum, where they are cleaved by signal peptidases to produce mature proteins that then enter their proper destinations. The diversity of signal sequence length and amino acid composition makes it difficult to accurately predict the cleavage site. For the polypeptide sequences disclosed herein, when referring to a signal sequence, polypeptide sequences in which no signal sequence is present or which have a partial signal sequence are also contemplated.

The role of the signal peptide is to prompt the cell to transfer proteins, usually to the cell membrane. In prokaryotes, the signal peptide directs newly synthesized proteins to the SecYEG protein conduction channel present in the plasma membrane. A homologous system exists in eukaryotes, in which the signal peptide directs newly synthesized proteins to the Sec6L channel, which has structural and sequence homology with SecYEG but is present in the endoplasmic reticulum (Rapoport TA, Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes, Nature. 450(7170):663-9(2007).). The SecYEG and Sec6L channels are often called transporters, and transport through the channel is called translocation. When a secreted protein passes through the channel, the transmembrane region may diffuse through the side gate in the translocon to be distributed into the surrounding membrane.

"MOI": "Multiplicity of Infection (MOI)" refers to the number of virus particles added to each cell during infection. For example, when one million virus particles are added to one million cells, MOI = 1.

"Operably": When a polynucleotide is in a functional relationship with another polynucleotide, the nucleic acid is "operably linked". For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or, if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the polynucleotides being linked are adjacent, and in the case of a secretory leader, adjacent and in the reading frame. However, enhancers do not have to be adjacent. Connection is achieved by connection at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or joints are used according to conventional practice.

"Transduction": As used herein, the terms "transfection", "transformation" and "transduction" are used synonymously and refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell, packaging cell. A "transfected", "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

Methods for introducing vectors such as viral particles or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical or biological methods.

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

Biological methods for introducing vectors or isolated nucleic acids into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

Chemical methods for introducing vectors or isolated nucleic acids into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome.

All publications, documents and patents mentioned herein are hereby incorporated by reference in their entirety, just as if each individual publication, document or patent not specifically and individually indicated to be incorporated by reference in its entirety herein. In the event of a conflict, the present 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 taken as an admission or any form of suggestion or that they constitute valid prior art or form part of the common general knowledge in any country in the world.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow cytometry result diagram of detecting the transduction efficiency of the LVV-V5-GFP and the wild-type LVV-GFP in transducing LDL-R ⁺ CD3 ⁺ Jurkat cells, LDL-R ⁺ CD3 ^- Nalm-6 cells and LDL-R ^- CD3 ⁺ human non-activated PBMCs, respectively, in Example 1;

Figure 2 is a flow cytometry result diagram of the expression efficiency of the CAR-19 in CD3 ⁺ T cells on Day 5 after the LVV-V5-CAR19 and the LVV-V7-CAR19 were transduced into human non-activated PBMCs respectively in Example 2;

Figure 3: In Example 3, the CD19-CAR-T cells prepared after the LVV-V5-CAR19 and the LVV-V7-CAR19 respectively transduced human non-activated PBMCs killed CD19 ⁺ Nalm-6 cells, Day 4, and the flow cytometry results of detecting the expression of CD19 in each group of mixed cells;

Figure 4 is a flow cytometry result diagram showing the killing of CD19 ⁺ Nalm-6 cells by CD19-CAR-T cells prepared by transducing human non-activated PBMCs with the LVV-V5-CAR19 and the LVV-V7-CAR19, respectively, on Day 7, and detecting the expression of CD19 in each group of mixed cells;

Figure 5: In Example 5, the LVV-V5-CAR19, LVV-V6-CAR19, LVV-V7-CAR19 and LVV-V8-CAR19 were respectively transduced into human non-activated PBMCs, and on Day 2, a bar graph comparing the expression efficiency of the CAR-19 in each group of cells was detected;

Figure 6 is a bar graph comparing the killing efficiency of CD19-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V5-CAR19, LVV-V6-CAR19, LVV-V7-CAR19 ^and LVV-V8-CAR19 on Day 5 in Example 5;

Figure 7: In Example 6, the LVV-V1-CAR19, LVV-V2-CAR19, LVV-V3-CAR19 and LVV-V4-CAR19 were respectively transduced into human non-activated PBMCs, and on Day 2, a bar graph comparing the expression efficiency of the CAR-19 in each group of cells was detected;

Figure 8 is a bar graph comparing the killing efficiency of CD19-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V1-CAR19, LVV-V2-CAR19, LVV-V3-CAR19 ^and LVV-V4-CAR19 on Day 5 in Example 6;

Figure 9 is a bar graph comparing the expression efficiency of CAR-20 in each group of cells, in which the LVV-V9-CAR20, LVV-V10-CAR20 and LVV-V11-CAR20 were transduced into human non-activated PBMCs, respectively, on Day 2 in Example 7;

Figure 10: In Example 7, the CD20-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V9-CAR20, LVV-V10-CAR20 and LVV-V11-CAR20 respectively killed CD20 ⁺ Dakiki cells, Day 5, and the flow cytometry results of detecting the expression of CD20 in each group of mixed cells;

Figure 11 is a bar graph comparing the expression efficiency of CAR-HER2 in each group of cells, in which the LVV-V13-CARHER2, LVV-V14-CARHER2 and LVV-V15-CARHER2 were respectively transduced into human non-activated PBMCs on Day 2 in Example 8;

Figure 12 is a bar graph comparing the killing efficiency of HER2-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V13-CARHER2, LVV-V14-CARHER2, and LVV-V15-CARHER2 respectively in killing HER-2 ⁺ OVCAR-3 cells on Day 2 in Example 8;

Figure 13 is a flow cytometry result diagram of the expression efficiency of the CAR-CEA in CD3 ⁺ T cells detected on Day 2 after the LVV-V16-CARCEA, LVV-V17-CARCEA and LVV-V18-CARCEA were transduced into human non-activated PBMCs in Example 9;

Figure 14 is a bar graph comparing the killing efficiency of CEA-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V16-CARCEA, LVV-V17-CARCEA, and LVV ^- V18-CARCEA on Day 2 in Example 9;

Figure 15 is a flow cytometry result diagram showing the expression efficiency of the CAR-33 in CD3 ⁺ T cells in Example 10, where the LVV-V19-CAR33, LVV-V20-CAR33 and LVV-V21-CAR33 were respectively transduced into human non-activated PBMCs on Day 2;

Figure 16 is a bar graph comparing the killing efficiency of CD33-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V19-CAR33, LVV-V20-CAR33 and LVV-V21-CAR33 respectively on Day 5 in killing CD33 ⁺ MOLM-13 cells in Example 10;

Figure 17 is a flow cytometry result diagram showing the expression efficiency of the CAR-BCMA in CD3 ⁺ T cells in Example 11, where the LVV-V22-CARBCMA, LVV-V23-CARBCMA and LVV-V24-CARBCMA were transduced into human non-activated PBMCs on Day 2;

Figure 18 is a bar graph comparing the killing efficiency of BCMA-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V22-CARBCMA, LVV-V23-CARBCMA and LVV-V24-CARBCMA respectively in Example 11, in killing BCMA ⁺ U266 cells on Day 5.

DETAILED DESCRIPTION

The following is a clear and complete description of the concept and technical effects of the present invention in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions known in the art, or according to the product specifications. The reagents and raw materials without specific components in the present invention are all commercially available.

Example 1

A targeted lentiviral vector V5-GFP (LVV-V5-GFP) was constructed that can target activated and transduce non-activated T cells.

1. Design of polynucleotide encoding membrane-expressed anti-CD3 antibody × anti-CD28 antibody structure

In this embodiment, the polynucleotides encoding membrane-expressed anti-CD3 antibody × anti-CD28 antibody (membrane-expressed CD3 × CD28 dual antibody) are, from 5' to 3' end: a polynucleotide encoding a human CD8α signal peptide, a polynucleotide encoding an anti-CD3 antibody (UCHT1-scFv), a polynucleotide encoding a human CD8α hinge region, a polynucleotide encoding a human CD8α transmembrane region, a polynucleotide encoding a FT2A peptide, a polynucleotide encoding a human CD8α signal peptide, a polynucleotide encoding an anti-CD28 antibody (15-E8-scFv), a polynucleotide encoding a human CD8α hinge region, and a polynucleotide encoding a human CD8α transmembrane region;

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

(2) The amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO:9;

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

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

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

(6) The amino acid sequence of the 15E8-scFv is shown in SEQ ID NO:13.

2. Packaging of LVV-V5-GFP for targeted activation of non-activated T cells

A. Prepare the following four plasmids : an envelope plasmid carrying a polynucleotide encoding a mutant VSV-G1 and a polynucleotide encoding the membrane-expressed CD3×CD28 dual antibody (envelope plasmid 1), a pMDLg/pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a lentivirus-GFP master plasmid carrying a GFP (Green Fluorescent Protein, "GFP") gene (pGClenti-GFP plasmid); the envelope plasmid 1 is synthesized by conventional molecular cloning methods;

The extracellular domain of the mutant VSV-G1 comprises the amino acid sequence shown in SEQ ID NO:3; relative to SEQ ID NO:1, SEQ ID NO:3 comprises a K47 deletion; the amino acid sequence shown in SEQ ID NO:1 is the amino acid sequence contained in the extracellular domain of the wild-type VSV-G; the amino acid sequence of the wild-type VSV-G full-length protein (including the VSV-G signal peptide) is shown in SEQ ID NO:22; the amino acid sequence of the full-length protein of the mutant VSV-G1 (including the VSV-G signal peptide) is shown in SEQ ID NO:29.

The mutant VSV-G1 lacks the 47th amino acid lysine in its extracellular domain, so that the ability of the mutant VSV-G1 to specifically bind to LDL-R is weakened or even lost, but the ability to mediate membrane fusion and endosomal/lysosomal escape is still retained; thereby, the lentiviral vector whose viral envelope contains the mutant VSV-G1, such as the LVV-V5-GFP, can target, activate and transduce non-activated T cells with improved specificity.

