Viral vectors and their uses

Abstract

A set of viral vectors is provided, comprising a first viral vector carrying a first nucleic acid molecule encoding an envelope protein, and a second viral vector carrying a second nucleic acid molecule encoding a fusion protein comprising a single chain antibody capable of binding to CD28 or CD3, and a C-terminal domain comprising the transmembrane and intracellular regions of the envelope protein. The C-terminus of the single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein, and the envelope protein and the fusion protein are in a non-fused form. Also provided are a method for obtaining lentivirus and the obtained lentivirus, a method for introducing lentivirus into unactivated T lymphocytes, a method for expressing a target gene, a method for obtaining CAR-T cells and the obtained CAR-T cells, a pharmaceutical composition, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to viral vectors and uses thereof, more specifically, the present invention relates to viral vectors, methods for obtaining lentiviruses, lentiviruses, methods for introducing lentiviruses into unactivated T lymphocytes, methods for expressing a gene of interest, and methods for obtaining CAR-T cells. [Background technology]

[0002] Gene therapy aims to treat or cure diseases by modifying gene expression in an organism, and is divided into in vivo gene therapy and ex vivo gene therapy according to the location where gene transfer occurs. In vivo therapeutic strategy is to assemble a therapeutic gene into a specific vector and transduce the therapeutic gene into cells in the human body through the vector. Currently, adeno-associated virus (AAV) vectors are mainly used. Ex vivo therapeutic strategy is to isolate cells from a patient, genetically modify the cells during ex vivo culture, and then inject the genetically modified cells back into the patient. Currently, lentiviral vectors (LV) are mainly used.

[0003] Comparing in vivo and ex vivo therapeutic strategies, the inventors know that the former drug is a gene transfer vector, which is a general-purpose product, and the production process of the product simply involves the production of gene transfer vectors. The latter drug is modified patient cells, which is an individualized product and is only suitable for treating diseases in which blood cells can be separated and ex vivo cultured. The production process of the product is complicated, including the production of gene transfer vectors, isolation of patient cells, ex vivo culture, cell gene modification, and cell reinfusion. In general, in vivo gene therapy products have the enormous advantages of a wide range of indications, a relatively simple and universal production process, mass production, and low cost.

[0004] However, current in vivo gene therapy using AAV vectors has certain problems in terms of efficacy and safety. Regarding efficacy, after transduction into the patient's cells, the gene does not contain the host genome in the form of microloops, and disappears as the cells divide and undergo apoptosis, resulting in a gradual attenuation of drug resistance. Regarding safety, AAV gene therapy has caused frequent fatalities during its introduction into the market and in early clinical trials, and the FDA has focused on safety issues related to liver toxicity, kidney damage, and neuronal loss of this treatment. Furthermore, the industry once viewed AAV transduced genes as very safe, not integrated into the host genome and not posing a risk of carcinogenesis. However, there are continuing reports that AAV transduced genes are integrated into the host genome and have carcinogenic risks.

[0005] Genes transduced by lentiviral vectors can penetrate the nuclear membrane and efficiently integrate into the host genome in both dividing and non-dividing cells, allowing therapeutic genes to replicate during cell division and persist and stably exist in transduced cells, which allows one-time administration and lifelong treatment to be achieved. At present, lentiviral vectors are widely used in ex vivo gene therapy, such as CAR-T cell production and hematopoietic stem cell modification. A huge amount of preclinical and clinical data has been accumulated, and there have been no reports of tumors caused by lentiviral vector transduced genes inserted into the host genome, providing the safety of lentivirus. However, lentiviral vectors have not yet made any breakthrough in in vivo gene therapy, the main reason being that transduction is non-targeted and may affect safety due to off-target. The construction of targeted lentiviral vectors may improve the corresponding safety. Although some papers have reported that conjugation of scFv or pre-designed ankyrin repeat proteins (DARPins) to the envelope protein of lentiviral vectors can enhance the transduction efficiency of the vectors to certain types of cells, the modified vectors still have problems such as high or unassessed off-target rates and significantly reduced production titers, making them unusable for industrial production.