B. Packaging and preparation of LVV-V5-GFP :

(1) Mixing the four plasmids and transfecting the four plasmids into the packaging cell line HEK-293T cell line using PEI reagent, the specific steps are as follows:

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

Day 0, 4.5×10 ⁶ HEK-293T cells were inoculated in a 10 cm culture dish. About 48 hours after inoculation, when the cell confluence was 80-90%, the four plasmids were transfected into the packaging cells HEK-293T cells using PEI reagent, including:

Add 9 μg of the main plasmid, 4 μg of pMDLg/pRRE packaging plasmid, 2 μg of pRSV-REV packaging plasmid and the 2 μg of the envelope plasmid 1 to 1 mL of Opti-MEM medium, shake well and add 64 μL of PEI reagent, pipet evenly and let stand for 10 minutes, then add to the culture medium of HEK-293T cells, refresh the culture medium after 6 hours, collect the culture supernatant 48 hours after transfection, filter with a 0.45 um filter membrane, centrifuge at 50,000 g for 2.5 h, discard the supernatant, resuspend the LVV-V5-GFP in 200 uL of F12 medium and store at -80°C.

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

HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

F12 medium: Brand: GIBCO, catalog number: #C11330500BT;

Syringe filter: Brand: SORFA, item number: #622120.

(2) Transduction of LDL-R ⁺ CD3 ⁺ Jurkat cells, LDL-R ⁺ CD3 ^- Nalm-6 cells and human non-activated PBMCs (MOI = 5):

On Day 0, 1×10 ⁵ LDL-R ⁺ CD3 ⁺ Jurkat cells, LDL-R ⁺ CD3 ^- Nalm-6 cells and LDL-R ^- CD3 ⁺ human non-activated PBMCs (non-activated PBMCs frozen from healthy person Donor 1 were resuscitated, and the resuscitation method is well known to those skilled in the art) were taken and resuspended in 200 μL of culture medium respectively; the culture medium for resuspending the Jurkat cells and Nalm-6 cells included 1640 (brand: ELGBIO, product number: #EH80809) culture medium and 10% FBS (brand: EXCELL, product number: #FSP500); the culture medium for resuspending the human non-activated PBMCs (PBMCs culture medium) included XVT culture medium (trade name PRIME-XV T cell CDM, brand: IRVINE (FUJIFILM), catalog number: #91154), IL-7 with a final concentration of 20 ng/mL (trade name: IL-7 Protein, Human, Recombinant, brand: Sino Biological, catalog number: #11821-HNAE) and IL-15 with a final concentration of 20 ng/mL (trade name: IL-15 Protein, Human, Recombinant (His Tag), brand: Sino Biological, catalog number: #10360-H07E).

According to MOI=5, the LVV-V5-GFP was added to the Jurkat cells, Nalm-6 cells and human non-activated PBMCs cell culture system respectively, mixed well, and placed in a 5% CO ₂ , 37° C. incubator for culture;

On Day 5, the expression of GFP in the Jurkat cells, Nalm-6 cells and PBMCs was detected. The results are shown in Figure 1 (Figure 1, lower column "Target CD3&CD28").

3. Packaging of wild-type VSV-G lentiviral vector in control group

(1) Referring to the above-mentioned method for packaging LVV-V5-GFP, a wild-type VSV-G lentiviral vector (wild-type LVV-GFP) was packaged. The specific packaging steps are as follows:

Prepare the following four plasmids: pMD2.G envelope plasmid (containing wild-type VSV-G gene), pMDLg/pRRE packaging plasmid, pRSV-REV packaging plasmid and the lentivirus-GFP main plasmid; mix the four plasmids, transfect the four plasmids into the packaging cell line HEK-293T cell line by PEI reagent, and package and prepare the wild-type LVV-GFP;

The wild-type VSV-G extracellular domain contains the amino acid sequence shown in SEQ ID NO:1; the viral envelope of the wild-type LVV-GFP does not contain anti-CD3 antibodies and anti-CD28 antibodies and still has the ability to specifically bind to LDL-R.

(2) Transduction of CD3 ⁺ Jurkat cells, CD3 ^- Nalm-6 cells and human non-activated PBMCs (MOI = 5)

On Day 0, referring to the above-mentioned LVV-V5-GFP transduction method for LDL-R ⁺ CD3 ⁺ Jurkat cells, LDL-R ⁺ CD3 ^- Nalm-6 cells and LDL-R ⁺ CD3 ⁺ human non-activated PBMCs, the wild-type LVV-GFP was used to transduce each group of cells, and the expression of GFP in each group of cells was detected on Day 5. The results are shown in Figure 1 (Figure 1 upper column "VSVG").

4. Transduction Results

As can be seen from Figure 1, the LVV-V5-GFP cannot effectively transduce LDL-R ⁺ CD3 ^- Nalm-6 cells, but can effectively transduce LDL-R ⁺ CD3 ⁺ Jurkat cells and LDL-R ^- CD3 ⁺ human non-activated PBMCs; while the wild-type LVV-GFP can effectively transduce LDL-R ⁺ CD3 ⁺ Jurkat cells and LDL-R ⁺ CD3 ^- Nalm-6 cells, but is difficult to transduce LDL-R ^- CD3 ⁺ human non-activated PBMCs.

Due to the first mutation of VSV-G, i.e., the deletion of lysine at position 47 of the extracellular domain of VSV-G (K47 deletion), the mutant VSV-G1 has a weakened or even lost ability to specifically bind to LDL-R, and the LVV-V5-GFP is difficult to transduce LDL-R ⁺ CD3 ^- Nalm-6 cells;

The LVV-V5-GFP, because its viral envelope surface contains primary and secondary signal molecules for T cell activation, i.e., the membrane expresses anti-CD3 antibodies and anti-CD28 antibodies, can bind to the endocytic receptor CD3 on the surface of non-activated T cells in Jurkat cells and non-activated human PBMCs, and while effectively activating non-activated T cells, it can also enter and effectively transduce CD3 ⁺ non-activated T cells in CD3 ⁺ Jurkat cells and non-activated human PBMCs through endocytosis;

The wild-type LVV-GFP can enter and transduce Jurkat cells and Nalm-6 cells by binding to LDL-R on the surface of Jurkat cells and Nalm-6 cells, and its specificity for targeted transduction of T cells is significantly lower than that of the LVV-V5-GFP; moreover, the wild-type LVV-GFP is difficult to effectively transduce non-activated T cells in human non-activated PBMCs that do not express or lowly express LDL-R.

Compared with the wild-type LVV-GFP, the ability of the LVV-V5-GFP to transduce LDL-R ⁺ cells is inhibited, and the specificity of targeted transduction and activation of CD3 ⁺ non-activated T cells is significantly improved, making it more suitable for use in applications that require targeted activation and transduction of non-activated T cells, such as in vivo CAR-T cell therapy.

Example 2

The expression efficiency of lentiviral vectors whose viral envelopes contain primary and secondary signaling molecules for T cell activation or whose membranes express anti-CD7 antibodies to transduce non-activated T cells and deliver CAR genes targeting CD19 was compared.

1. Design of chimeric antigen receptor CAR-19

In this embodiment, a chimeric antigen receptor (CAR-19) targeting human CD19 is designed; human CD19: Uniprot ID: P15391; CD19 is an effective target for treating B cell malignancies, especially B cell lymphoma and acute lymphoblastic leukemia.

The structure of the CAR-19 from N-terminus to C-terminus is, in order, an antigen binding region targeting human CD19, a hinge region of human CD8α, a transmembrane region of human CD8α, a human 4-1BB co-stimulatory signal transduction domain, and a human CD3ζ intracellular signal transduction domain;

The antigen binding region targeting human CD19 is a scFv (FMC63-scFv) derived from the monoclonal antibody FMC-63 that can specifically bind to human CD19, wherein the heavy chain variable region (VH) of the FMC63-scFv is connected to the light chain variable region (VL) of the scFv via a (G ₄ S) ₃ connecting peptide.

The 5' end of the polynucleotide encoding the CAR-19 is operably linked to the 3' end of the polynucleotide encoding the human CD8α signal peptide, and the human CD8α signal peptide is located at the N-terminus of the CAR-19;

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

(2) The amino acid sequence of the VH region of the FMC63-scFv is shown in SEQ ID NO: 14, and the amino acid sequence of the VL region of the FMC63-scFv is shown in SEQ ID NO: 15; the VH region and the VL region are connected by a (G ₄ S) ₃ connecting peptide, and the amino acid sequence of the (G ₄ S) ₃ connecting peptide is shown in SEQ ID NO: 16; the amino acid sequence of the FMC63-scFv is shown in SEQ ID NO: 30; the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 31-33, respectively, and the amino acid sequences of the LCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 34-36, respectively;

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

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

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

(6) The amino acid sequence of the intracellular signaling domain of human CD3ζ is shown in SEQ ID NO:18.

2. Packaging LVV-V5-CAR19

Prepare the following four plasmids: the envelope plasmid 1, the pMDLg/pRRE packaging plasmid, the pRSV-REV packaging plasmid, and a lentiviral main plasmid (CAR-19 main plasmid) carrying the polynucleotide encoding the CAR-19 (CAR-19 gene); the CAR-19 main plasmid is synthesized by a conventional molecular cloning method;

Referring to the above-mentioned packaging method for packaging the LVV-V5-GFP, a lentiviral vector LVV-V5-CAR19 carrying the CAR-19 gene was packaged;

3. Packaging LVV-V7-CAR19

Prepare the following four plasmids: an envelope plasmid 2 carrying a polynucleotide encoding the mutant VSV-G1 and a polynucleotide encoding a membrane-expressed anti-CD7 antibody, a pMDLg/pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the CAR-19 main plasmid; the envelope plasmid 2 is synthesized by a conventional molecular cloning method;

The structures of the membrane-expressed anti-CD7 antibody from N-terminus to C-terminus are scFv (TH69-scFv) derived from monoclonal antibody TH-69 that can specifically bind to human CD7, the human CD8α hinge region, and the human CD8α transmembrane region; human CD7: Uniprot ID: P15391.

The amino acid sequence of the VH region of the TH69-scFv is shown in SEQ ID NO: 19; the amino acid sequence of the VH region of the TH69-scFv is shown in SEQ ID NO: 19; the amino acid sequence of the TH69-scFv is shown in SEQ ID NO: 37; the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NO: 38-40, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NO: 41-43, respectively;

The 5' end of the polynucleotide encoding the membrane-expressed anti-CD7 antibody (membrane-expressed TH69-scFv) is operably connected to the 3' end of the polynucleotide encoding the human CD8α signal peptide, and the CD8α signal peptide is located at the N-terminus of the membrane-expressed anti-CD7 antibody;

Referring to the packaging method of the LVV-V5-GFP, LVV-V7-CAR19 carrying the CAR-19 gene is packaged.

4. Comparison of transduction efficiency of LVV-V5-CAR19 and LVV-V7-CAR19 in transducing non-activated T cells

Referring to the transduction method of LVV-V5-GFP transducing human non-activated PBMCs, at an MOI of 5, the LVV-V5-CAR19 and LVV-V7-CAR19 were used to transduce human non-activated PBMCs, respectively. On Day 5, flow cytometry was used to detect the expression efficiency of CAR-19 in CD3 ⁺ T cells of each group of PBMCs. The results are shown in Figure 2.

As can be seen from the middle figure of Figure 2 (i.e., “Target CD7”), after the LVV-V7-CAR19 transduced human non-activated PBMCs, the expression efficiency of the CAR-19 molecule in CD3 ⁺ T cells was approximately 39.22%;

As can be seen from the rightmost figure in Figure 2 (i.e., “Target CD3&CD28”), after the LVV-V5-CAR19 targeted activation and transduction of non-activated T cells, the expression efficiency of the CAR-19 molecule in CD3 ⁺ T cells was approximately 46.18%;

It can be seen that the viral envelope contains primary and secondary signal molecules for T cell activation, that is, the LVV-V5-CAR19 whose membrane expresses anti-CD3 antibodies and anti-CD28 antibodies is significantly better than the LVV-V7-CAR19 whose viral envelope does not contain primary and secondary signal molecules for T cell activation and only contains targeting molecules and membranes expressing anti-CD7 antibodies.

Flow cytometry antibodies used in the assay:

Flow cytometry antibody for detecting CD3: Product name: FITC Mouse Anti-Human CD3; Brand: BIOLEGEND, item number: #555339.

Flow cytometry antibody for detecting CAR-19 molecules: Product name: PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (0.03% Proclin) DMF Filed, Brand: Acro, Item number: #FM3-PY54A2-200 tests.

Example 3

Detect the killing efficiency of CD19-CAR-T cells in vitro.

Day 0: 1×10 ⁷ human non-activated PBMCs were taken respectively, and the human non-activated PBMCs were transduced with the LVV-V5-CAR19 and the LVV-V7-CAR19 at an MOI of 5 to prepare two groups of CD19-targeted CAR-T cells (CD19-CAR-T cells);

Day 2: The two groups of CD19-CAR-T cells were counted using a cell counter (brand: COUNTERSTAR, model: Rigel S2). According to the effector-target ratio (E:T) = 1:1, 1×10 ⁶ CD19 ⁺ target cells Nalm-6 cells (human B lymphoid leukemia cells) were added to the two groups of CD19-CAR-T cells respectively;

Day 4: Flow cytometry was used to detect the killing efficiency of the two groups of CD19-CAR-T cells in killing Nalm-6 cells. The results are shown in Figure 3.

As can be seen from Figure 3, on Day 4, in the mixed cells added with the LVV-V5-CAR19, the expression of CD19 was only 8.84%; while in the mixed cells added with the LVV-V7-CAR19, the expression of CD19 was still 17.97%; this shows that on Day 4, the killing efficiency of the activated CD19-CAR-T cells prepared by the CD3 ⁺ T cells in the non-activated PBMCs of humans transduced with the LVV-V5-CAR19 was significantly better than that of the non-activated or relatively under-activated CD19-CAR-T cells prepared by the non-activated PBMCs of humans transduced with the LVV-V7-CAR19.

Example 4

Referring to the method for packaging the LVV-V5-CAR19 and LVV-V7-CAR19 in Example 2, the LVV-V5-CAR19 and LVV-V7-CAR19 were packaged in the same batch.

Day 0: 1×10 ⁷ human non-activated PBMCs were taken respectively, and the human non-activated PBMCs were transduced with the LVV-V5-CAR19 and the LVV-V7-CAR19 at an MOI of 5 to prepare two groups of CD19-CAR-T cells;

Day 2: The two groups of CD19-CAR-T cells were counted using a cell counter, and 1×10 ⁶ CD19 ⁺ target cells Nalm-6 cells were added to each of the two groups of CD19-CAR-T cells at an effector-target ratio (E:T) of 1:1;

Day 7: Flow cytometry was used to detect the killing efficiency of the two groups of CD19-CAR-T cells in killing Nalm-6 cells. The results are shown in Figure 4.

As shown in Figure 4, on Day 7, the sustained killing efficiency of the two groups of CD19-CAR-T cells was comparable.

Example 5

1. Design of polynucleotide encoding membrane-expressed CD3 antibody × membrane-expressed CD86 structure

Constructing a membrane-expressed CD86, wherein the membrane-expressed CD86 sequentially comprises a human CD86 extracellular domain and a human CD86 transmembrane region from the N-terminus to the C-terminus (CD86-ECD+TM);

The 5' end of the polynucleotide encoding the human CD86-ECD+TM is operably linked to the 3' end of the polynucleotide encoding the human CD86 signal peptide;

The amino acid sequence of the human CD86-ECD+TM is shown in SEQ ID NO: 79; the amino acid sequence of the human CD86-ECD+TM containing the human CD86 signal peptide is shown in SEQ ID NO: 80;

Human CD86-ECD+TM:

Human CD86 signal peptide + human CD86-ECD + TM:

Among them, positions 1 to 23 (underlined) of SEQ ID NO:80 are the amino acid sequence of the human CD86 signal peptide: MDPQCTMGLSNILFVMAFLLSGA (SEQ ID NO:96) .

In this embodiment, the polynucleotide encoding membrane expressed anti-CD3 antibody (the membrane expressed UCHT1-scFv) × membrane expressed CD86 includes, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the UCHT1-scFv, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the FT2A peptide, a polynucleotide encoding the human CD86 signal peptide, and a polynucleotide encoding the human CD86-ECD+TM.

When transfected into packaging cells, the membrane-expressed UCHT1-scFv and the membrane-expressed CD86-ECD+TM can be separated and expressed on the cell membrane of the packaging cells under the action of the self-cleaving peptide FT2A peptide.

2. Packaging multiple sets of lentiviral vectors

Referring to the method for packaging the LVV-V5-CAR19 in Example 2, multiple groups of lentiviral vectors were packaged in the same batch: the LVV-V5-CAR19, LVV-V6-CAR19, the LVV-V7-CAR19 and LVV-V8-CAR19;

The LVV-V6-CAR19: (a) the viral envelope contains (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv, and a secondary signaling molecule for T cell activation, i.e., the membrane expresses CD86-ECD+TM; and (ii) the mutant VSV-G1; and (b) contains the CAR-19 gene;

The LVV-V8-CAR19: (a) the viral envelope contains (i) a primary signal molecule for T cell activation, the membrane expresses UCHT1-scFv and (ii) the mutant VSV-G1; and (b) contains the CAR-19 gene.

3. Transduce human non-activated PBMCs and detect the expression efficiency of CAR-19

Referring to the method of using the LVV-V5-CAR19 to transduce human non-activated PBMCs in Example 2, the LVV-V5-CAR19, LVV-V6-CAR19, LVV-V7-CAR19 and LVV-V8-CAR19 were used to transduce the non-activated PBMCs of Donor 1 at MOI=1; Day 2, flow cytometry was used to detect the expression efficiency of the CAR-19 in each group of PBMCs, and the results are shown in Figure 5.

As can be seen from Figure 5, the efficiency of expressing the CAR-19 in human non-activated PBMCs transduced with the LVV-V5-CAR19 and the LVV-V6-CAR19 is comparable; but both are significantly better than the transduction efficiency of the LVV-V7-CAR19 and LVV-V8-CAR19; this may be because the anti-CD7 antibody does not have the ability to activate and stimulate non-activated T cells, so the efficiency of non-activated T cells expressing CAR-19 is low; and in the absence of T cell activation secondary signal molecules, the single T cell activation primary signal molecules are relatively unable to fully activate and stimulate non-activated T cells, resulting in a low expression efficiency of CAR-19.

In summary, constructing T cell activation primary signal molecules and T cell activation secondary signal molecules on the surface of a lentiviral vector containing a mutant VSV-G1 with weakened or lost ability to specifically bind to LDL-R can not only improve the specificity of lentiviral vector targeted transduction of T cells to improve the transduction efficiency, but also fully activate and stimulate non-activated T cells to further improve the transduction efficiency.

4. Detection of the killing efficiency of PBMCs transduced with lentiviral vectors in each group to prepare CAR-T cells

Day 0: 1×10 ⁶ human non-activated PBMCs from 5 groups of Donor 1 were taken respectively, mixed with Nalm-6 cells at an effector-target ratio of E:T=1:1, and added to the culture medium (1640+10% FBS); at an MOI=1, the LVV-V5-CAR19, LVV-V6-CAR19, LVV-V7-CAR19 and LVV-V8-CAR19 were added to 4 groups of mixed cells respectively; the control group was a mixed cell group to which no lentiviral vector was added (CTR group).

Day 5: Flow cytometry was used to detect the expression of CD19 in each group of mixed cells in order to detect the killing efficiency of CD19-CAR-T cells prepared from non-activated human PBMCs transduced with lentiviral vectors. The specific method was as follows: the blank group of Nalm-6 cells without PBMCs and lentiviral vectors was used as the background value, the percentage of remaining Nalm-6 cells in each group of mixed cells was calculated, and then the killing ratio was calculated; the results are shown in Figure 6.