[0006] Therefore, in order to solve the above problems, a new technique needs to be developed. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to solve at least to some extent one of the technical problems in the related art. To this end, the present invention provides a viral vector with target infectivity and significantly improved viral titer. [Means for solving the problem]

[0008] In a first aspect of the present invention, a group of viral vectors is provided herein. According to an embodiment of the present invention, the group of viral vectors comprises a first viral vector carrying a first nucleic acid molecule encoding an envelope protein; at least one second viral vector carrying a second nucleic acid molecule encoding at least one fusion protein, the fusion protein comprises at least one single chain antibody and a C-terminal domain of the envelope protein, the single chain antibody can bind to CD28 or CD3, the C-terminal domain of the envelope protein comprises the transmembrane domain and the intracellular domain of the envelope protein, and the C-terminus of the at least one single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein; the first nucleic acid molecule and the second nucleic acid molecule are arranged to express the envelope protein and the fusion protein, and the envelope protein and the fusion protein are in unfused form. After the viral vector according to the embodiment of the present invention is introduced into the recipient cells, a virus with high viral titer can be packaged, the single chain antibody expressed by the virus achieves specific targeting of the virus to immune cells via binding to CD28 or CD3, and then specific infection of immune T cells by the virus, and the packaged virus of the present invention can directly transduce non-pre-activated or activated T cells in vitro and ex vivo.

[0009] According to an embodiment of the present invention, the viral vector may further include at least one of the following additional technical features:

[0010] According to an embodiment of the present invention, the viral vector is a retroviral vector, a lentiviral vector, or another enveloped viral vector.

[0011] According to an embodiment of the present invention, the enveloped virus is selected from the group consisting of Bornaviridae, Nyamaviridae, Arenaviridae, Filoviridae, Hantaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Phenuiviridae, Lavroviridae, and the like. The virus includes at least one selected from the group consisting of Rhabdoviridae, Arteriviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepadnaviridae, Spumavirus, Iridoviridae, Herpesviridae, Poxviridae, and Deltavirus.

[0012] According to an embodiment of the present invention, the envelope protein is the envelope G glycoprotein (VSV-G) from Vesicular Stomatitis Virus, which belongs to the Rhabdoviridae family, or its mutant variant.The envelope G glycoprotein of Vesicular Stomatitis Virus has cell membrane attachment and fusion abilities.Therefore, the virus packaged by the virus vector provided herein has cell attachment and infection abilities.

[0013] According to an embodiment of the invention, the envelope G glycoprotein (VSV-G) has the amino acid sequence shown in SEQ ID NO:1.

[0014] [ka]

[0015] According to an embodiment of the present invention, the mutant envelope protein has a mutation that reduces the attachment ability. According to a specific embodiment of the present invention, the mutant envelope protein has a reduced cell membrane attachment ability, which reduces the non-specific cell attachment ability of the packaged virus, but does not affect its membrane fusion ability, so that the virus still has the ability to infect cells.

[0016] According to an embodiment of the present invention, the mutant envelope G glycoprotein has K47Q and R354Q mutations. The envelope G glycoprotein mutant has a reduced ability to attach to a cell membrane, but its membrane fusion ability is not affected. Thus, the envelope G glycoprotein mutant has K47Q and R354Q mutations attenuates the non-specific cell attachment ability of the virus obtained by packaging, but does not affect the ability of the virus to infect cells.

[0017] According to an embodiment of the invention, the mutant envelope G glycoprotein has the amino acid sequence shown in SEQ ID NO:2.

[0018] [ka]

[0019] According to an embodiment of the invention, the single chain antibody is capable of binding to CD28. According to an embodiment of the invention, the single chain antibody is capable of specifically targeting and binding to CD28 and is an anti-CD28 single chain antibody.

[0020] According to embodiments of the invention, the single chain antibody has the amino acid sequence shown in SEQ ID NO: 3 or 4. Single chain antibodies according to embodiments of the invention can target and bind CD28 positive cells, such as T cells.

[0021] [ka]

[0022] [ka]

[0023] According to an embodiment of the invention, the single chain antibody is capable of binding to CD3. According to an embodiment of the invention, the single chain antibody is capable of specifically targeting and binding to CD3 and is an anti-CD3 single chain antibody.