As can be seen from Figure 6, on Day 5, the CD19-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V5-CAR19, LVV-V6-CAR19, LVV-V7-CAR19 and LVV-V8-CAR19 can effectively kill Nalm-6 cells.

Example 6

1. Packaging of each group of lentiviral vectors containing mutant VSV-G2

Referring to the method for packaging the LVV-V5-CAR19 in Example 2, multiple groups of lentiviral vectors containing mutant VSV-G2 in the same batch of packaging virus envelopes: LVV-V1-CAR19, LVV-V2-CAR19, LVV-V3-CAR19 and LVV-V4-CAR19;

The extracellular domain of the mutant VSV-G2 comprises the amino acid sequence shown in SEQ ID NO:4; relative to SEQ ID NO:1, SEQ ID NO:4 comprises R354Q, which weakens or loses the ability of the mutant VSV-G2 to specifically bind to LDL-R, but at the same time retains the ability to mediate membrane fusion and endosomal/lysosomal escape.

The LVV-V1-CAR19: (a) the viral envelope contains (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv, and a secondary signaling molecule for T cell activation, i.e., the membrane expresses 15E8-scFv; and (ii) the mutant VSV-G2; and (b) contains the CAR-19 gene;

The LVV-V2-CAR19: (a) the viral envelope contains (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv, and a secondary signaling molecule for T cell activation, i.e., the membrane expresses CD86-ECD+TM; and (ii) the mutant VSV-G2; and (b) contains the CAR-19 gene;

The LVV-V3-CAR19: (a) the viral envelope comprises (i) a membrane-expressed anti-CD7 antibody, i.e., the membrane expresses TH69-scFv and (ii) the mutant VSV-G2; and (b) comprises the CAR-19 gene;

The LVV-V4-CAR19: (a) the viral envelope contains (i) T cell activation primary signal molecule anti-CD3 antibody, that is, the membrane expresses UCHT1-scFv and (ii) the mutant VSV-G2; and (b) contains the CAR-19 gene.

2. Transduce human non-activated PBMCs and detect the expression efficiency of CAR-19 (MOI = 1)

Referring to the method of using the LVV-V5-CAR19 to transduce human non-activated PBMCs in Example 2, the LVV-V1-CAR19, LVV-V2-CAR19, LVV-V3-CAR19 and LVV-V4-CAR19 were used to transduce the non-activated PBMCs of Donor 1 at MOI=1; Day 2, flow cytometry was used to detect the expression efficiency of the CAR-19 in each group of PBMCs, and the results are shown in Figure 7.

As can be seen from Figure 7, the efficiency of expressing the CAR-19 in human non-activated PBMCs transduced by the LVV-V1-CAR19 and the LVV-V2-CAR19 is comparable; but both are significantly better than the transduction efficiency of the LVV-V3-CAR19 and LVV-V4-CAR19.

3. Detection of the killing efficiency of PBMCs transduced with lentiviral vectors in each group to prepare CAR-T cells

Day 0: Referring to the method for detecting the killing efficiency of CD19-CAR-T cells prepared by lentiviral vector-transduced human non-activated PBMCs in each group in Example 4, the LVV-V1-CAR19, LVV-V2-CAR19, LVV-V3-CAR19 and LVV-V4-CAR19 were respectively added to 4 groups of mixed cell culture media; the control group was a mixed cell group to which no lentiviral vector was added (CTR group).

Day 5: Flow cytometry was used to detect the expression of CD19 in each group of mixed cells to calculate the killing efficiency of each group of CD19-CAR-T cells; the results are shown in Figure 8.

As can be seen from Figure 8, on Day 5, the CD19-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V1-CAR19, LVV-V2-CAR19, LVV-V3-CAR19 and LVV-V4-CAR19 can effectively kill Nalm-6 cells.

Example 7

1. Design of CAR targeting CD20

Design a CAR targeting human CD20 (CAR-20); the polynucleotide encoding the CAR-20 (CAR-20 gene) comprises, from the 5' end to the 3' end:

A polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding an antigen binding region targeting human CD20, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the human 4-1BB co-stimulatory signaling domain, and a polynucleotide encoding the human CD3ζ intracellular signaling domain;

Human CD20: Uniprot ID: P11836. CD20 is an effective target for the treatment of B-cell malignancies such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia.

The antigen binding region targeting human CD20 is a scFv (2f2-scFv) derived from the monoclonal antibody 2f2, and the amino acid sequence of the 2f2-scFv is shown in SEQ ID NO:44; the amino acid sequences of the HCDR1-3 regions of the 2f2-scFv are shown in SEQ ID NO:45-47, respectively; and the amino acid sequences of the LCDR1-3 regions of the -scFv are shown in SEQ ID NO:48-50, respectively.

2. Packaging multiple sets of lentiviral vectors containing the CAR-20 gene

Referring to the method for packaging the LVV-V5-CAR19, multiple groups of lentiviral vectors LVV-V9-CAR20, LVV-V10-CAR20, and LVV-V11-CAR20 were packaged in the same batch;

The LVV-V9-CAR20: (a) the viral envelope contains (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv and a secondary signaling molecule for T cell activation, i.e., the membrane expresses 15E8-scFv; and (ii) the mutant VSV-G1; and (b) contains the CAR-20 gene;

The LVV-V10-CAR20: (a) the viral envelope comprises (i) a membrane-expressed anti-CD7 antibody, i.e., the membrane expresses TH69-scFv and (ii) the mutant VSV-G1; and (b) comprises the CAR-20 gene;

The LVV-V11-CAR20: (a) the viral envelope contains (i) the primary signal molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv and (ii) the mutant VSV-G1; and (b) contains the CAR-20 gene.

3. Transduce human non-activated PBMCs and detect the expression efficiency of CAR-20 (MOI = 1)

On Day 0, at MOI = 1, the LVV-V9-CAR20, LVV-V10-CAR20 and LVV-V11-CAR20 were added to the non-activated PBMCs culture system of Donor 1 in each group. On Day 2, flow cytometry was used to detect the expression efficiency of the CAR-20 in the PBMCs of each group. The results are shown in Figure 9.

As can be seen from Figure 9, the transduction efficiency of the LVV-V9-CAR20 in transducing human non-activated PBMCs and delivering the CAR-20 gene is significantly better than that of the LVV-V10-CAR20 and LVV-V11-CAR20.

Flow cytometry detection antibody: Anti-G4Slinker, brand: Heyousheng, product number: #GS-ARPE100.

4. Detection of the killing efficiency of CD20-CAR-T cells prepared by PBMCs transduced with lentiviral vectors in each group

Day 0: 1×10 ⁶ human non-activated PBMCs from 4 groups of Donor 1 were taken, mixed with CD20 ⁺ Dakiki cells (human B lymphocytes) at an effector-target ratio of E:T=1:1, and added to the culture medium (1640+10% FBS); at an MOI=1, the LVV-V9-CAR20, LVV-V10-CAR20 and LVV-V11-CAR20 were added to the mixed cell culture medium of 3 groups respectively; the control group (CTR group) was a mixed cell group that did not contain any lentiviral vector.

Day 5: A cell counter was used to count the number of mixed cells in each group, and flow cytometry was used to detect the expression of CD20 in each group of mixed cells. The results are shown in Figure 10.

As can be seen from Figure 10, on Day 5, the expression level of CD20 in the mixed cells of each group was significantly reduced compared with that of the control group. The CD20-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V9-CAR20, LVV-V10-CAR20 and LVV-V11-CAR20 can effectively kill CD20 ⁺ Dakiki cells.

Example 8

1. Design of CAR targeting HER-2

A CAR targeting human HER-2 (CAR-HER2) is designed, and the polynucleotide encoding the CAR-HER2 (CAR-HER2 gene) comprises, from the 5' end to the 3' end:

A polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding an antigen binding region targeting human HER-2, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the human 4-1BB co-stimulatory signaling domain, and a polynucleotide encoding the human CD3ζ intracellular signaling domain;

Human HER-2: Uniprot ID: P04626. HER-2 is an effective target for the treatment of solid cancers such as HER-2 ⁺ breast cancer and gastric cancer.

The antigen binding region targeting human HER-2 is a scFv (Pertuzumab-scFv) derived from the monoclonal antibody Pertuzumab, and the amino acid sequence of the Pertuzumab-scFv is shown in SEQ ID NO:51; the amino acid sequences of the HCDR1-3 regions of the Pertuzumab-scFv are shown in SEQ ID NO:52-54, respectively; the amino acid sequences of the LCDR1-3 regions of the Pertuzumab-scFv are shown in SEQ ID NO:55-57, respectively.

2. Packaging multiple sets of lentiviral vectors containing the CAR-HER2 gene

Referring to the method for packaging the LVV-V5-CAR19, multiple groups of lentiviral vectors LVV-V13-CARHER2, LVV-V14-CARHER2, and LVV-V15-CARHER2 were packaged in the same batch;

The LVV-V13-CARHER2: (a) the viral envelope comprises (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv, and a secondary signaling molecule for T cell activation, i.e., the membrane expresses 15E8-scFv; and (ii) the mutant VSV-G1; and (b) comprises the CAR-HER2 gene;

The LVV-V14-CARHER2: (a) the viral envelope comprises (i) a membrane-expressed anti-CD7 antibody, i.e., the membrane expresses TH69-scFv, and (ii) the mutant VSV-G1; and (b) comprises the CAR-HER2 gene;

The LVV-V15-CARHER2: (a) the viral envelope contains (i) the primary signal molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv, and (ii) the mutant VSV-G1; and (b) contains the CAR-HER2 gene.

3. Transduce human non-activated PBMCs and detect the expression efficiency of CAR-HER2 (MOI = 1)

On Day 0, at MOI = 1, the LVV-V13-CARHER2, LVV-V14-CARHER2, and LVV-V15-CARHER2 were added to the non-activated PBMCs culture system of Donor 1 in each group. On Day 2, flow cytometry was used to detect the expression efficiency of the CAR-HER2 in the PBMCs of each group. The results are shown in Figure 11.

As can be seen from Figure 11, the LVV-V13-CARHER2 transduced human non-activated PBMCs, and the transduction efficiency of delivering the CAR-HER2 gene was about 28%, which was significantly better than the LVV-V14-CARHER2 (about 18%) and LVV-V15-CARHER2 (about 15%).

Flow cytometry detection antibody: Anti-G4Slinker, brand: Heyousheng, product number: #GS-ARPE100.

4. Detection of the killing efficiency of HER2-CAR-T cells prepared by PBMCs transduced with lentiviral vectors in each group

Day 0: 1×10 ⁶ human non-activated PBMCs from 4 groups of Donor 1 were taken and mixed with HER-2 ⁺ OVCAR-3 cells (human ovarian cancer cells) transduced with luciferase gene according to the effector-target ratio E:T=1:1 and added to the culture medium (1640+10% FBS); at MOI=1, the LVV-V13-CARHER2, LVV-V14-CARHER2 and LVV-V15-CARHER2 were added to the culture medium of 3 groups of mixed cells respectively; the control group did not contain any lentiviral vector, but only contained the mixed cells (CTR group);

Day 2: An ELISA instrument was used to detect and calculate the killing efficiency of each group of HER2-CAR-T cells. The specific method was: the fluorescence value of the blank group without PBMCs and LVV but only containing target cells OVCAR-3 cells was taken as the background value, which was the total fluorescence value of the original tumor cell number. The tumor fluorescence value remaining after each group of HER2-CAR-T cells killed OVCAR-3 cells was taken as the residual fluorescence value. The killing efficiency was calculated as follows: Killing efficiency (%) = (total fluorescence value - residual fluorescence value) / total fluorescence value × 100%; the calculation results are shown in Figure 12.

As can be seen from Figure 12, on Day 2, the HER2-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V13-CARHER2, LVV-V14-CARHER2 and LVV-V15-CARHER2 can effectively kill HER-2 ⁺ OVCAR-3 cells.

Example 9

1. Design of CAR targeting CEA

A CAR targeting human CEA (CAR-CEA) is designed, and the polynucleotide encoding the CAR-CEA (CAR-CEA gene) sequentially comprises from the 5' end to the 3' end:

A polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding an antigen binding region targeting human CEA, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the human 4-1BB co-stimulatory signaling domain, and a polynucleotide encoding the human CD3ζ intracellular signaling domain;

Human CEA: Uniprot ID: P06731. CEA is an effective target for the treatment of CEA ⁺ solid cancers such as colorectal cancer, gastric cancer, pancreatic cancer and lung cancer.

The antigen binding region targeting human CEA is an anti-human CEA scFv (anti-CEA-scFv), and the amino acid sequence of the anti-CEA-scFv is shown in SEQ ID NO:58; the amino acid sequences of the HCDR1-3 regions of the anti-CEA-scFv are shown in SEQ ID NO:59-61, respectively; the amino acid sequences of the LCDR1-3 regions of the anti-CEA-scFv are shown in SEQ ID NO:62-64, respectively.

2. Packaging multiple sets of lentiviral vectors containing the CAR-CEA gene

Referring to the method for packaging the LVV-V5-CAR19, multiple groups of lentiviral vectors LVV-V16-CARCEA, LVV-V17-CARCEA, and LVV-V18-CARCEA were packaged in the same batch;

The LVV-V16-CARCEA: (a) the viral envelope comprises (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv, and a secondary signaling molecule for T cell activation, i.e., the membrane expresses 15E8-scFv; and (ii) the mutant VSV-G1; and (b) comprises the CAR-CEA gene;

The LVV-V17-CARCEA: (a) the viral envelope comprises (i) a membrane-expressed anti-CD7 antibody, i.e., the membrane expresses TH69-scFv, and (ii) the mutant VSV-G1; and (b) comprises the CAR-CEA gene;

The LVV-V18-CARCEA: (a) the viral envelope contains (i) the primary signal molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv, and (ii) the mutant VSV-G1; and (b) contains the CAR-CEA gene.

3. Transduce human non-activated PBMCs and detect the expression efficiency of CAR-CEA (MOI = 1)

On Day 0, at MOI=1, the LVV-V16-CARCEA, LVV-V17-CARCEA, and LVV-V18-CARCEA were added to the non-activated PBMCs culture system of Donor 1 in each group. On Day 2, flow cytometry was used to detect the expression efficiency of the CAR-CEA in the CD3 ⁺ T cells of the PBMCs in each group. The results are shown in FIG13 .

As can be seen from Figure 13, the LVV-V16-CARCEA transduced CD3 ⁺ T cells in human non-activated PBMCs, and the transduction efficiency of delivering the CAR-CEA gene was about 67.39%, which was significantly better than the LVV-V17-CARCEA (about 33.38%) and LVV-V18-CARCEA (about 26.69%).

Flow cytometry detection antibody: Anti-G4Slinker, brand: Heyousheng, product number: #GS-ARPE100.

4. Detection of the killing efficiency of CEA-CAR-T cells prepared by PBMCs transduced with lentiviral vectors in each group

Day 0: 1×10 ⁶ human non-activated PBMCs from 4 groups of Donor 1 were taken and mixed with CEA ⁺ T-84 cells (human colon adenocarcinoma cells) transduced with luciferase gene according to the effector-target ratio E:T=1:1 and added to the culture medium (1640+10% FBS); at MOI=1, the LVV-V16-CARCEA, LVV-V17-CARCEA and LVV-V18-CARCEA were added to the culture medium of 3 groups of mixed cells respectively; the control group (CTR) was the mixed cells without any lentiviral vector.

Day 2: Use an ELISA instrument to detect and calculate the killing efficiency of CEA-CAR-T cells in each group. The calculation results are shown in Figure 14.

As shown in Figure 14, on Day 2, the CEA-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V16-CARCEA, LVV-V17-CARCEA and LVV-V18-CARCEA can effectively kill CEA ⁺ T-84 cells.

Example 10

1. Design of CAR targeting CD33

A CAR targeting human CD33 (CAR-33) is designed, and the polynucleotide encoding the CAR-33 (CAR-33 gene) comprises, from the 5' end to the 3' end:

A polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding an antigen binding region targeting human CD33, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the human 4-1BB co-stimulatory signaling domain, and a polynucleotide encoding the human CD3ζ intracellular signaling domain;

Human CD33: Uniprot ID: P20138. CD33 is an effective target for the treatment of blood cancers such as acute myeloid leukemia.

The antigen binding region targeting human CD33 is a scFv (Gemtuzumab-scFv) derived from the monoclonal antibody Gemtuzumab, and the amino acid sequence of the Gemtuzumab-scFv is shown in SEQ ID NO:65; the amino acid sequences of the HCDR1-3 regions of the Gemtuzumab-scFv are shown in SEQ ID NO:66-68, respectively; the amino acid sequences of the LCDR1-3 regions of the Gemtuzumab-scFv are shown in SEQ ID NO:69-71, respectively.

2. Packaging multiple sets of lentiviral vectors containing the CAR-33 gene

Referring to the method for packaging the LVV-V5-CAR19, multiple groups of lentiviral vectors LVV-V19-CAR33, LVV-V20-CAR33, and LVV-V21-CAR33 were packaged in the same batch;

The LVV-V19-CAR33: (a) the viral envelope contains (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv and a secondary signaling molecule for T cell activation, i.e., the membrane expresses 15E8-scFv; and (ii) the mutant VSV-G1; and (b) contains the CAR-33 gene;

The LVV-V20-CAR33: (a) the viral envelope comprises (i) a membrane-expressed anti-CD7 antibody, i.e., the membrane expresses TH69-scFv and (ii) the mutant VSV-G1; and (b) comprises the CAR-33 gene;

The LVV-V21-CAR33: (a) the viral envelope contains (i) the primary signal molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv and (ii) the mutant VSV-G1; and (b) contains the CAR-33 gene.

3. Transduce human non-activated PBMCs and detect the expression efficiency of CAR-33 (MOI = 1)

Day 0, at MOI=1, the LVV-V19-CAR33, LVV-V20-CAR33 and LVV-V21-CAR33 were added to the non-activated PBMCs culture system of Donor 1 in each group; Day 2, flow cytometry was used to detect the expression efficiency of the CAR-33 in the CD3 ⁺ T cells of each group of PBMCs. The results are shown in Figure 15.

As can be seen from Figure 15, the LVV-V19-CAR33 transduced CD3 ⁺ T cells in human non-activated PBMCs, and the transduction efficiency of delivering the CAR-33 gene (about 32.14%) was significantly better than that of the LVV-V20-CAR33 (about 27.86%) and LVV-V21-CAR33 (about 19.84%).

Flow cytometry detection antibody: PE-Labeled Human Siglec-3/CD33 Protein, brand: Acro, product number: #CD3-HP2E3.

4. Detection of the killing efficiency of CD33-CAR-T cells prepared by PBMCs transduced with lentiviral vectors in each group

Day 0: 1×10 ⁶ human non-activated PBMCs from 4 groups of Donor 1 were taken, mixed with CD33 ⁺ MOLM-13 cells (human acute myeloid leukemia cells) at an effector-target ratio of E:T=1:1, and added to the culture medium (1640+10% FBS); at MOI=1, the LVV-V19-CAR33, LVV-V20-CAR33 and LVV-V21-CAR33 were added to the culture medium of 3 groups of mixed cells respectively; the control group (CTR) did not contain any lentiviral vector, but only contained the mixed cells.

Day 5: A cell counter was used to count the number of mixed cells in each group, and flow cytometry was used to detect the expression of CD33 in each group of mixed cells to calculate the killing efficiency. The results are shown in Figure 16.

As can be seen from Figure 16, on Day 5, compared with the control group, the CD33-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V19-CAR33, LVV-V20-CAR33 and LVV-V21-CAR33 can effectively kill CD33 ⁺ MOLM-13 cells.

Embodiment 11

1. Design of CAR targeting BCMA

A CAR targeting human BCMA (CAR-BCMA) is designed, and the polynucleotide encoding the CAR-BCMA (CAR-BCMA gene) comprises, from the 5' end to the 3' end:

A polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the antigen binding region targeting human BCMA, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the human 4-1BB co-stimulatory signaling domain, and a polynucleotide encoding the human CD3ζ intracellular signaling domain;

Human BCMA: Uniprot ID: Q02223. BCMA is an effective target for the treatment of multiple myeloma.