[0024] According to embodiments of the invention, the single chain antibody has the amino acid sequence shown in SEQ ID NO: 5 or 6. Single chain antibodies according to embodiments of the invention can target and bind CD3 positive cells, such as T cells.

[0025] [ka]

[0026] [ka]

[0027] According to an embodiment of the present invention, the fusion protein comprises a first single chain antibody, a second single chain antibody, and a C-terminal domain of an envelope protein. The first single chain antibody can bind to CD28, and the second single chain antibody can bind to CD3. The C-terminus of the first single chain antibody is linked to the N-terminus of the second single chain antibody, and the C-terminus of the second single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein; or the C-terminus of the second single chain antibody is linked to the N-terminus of the first single chain antibody, and the C-terminus of the first single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein. The fusion protein according to an embodiment of the present invention can specifically target and bind CD28 and CD3. The fusion protein according to an embodiment of the present invention can specifically target and bind CD28 and CD3 positive cells, such as T cells.

[0028] According to an embodiment of the invention, the first single chain antibody has the amino acid sequence shown in SEQ ID NO:3 or 4.

[0029] According to an embodiment of the invention, the second single chain antibody has the amino acid sequence shown in SEQ ID NO:5 or 6.

[0030] According to an embodiment of the invention, the C-terminal domain of the envelope protein further comprises at least a portion of the extracellular region of the envelope protein.

[0031] According to an embodiment of the invention, the fusion protein further comprises a first binding peptide, the N-terminus of the first binding peptide being linked to the C-terminus of the first single chain antibody and the C-terminus of the first binding peptide being linked to the N-terminus of the second single chain antibody; or the N-terminus of the first binding peptide being linked to the C-terminus of the second single chain antibody and the C-terminus of the first binding peptide being linked to the N-terminus of the first single chain antibody.

[0032] According to an embodiment of the present invention, the first binding peptide has any one of the amino acid sequences shown in SEQ ID NOs:7-11.

[0033] GGGGSGGGGSGGGGSGGGGSGGGGS (sequence number 7).

[0034] GGGGSGGGGSGGGGSGGGGS (sequence number 8).

[0035] GGGGSGGGGSGGGGS (sequence number 9).

[0036] EAAAKEAAAKEAAAKE (sequence number 10).

[0037] EAAAKEAAAKEAAAKEAAAK (sequence number 11).

[0038] According to an embodiment of the invention, the fusion protein further comprises a second binding peptide, the N-terminus of the second binding peptide being linked to the C-terminus of the at least one single chain antibody and the C-terminus of the second binding peptide being linked to the N-terminus of the C-terminal domain of the envelope protein.

[0039] According to an embodiment of the invention, the second binding peptide has the amino acid sequence shown in SEQ ID NO: 12. This second binding peptide separates the single-chain antibody region from the C-terminal region of the envelope protein, alleviating functional interference between the two.

[0040] AAATTT (sequence number 12).

[0041] According to an embodiment of the invention, the C-terminal domain of the envelope protein comprises a peptide chain ranging from amino acid 386 to amino acid 434 to amino acid 495 of the envelope protein.

[0042] According to an embodiment of the present invention, the first amino acid at the N-terminus of the envelope protein is used as the first amino acid. For example, for an envelope G glycoprotein having the amino acid sequence shown in SEQ ID NO: 1 or 2, the C-terminal domain of the envelope protein comprises a peptide chain between amino acid 386 and amino acid 434 to 495 (the first amino acid at the C-terminus) of the envelope protein, i.e., when the length of the C-terminal domain of the envelope protein is longer than 61 amino acids and shorter than 111 amino acids (i.e., 62 amino acids or more and 110 amino acids or less), the packaged lentiviral vector has the highest transduction efficiency.

[0043] According to an embodiment of the invention, the C-terminal domain of the envelope protein comprises a peptide chain starting at an amino acid between amino acids 395 and 425 up to and including amino acid 495 of the envelope protein.

[0044] According to an embodiment of the present invention, the C-terminal domain of the envelope protein comprises amino acids 425 to 495, 415 to 495, 405 to 495, or 395 to 495 of the VSV-G protein.