The antigen binding region targeting human BCMA is an anti-human BCMA scFv (anti-BCMA-scFv), and the amino acid sequence of the anti-BCMA-scFv is shown in SEQ ID NO:72; the amino acid sequences of the HCDR1-3 regions of the anti-BCMA-scFv are shown in SEQ ID NO:73-75, respectively; the amino acid sequences of the LCDR1-3 regions of the anti-BCMA-scFv are shown in SEQ ID NO:76-78, respectively.

2. Packaging multiple sets of lentiviral vectors containing the CAR-BCMA gene

Referring to the method for packaging the LVV-V5-CAR19, multiple groups of lentiviral vectors LVV-V22-CARBCMA, LVV-V23-CARBCMA, and LVV-V24-CARBCMA were packaged in the same batch;

The LVV-V22-CARBCMA: (a) the viral envelope contains (i) a primary signaling molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv and a secondary signaling molecule for T cell activation, i.e., the membrane expresses 15E8-scFv; and (ii) the mutant VSV-G1; and (b) contains the CAR-BCMA gene;

The LVV-V23-CARBCMA: (a) the viral envelope comprises (i) a membrane-expressed anti-CD7 antibody, i.e., the membrane expresses TH69-scFv and (ii) the mutant VSV-G1; and (b) comprises the CAR-BCMA gene;

The LVV-V24-CARBCMA: (a) the viral envelope contains (i) the primary signal molecule for T cell activation, i.e., the membrane expresses UCHT1-scFv and (ii) the mutant VSV-G1; and (b) contains the CAR-BCMA gene.

3. Transduce human non-activated PBMCs and detect the expression efficiency of CAR-BCMA (MOI = 1)

Day 0, at MOI = 1, the LVV-V22-CARBCMA, LVV-V23-CARBCMA and LVV-V24-CARBCMA were added to the non-activated PBMCs culture system of Donor 1 in each group; Day 2, flow cytometry was used to detect the expression efficiency of the CAR-BCMA in the CD3 ⁺ T cells of each group of PBMCs. The results are shown in Figure 17.

As can be seen from Figure 17, the LVV-V22-CARBCMA transduced CD3 ⁺ T cells in human non-activated PBMCs, and the transduction efficiency of delivering the CAR-BCMA gene (about 19.4%) was significantly better than that of the LVV-V23-CARBCMA (about 15.32%) and LVV-V24-CARBCMA (about 9.36%).

Flow cytometry detection antibody: Anti-G4Slinker for detection, brand: Heyousheng, item number: #GS-ARPE100.

4. Detection of the killing efficiency of BCMA-CAR-T cells prepared by PBMCs transduced with lentiviral vectors in each group

Day 0: 1×10 ⁶ human non-activated PBMCs from 4 groups of Donor 1 were taken, mixed with BCMA ⁺ U266 cells (human multiple myeloma cells) at an effector-target ratio of E:T=1:1, and added to the culture medium (1640+10% FBS); at an MOI=1, the LVV-V22-CARBCMA, LVV-V23-CARBCMA and LVV-V24-CARBCMA were added to the culture medium of 3 groups of mixed cells respectively; the control group did not contain any lentiviral vector, but only contained the mixed cells.

Day 5: A cell counter was used to count the number of mixed cells in each group, and flow cytometry was used to detect the expression of BCMA in each group of mixed cells to calculate the killing efficiency. The results are shown in Figure 18.

As can be seen from Figure 18, on Day 5, compared with the control group, the BCMA-CAR-T cells prepared by transducing human non-activated PBMCs with LVV-V22-CARBCMA, LVV-V23-CARBCMA and LVV-V24-CARBCMA can effectively kill BCMA ⁺ U266 cells.

Claims (102)

A virus particle, characterized in that (a) the surface of the virus particle contains T cell activation primary signal molecules and T cell activation secondary signal molecules; and (b) The surface of the virus particle comprises a glycoprotein, and the glycoprotein undergoes a first mutation, thereby weakening or losing the ability of the glycoprotein to bind to a glycoprotein receptor relative to before the first mutation occurs. The viral particle according to claim 1, characterized in that the viral particle targets activated and transduces non-activated T cells. The viral particle according to claim 1 or 2, characterized in that the viral particle is a lentiviral vector (LVV) or a retroviral vector (RVV). The virus particle according to any one of claims 1-3 is characterized in that the T cell activation primary signal molecule binds to the TCR/CD3 complex or the TCR/CD3 complex subunit; the TCR/CD3 complex subunit is selected from CD3ε, CD3γ, CD3δ, TCRα and TCRβ. The viral particle according to claim 4, characterized in that the TCR/CD3 complex or TCR/CD3 complex subunit is a human TCR/CD3 complex or TCR/CD3 complex subunit. The viral particle according to claim 4 or 5, characterized in that the T cell activation primary signal molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof. The virus particle according to claim 6 is characterized in that the anti-CD3 antibody is a scFv (UCHT1-scFv) derived from UCHT1, and the amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO:9; the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO:81-83, respectively, and the LCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO:84-86, respectively. The viral particle according to any one of claims 1-7, characterized in that the T cell activation secondary signal molecule binds to CD28. The viral particle according to claim 8, characterized in that the CD28 is human CD28. The viral particle according to claim 8 or 9, characterized in that the T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof. The viral particle according to claim 10, characterized in that the CD28 ligand or its receptor binding fragment comprises CD80 or its receptor binding fragment and CD86 or its receptor binding fragment. The viral particle according to claim 11, characterized in that the CD80 receptor binding fragment is the CD80 extracellular domain; the CD86 receptor binding fragment is the CD86 extracellular domain. The viral particle according to claim 8, characterized in that the T cell activation secondary signal molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof. The virus particle according to claim 13 is characterized in that the anti-CD28 antibody or its antigen-binding fragment is a scFv (15E8-scFv) derived from 15E8, and the amino acid sequence of the 15E8-scFv is as shown in SEQ ID NO: 13; the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are as shown in SEQ ID NO: 87-89, respectively, and the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are as shown in SEQ ID NO: 90-92, respectively. The virus particle according to any one of claims 8 to 14, characterized in that the T cell activation secondary signal molecule further comprises at least one ligand or its receptor binding fragment selected from ICOS ligand (ICOSL) or its receptor binding fragment, 4-1BB ligand (4-1BBL) or its receptor binding fragment and OX40 ligand (OX40L) or its receptor binding fragment. The virus particle according to any one of claims 1-15 is characterized in that the T cell activation primary signal molecule and/or T cell activation secondary signal molecule is directly or indirectly connected to a transmembrane polypeptide (Transmembrane Polypeptide) and displayed on the surface of the virus particle. The virus particle according to claim 16, characterized in that the transmembrane polypeptide is selected from the transmembrane region of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD 37. CD40, CD45, CD64, CD80, CD86, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-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; Preferably, the transmembrane polypeptide is the CD8α transmembrane region. The virus particle according to claim 16 or 17, characterized in that the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule are indirectly connected to the transmembrane polypeptide through a connecting domain and displayed on the surface of the virus particle; Preferably, the linking domain is selected from: (a) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge region; (b) a hinge region 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 an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; (d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (e) flexible linker peptide; More preferably, the connecting domain is the CD8α hinge region. The virus particle according to any one of claims 16 to 18, characterized in that the T cell activation primary signal molecule comprises the anti-CD3 antibody or its antigen-binding fragment, the CD8α hinge region and the CD8α transmembrane region from the N-terminus to the C-terminus; preferably, the anti-CD3 antibody is the UCHT1-scFv. The viral particle according to any one of claims 16 to 19, characterized in that the T cell activation secondary signal molecule comprises an anti-CD28 antibody or its antigen-binding fragment, a CD8α hinge region and a CD8α transmembrane region from the N-terminus to the C-terminus; preferably, the anti-CD28 antibody is the 15E8-scFv. The viral particle according to any one of claims 1 to 19, characterized in that the T cell activation secondary signal molecule is selected from at least one of human CD80 or its receptor binding fragment and human CD86 or its receptor binding fragment; Preferably, the amino acid sequence of human CD80 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:93; Preferably, the amino acid sequence of human CD86 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:95. The viral particle according to claim 21, characterized in that the receptor binding fragment of human CD80 comprises the extracellular domain and transmembrane region of human CD80; the receptor binding fragment of human CD86 comprises the extracellular domain and transmembrane region of human CD86; Preferably, the amino acid sequences of the extracellular domain and transmembrane region of human CD80 are at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:94; Preferably, the amino acid sequences of the extracellular domain and transmembrane region of human CD86 are at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:79. The viral particle according to claim 16, characterized in that the transmembrane polypeptide is the glycoprotein, and the glycoprotein is directly or indirectly connected to the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule. The virus particle according to claim 23 is characterized in that the glycoprotein is indirectly connected to the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule through the first polypeptide linker (The First Polypeptide Linker). The viral particle according to claim 23 or 24, characterized in that the T cell activation primary signal molecule is directly or indirectly connected to the T cell activation secondary signal molecule. The virus particle according to claim 25 is characterized in that the T cell activation primary signal molecule is indirectly connected to the T cell activation secondary signal molecule through a second polypeptide linker (The Second Polypeptide Linker). The virus particle according to any one of claims 24-26, characterized in that the polypeptide linker is a flexible linker peptide. The virus particle according to claim 27, characterized in that the flexible connecting peptide is selected from (G ₄ S) _n connecting peptide, linker 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 97) and linker 2: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 98); wherein n=1 to 4. The virus particle according to any one of claims 24 to 28, characterized in that (a) the glycoprotein is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( _G4S ) _n connecting peptide, and the anti-CD3 antibody or antigen-binding fragment thereof is indirectly linked to the anti-CD28 antibody or antigen-binding fragment thereof, human CD86 extracellular domain or human CD80 extracellular domain via a ( _G4S ) _n connecting peptide; and/or (b) the glycoprotein is indirectly linked to the anti-CD28 antibody or antigen-binding fragment thereof, the extracellular domain of human CD86 or the extracellular domain of human CD80 via a (G ₄ S) _n connecting peptide, and the anti-CD28 antibody or antigen-binding fragment thereof, the extracellular domain of human CD86 or the extracellular domain of human CD80 is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a (G ₄ S) _n connecting peptide; Preferably, n=3; Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is the UCHT1-scFv; Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is the 15E8-scFv. The virus particle according to any one of claims 1-29 is characterized in that the glycoprotein is selected from at least one of the envelope glycoproteins of vesicular stomatitis virus strains and their variants, the envelope glycoproteins of baboon endogenous retrovirus BaEV and their variants, the envelope glycoproteins RD114 and their variants of feline endogenous retroviruses, and the envelope glycoproteins GALV and their variants of gibbon ape leukemia virus. The virus particle according to claim 30, characterized in that the glycoprotein is selected from at least one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof; Preferably, the envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain include the following envelope glycoproteins and variants thereof: envelope glycoprotein and variants of Indiana strain of vesicular stomatitis virus, envelope glycoprotein and variants of Cocal strain of vesicular stomatitis virus, envelope glycoprotein and variants of Maraba strain of vesicular stomatitis virus, envelope glycoprotein and variants of Morreton strain of vesicular stomatitis virus, envelope glycoprotein and variants of Alagoas strain of vesicular stomatitis virus, envelope glycoprotein and variants of New Jersey strain of vesicular stomatitis virus, envelope glycoprotein and variants of Carajas strain of vesicular stomatitis virus, envelope glycoprotein and variants of Chandipura strain of vesicular stomatitis virus and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants. The virus particle according to claim 31 is characterized in that the glycoprotein is the envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the glycoprotein receptor is the low-density lipoprotein receptor LDL-R; the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, or has at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2. The viral particle according to claim 32, characterized in that the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution or deletion of the amino acid at position 8, substitution or deletion of the amino acid at position 9, substitution or deletion of the amino acid at position 10, substitution or deletion of the amino acid at position 47, substitution or deletion of the amino acid at position 50, substitution or deletion of the amino acid at position 51, substitution or deletion of the amino acid at position 183, substitution or deletion of the amino acid at position 179, substitution or deletion of the amino acid at position 180, substitution or deletion of the amino acid at position 182, substitution or deletion of the amino acid at position 184, substitution or deletion of the amino acid at position 209 of SEQ ID NO:1 or SEQ ID NO:2. 347, substitution or deletion of amino acid 350, substitution or deletion of amino acid 352, substitution or deletion of amino acid 353, substitution of amino acid 354, 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; (b) after optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184 The present invention relates to a substitution or deletion of the amino acid at position 350, a substitution or deletion of the amino acid at position 352, a substitution or deletion of the amino acid at position 353, a substitution or deletion of the amino acid at position 354, a deletion of the amino acids at positions 1-18, a deletion of the amino acids at positions 19-36, a deletion of the amino acids at positions 37-51, a deletion of the amino acids at positions 314-384, a deletion of the amino acids at positions 321-374, a deletion of the amino acids at positions 331-364, a deletion of the amino acids at positions 344-354, and a deletion of the amino acids at positions 345-353. The viral particle according to claim 33, characterized in that the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) deletion of amino acids 331 to 364, deletion of amino acids 344 to 354, substitution of K47, deletion of K47, or substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletion at positions 331 to 364, amino acid deletion at positions 344 to 354, replacement of K47, deletion of K47, and replacement of R354 are located at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) The amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 in SEQ ID NO: 1 or SEQ ID NO: 2; (b) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 are located corresponding to SEQ ID NO:1 or SEQ ID NO:2. The virus particle according to any one of claims 32 to 34, characterized in that the first mutation includes a mutation in which the amino acid sequence comprises the following amino acids: (a) K47 deletion located at SEQ ID NO:1 or SEQ ID NO:2; (b) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the K47 deletion is located at the position equivalent to SEQ ID NO:1 or SEQ ID NO:2. The viral particle according to claim 32 or 33, characterized in that the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of K47, deletion of K47, substitution of I182, substitution of R354, and substitution of Y209 in SEQ ID NO: 1; (b) After optimal global alignment with SEQ ID NO: 1, the substitutions at K47, K47 deletion, I182 substitution, R354 substitution, and Y209 substitution are equivalent to those in SEQ ID NO: 1; (c) substitution of K47, deletion of K47, substitution of V182, substitution of R354, and substitution of Y209 at SEQ ID NO:2; (d) After optimal global alignment with SEQ ID NO: 2, the substitution of K47, K47 deletion, V182 substitution, R354 substitution, and Y209 substitution are located at positions equivalent to SEQ ID NO: 2; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q located in SEQ ID NO:1; (b) After optimal global alignment with SEQ ID NO:1, K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q are located at the positions equivalent to SEQ ID NO:1; (c) K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q located in SEQ ID NO:2; (d) After optimal global alignment with SEQ ID NO:2, it is located at K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q equivalent to SEQ ID NO:2. The virus particle according to any one of claims 1-36 is characterized in that the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 23 or SEQ ID NO: 24. The virus particle according to any one of claims 1-37 is characterized in that the glycoprotein also undergoes a second mutation, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced or not inactivated by complement relative to before the second mutation occurs. The virus particle according to claim 38 is characterized in that the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, or has at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2. The viral particle according to claim 39, characterized in that the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 214th amino acid corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 352nd amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at the 50th amino acid equivalent to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at the 146th amino acid corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the amino acid mutation is selected from at least one of amino acid deletion, insertion and substitution; More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids: (a) amino acid 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 214th amino acid corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 352nd amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at the 50th amino acid equivalent to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 146th amino acid corresponding to SEQ ID NO: 1 or SEQ ID NO: 2. The viral particle of claim 40, wherein the second mutation comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1, comprising at least one of the following site mutations: (a) substitution of T214, T352, K50 and S146 in SEQ ID NO: 1; (b) After optimal global alignment with SEQ ID NO: 1, the substitutions at T214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 1; Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a) T214N, T352A, K50T, S146T located in SEQ ID NO: 1; (b) After optimal global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO:1. The viral particle according to claim 41, characterized in that the second mutation comprises a combination of any of the following site mutations in the amino acid sequence: (a) substitution of (1) T214 and T352; or (2) substitution of T214, T352, K50 and S146 at SEQ ID NO:1; (b) after optimal global alignment with SEQ ID NO:1, substitutions at positions corresponding to (1) T214 and T352 of SEQ ID NO:1; or (2) substitutions at T214, T352, K50, and S146; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T located at SEQ ID NO:1; (b) After optimal global alignment with SEQ ID NO:1, located at (1) T214N and T352A equivalent to SEQ ID NO:1; or (2) T214N, T352A, K50T and S146T. The viral particle of claim 40, wherein the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 2, comprising at least one of the following site mutations: (a) substitution of K214, T352, K50 and S146 in SEQ ID NO: 2; (b) After optimal global alignment with SEQ ID NO: 2, the substitutions at K214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 2; Preferably, the second mutation includes that the amino acid sequence comprises at least one of the following site mutations: (a) K214N, T352A, K50T, S146T located in SEQ ID NO: 2; (b) After optimal global alignment with SEQ ID NO:2, K214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO:2. The viral particle according to claim 43, characterized in that the second mutation comprises a combination of any of the following site mutations in the amino acid sequence: (a) replacement of (1) K214 and T352; or (2) replacement of K214, T352, K50 and S146 at SEQ ID NO:2; (b) after optimal global alignment with SEQ ID NO:2, the position is equivalent to (1) replacement of K214 and T352 of SEQ ID NO:2; or (2) replacement of K214, T352, K50 and S146; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T located at SEQ ID NO:2; (b) After optimal global alignment with SEQ ID NO:2, located at (1) K214N and T352A; or (2) K214N, T352A, K50T and S146T equivalent to SEQ ID NO:2. The virus particle according to any one of claims 38-44 is characterized in that the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28. The viral particle according to any one of claims 1-45, characterized in that the viral particle contains an exogenous load gene. The viral particle according to claim 46 is characterized in that the exogenous load gene encodes a therapeutic protein or polypeptide, and the therapeutic protein or polypeptide is selected from at least one of antibody-based drugs, chimeric antigen receptors, T cell receptors, cytokine receptors, cytokines, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytic agents. The viral particle according to claim 47, characterized in that the exogenous load gene encodes a chimeric antigen receptor (CAR). The viral particle according to claim 48 is characterized in that the chimeric antigen receptor comprises an extracellular antigen binding region, a transmembrane region and an intracellular signaling domain. The viral particle according to claim 49, characterized in that the extracellular antigen binding region binds to an antigen associated with the disease. The viral particle according to claim 50, characterized in that the antigen is selected from: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD3 8. CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD 138, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain , TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD17 1. CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin (MSLN), IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, 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, bean curd protein, HPV E6/E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PC At least one of TA-1/Galectin 8, MelanA/MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2 ETS fusion gene/ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, and NKG2D. The viral particle according to claim 51, characterized in that the antigen is CD19. The viral particle according to claim 51, characterized in that the antigen is selected from one or more of CD19, CD20, BCMA, CD33, HER2 and CEA. The virus particle according to any one of claims 49-53 is characterized in that the extracellular antigen binding region comprises an antibody or an antigen binding fragment thereof and/or a ligand or a receptor binding fragment thereof, and the antibody or its antigen binding fragment is selected from at least one of an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab')2, an Fv fragment, a single-chain variable region fragment (scFv), a disulfide bond-stabilized antibody (dsFv), an antibody heavy chain variable region (VH) or a light chain variable region (VL), an Fd fragment composed of a VH and a CH1 domain, a linear antibody, and a single-domain antibody (nanoantibody). The viral particle according to any one of claims 49 to 54, characterized in that the transmembrane region is derived from the transmembrane region of at least one of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-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; Preferably, the transmembrane region is derived from the transmembrane region of CD8α. The viral particle according to any one of claims 49 to 55, characterized in that the intracellular signaling domain is derived from the intracellular signaling domain of at least one of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FcεRlγ, FcεRβ, FcγRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12 and other intracellular signaling domains of proteins containing at least one ITAM; Preferably, the intracellular signaling domain is derived from the intracellular signaling domain of CD3ζ. The virus particle according to any one of claims 49 to 56, characterized in that the chimeric antigen receptor further comprises a hinge region; the hinge region sequentially connects the extracellular antigen binding region and the transmembrane region; Preferably, the hinge region is derived from the hinge region of at least one of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154; More preferably, the hinge region is derived from the hinge region of CD8α. The viral particle according to any one of claims 49 to 57, characterized in that the chimeric antigen receptor further comprises a co-stimulatory signaling domain; Preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRlγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-l, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88; More preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of 4-1BB. The viral particle according to any one of claims 49 to 58, characterized in that the CAR further comprises a signal peptide; Preferably, the signal peptide is derived from the CD8α signal peptide. The viral particle according to any one of claims 1-59 is characterized in that the efficiency of transducing non-activated T cells by the viral particle is improved compared with a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules. The virus particle according to any one of claims 46-60 is characterized in that, compared with a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules, after the virus particles contact non-activated T cells, the efficiency of expression of the exogenous load gene in T cells is improved. The virus particle according to any one of claims 48-61 is characterized in that, compared with a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules, after the virus particles contact non-activated T cells, the efficiency of the chimeric antigen receptor in the membrane expression of T cells is improved. The virus particle according to any one of claims 48-62 is characterized in that, compared with a control vector whose surface does not contain T cell activation primary signal molecules and T cell activation secondary signal molecules, the killing efficiency of the prepared CAR-T cells is enhanced after the virus particles contact non-activated T cells. The viral particle according to any one of claims 60-63 is characterized in that the surface of the control vector contains at least one T cell targeting molecule, and the T cell targeting molecule binds to a T cell endocytosis receptor. The viral particle according to claim 64, characterized in that the T cell endocytosis receptor is selected from at least one of CD5 and CD7. The viral particle of claim 65, wherein the T cell targeting molecule binds to CD7. The viral particle according to claim 66, characterized in that the T cell targeting molecule is selected from at least one of an anti-CD7 antibody or an antigen-binding fragment thereof and a CD7 ligand or a receptor-binding fragment thereof; Optionally, the anti-CD7 antibody or its antigen-binding fragment is a scFv (TH69-scFv) derived from the monoclonal antibody TH-69; the amino acid sequence of the TH69-scFv is as shown in SEQ ID NO:37; the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO:38-40, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO:41-43, respectively. The virus particle according to any one of claims 60 to 63, characterized in that the surface of the control vector only contains the T cell activation primary signal molecule, and does not contain the T cell activation secondary signal molecule; Optionally, the T cell activation primary signal molecule binds to the TCR/CD3 complex or a TCR/CD3 complex subunit; the TCR/CD3 complex subunit is selected from CD3ε, CD3γ, CD3δ, TCRα and TCRβ; preferably, the T cell activation primary signal molecule binds to the human TCR/CD3 complex or a TCR/CD3 complex subunit; more preferably, the T cell activation primary signal molecule includes an anti-CD3 antibody or an antigen-binding fragment thereof against human CD3. An engineered T cell, characterized in that the engineered T cell expresses a chimeric antigen receptor, and the engineered T cell is prepared by contacting a T cell with a polynucleotide encoding a chimeric antigen receptor carried by a virus particle described in any one of claims 1 to 68. The engineered T cells according to claim 69, characterized in that the T cells include non-activated T cells and activated T cells. The engineered T cell according to claim 69 or 70, characterized in that the contact occurs in vivo and/or in vitro in a subject; the subject is an individual who is administered the engineered T cell and/or the viral particle according to any one of claims 1-68 carrying a polynucleotide encoding a chimeric antigen receptor. The engineered T cells according to claim 71 are characterized in that the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device administration. The engineered T cell according to any one of claims 69 to 72, characterized in that the T cell is a non-activated T cell, and the efficiency of expressing CAR by the engineered T cell is improved; The improvement in expression efficiency is relative to the engineered T cells prepared by contacting a control vector not containing the T cell activation primary signal molecule and the T cell activation secondary signal molecule on its surface with the non-activated T cells. The engineered T cell according to any one of claims 69 to 73, characterized in that the T cell is a non-activated T cell, and the killing efficiency of the engineered T cell is improved; The improvement in killing efficiency is relative to the engineered T cells prepared by contacting a control vector whose surface does not contain the T cell activation primary signal molecule and the T cell activation secondary signal molecule with the non-activated T cells. The engineered T cell according to claim 73 or 74 is characterized in that the surface of the control vector contains at least one T cell targeting molecule, and the T cell targeting molecule binds to a T cell endocytosis receptor. The engineered T cell according to claim 75, characterized in that the T cell endocytosis receptor is selected from at least one of CD5 and CD7. The engineered T cell of claim 76, wherein the T cell targeting molecule binds to CD7. The engineered T cell according to claim 77, characterized in that the T cell targeting molecule is selected from at least one of an anti-CD7 antibody or an antigen-binding fragment thereof and a CD7 ligand or a receptor-binding fragment thereof; Optionally, the anti-CD7 antibody or its antigen-binding fragment is a scFv (TH69-scFv) derived from the monoclonal antibody TH-69; the amino acid sequence of the TH69-scFv is as shown in SEQ ID NO:37; the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO:38-40, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO:41-43, respectively. The engineered T cell according to claim 73 or 74, characterized in that the surface of the control carrier only contains the T cell activation primary signal molecule, and does not contain the T cell activation secondary signal molecule; Optionally, the T cell activation primary signal molecule binds to the TCR/CD3 complex or a TCR/CD3 complex subunit; the TCR/CD3 complex subunit is selected from CD3ε, CD3γ, CD3δ, TCRα and TCRβ; preferably, the T cell activation primary signal molecule binds to the human TCR/CD3 complex or a TCR/CD3 complex subunit; more preferably, the T cell activation primary signal molecule includes an anti-CD3 antibody or an antigen-binding fragment thereof against human CD3. A composition, characterized in that the composition comprises a pharmaceutically acceptable excipient or carrier and any one of the following components: the virus particles described in any one of claims 1-68 and the engineered T cells described in any one of claims 69-79. Use of the viral particles described in any one of claims 1 to 68, the engineered T cells described in any one of claims 69 to 79, or the composition described in claim 80 in the preparation of a medicament for preventing and/or treating a disease. The use according to claim 81 is characterized in that the disease is an autoimmune disease or cancer, and the cancer includes solid cancer and blood cancer. The use according to claim 82 is characterized in that the cancer is blood cancer. The use according to claim 83 is characterized in that the blood cancer is B lymphocyte cancer, B lymphocyte leukemia, Hodgkin's lymphoma or multiple myeloma. The use according to claim 83 is characterized in that the blood cancer is selected from non-Hodgkin lymphoma (NHL), acute B-cell lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), One or more of follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma/leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), primary plasma cell leukemia (pPCL), peripheral T-cell lymphoma (PTCL-NHL), NK/T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, T-large granular lymphocytic leukemia (T-LGL) and germinal center T-cell lymphoma (FTCL). The use according to claim 82 is characterized in that the solid cancer is selected from one or more of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary cancer and gastrointestinal cancer. [Corrected 08.02.2025 in accordance with Rule 26]
The use according to claim 86 is characterized in that the cancer is selected from one or more of CD19 ⁺ cancer, CD20 ⁺ cancer, BCMA ⁺ cancer, CD33 ⁺ cancer, HER2 ⁺ cancer and CEA ⁺ cancer. A method for treating cancer in a subject or killing cancer cells in a subject, characterized in that it comprises administering to the subject the viral particles described in any one of claims 1-68, the engineered T cells described in any one of claims 69-79, or the composition described in claim 80; the viral particles or the composition further comprise an exogenous load gene. The method for treating cancer in a subject or killing cancer cells in a subject according to claim 88 is characterized in that the exogenous load gene encodes a therapeutic protein or polypeptide; the therapeutic protein or polypeptide is selected from at least one of antibody-based drugs, chimeric antigen receptors, T cell receptors, cytokine receptors, cytokines, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytic agents. The method for treating cancer in a subject or killing cancer cells in a subject according to claim 89, characterized in that the exogenous load gene encodes a chimeric antigen receptor. The method according to any one of claims 88-90 is characterized in that the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device administration. A cell, characterized in that the cell is configured to produce the viral particles described in any one of claims 1-68. The cell according to claim 92, characterized in that the cell is selected from at least one of: NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK cells, BHK cells and MDCKⅡ cells; Preferably, the cells are HEK-293T cells. A method for preparing viral particles, characterized in that the cells described in any one of claims 92 or 93 are cultured to a degree sufficient to produce the viral particles. A method for transducing T cells, characterized in that the virus particles described in any one of claims 1 to 68 are used to carry exogenous load genes and contact T cells. The method according to claim 95 is characterized in that the exogenous load gene encodes a therapeutic protein or polypeptide; the therapeutic protein or polypeptide is selected from at least one of antibody-based drugs, chimeric antigen receptors, T cell receptors, cytokine receptors, cytokines, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytic agents. The method according to claim 96 is characterized in that the exogenous cargo gene encodes a chimeric antigen receptor. The method according to any one of claims 95-97 is characterized in that the T cells include activated T cells and non-activated T cells. The method according to any one of claims 95-98 is characterized in that the contact occurs in vivo and/or in vitro in a subject; the subject is an individual who is given T cells transduced by the T cell transduction method and/or the viral particles according to any one of claims 1-68 carrying exogenous load genes. The method according to claim 99 is characterized in that the contacting occurs in a subject, wherein the subject is an individual who is administered the viral particles of any one of claims 1-68 carrying an exogenous load gene. The method according to claim 100, characterized in that the virus particle is the virus particle of any one of claims 48-68. The method according to any one of claims 95-101 is characterized in that the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device administration.