[0045] According to an embodiment of the invention, the C-terminal domain of the envelope protein has the amino acid sequence shown in SEQ ID NO: 13, 39, 40 or 41.

[0046] [ka]

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[0050] According to an embodiment of the invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 14, 15, 16, 17, 18, 19 or 20.

[0051] [ka]

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[0058] However, the fusion protein having the amino acid sequence set forth in SEQ ID NO:14 is a fusion protein comprising a single chain antibody capable of binding to CD28, and in the present application, the code corresponding to the expression of the fusion protein having the amino acid sequence set forth in SEQ ID NO:14 is S1.

[0059] The fusion protein having the amino acid sequence set forth in SEQ ID NO:18 is a fusion protein comprising a single chain antibody capable of binding to CD28, and in the present application, the code corresponding to the expression of the fusion protein having the amino acid sequence set forth in SEQ ID NO:18 is S3.

[0060] The fusion protein having the amino acid sequence set forth in SEQ ID NO:15 is a fusion protein comprising a single chain antibody capable of binding to CD3, and in the present application the code corresponding to the expression of the fusion protein having the amino acid sequence set forth in SEQ ID NO:15 is S2.

[0061] The fusion protein having the amino acid sequence set forth in SEQ ID NO:19 is a fusion protein comprising a single chain antibody capable of binding to CD3, and in the present application the code corresponding to the expression of the fusion protein having the amino acid sequence set forth in SEQ ID NO:19 is S4.

[0062] The fusion protein having the amino acid sequence set forth in SEQ ID NO:16 is a fusion protein comprising a single chain antibody capable of binding to CD28 and CD3, and in the present application the code corresponding to the expression of the fusion protein having the amino acid sequence set forth in SEQ ID NO:16 is S12.

[0063] The fusion protein having the amino acid sequence set forth in SEQ ID NO:20 is a fusion protein comprising a single chain antibody capable of binding to CD28 and CD3, and in the present application the code corresponding to the expression of the fusion protein having the amino acid sequence set forth in SEQ ID NO:20 is S34.

[0064] The fusion protein having the amino acid sequence shown in SEQ ID NO:17 is a fusion protein comprising a single chain antibody capable of binding to CD28 and CD3.

[0065] According to an embodiment of the present invention, the viral vector further comprises a first promoter operably linked to the first nucleic acid molecule and a second promoter operably linked to the second nucleic acid molecule, wherein the first nucleic acid molecule and the second nucleic acid molecule are under the control of the first promoter and the second promoter, respectively, to achieve highly efficient expression of the first nucleic acid molecule and the second nucleic acid molecule.

[0066] According to an embodiment of the invention, each of the first promoter and the second promoter is independently selected from a CMV, EF-1, or RSV promoter.

[0067] According to an embodiment of the invention, the first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 21 or 35.

[0068] [ka]

[0069] [ka]

[0070] According to an embodiment of the invention, the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 22, 23, 24, 25, 32, 33 or 34.

[0071] However, the fusion protein encoded by the second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:22 is a fusion protein comprising a single chain antibody capable of binding to CD28, and in the present application, the code for the fusion protein encoded by the nucleotide sequence set forth in SEQ ID NO:22 is S1.

[0072] The fusion protein encoded by the second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:23 is a fusion protein comprising a single chain antibody capable of binding to CD3, and in the present application, the code for the fusion protein encoded by the nucleotide sequence set forth in SEQ ID NO:23 is S2.

[0073] The fusion protein encoded by the second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:24 is a fusion protein comprising a single chain antibody capable of binding to CD28 and CD3, and in the present application, the code for the fusion protein encoded by the nucleotide sequence set forth in SEQ ID NO:24 is S12.

[0074] The fusion protein encoded by the second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:25 is a fusion protein comprising a single chain antibody capable of binding to CD3 and CD28. In the present application, the nucleotide sequence set forth in SEQ ID NO:25 encodes a fusion protein having the amino acid sequence set forth in SEQ ID NO:17.

[0075] The fusion protein having the nucleotide sequence set forth in SEQ ID NO:32 is a fusion protein comprising a single chain antibody capable of binding to CD28, and in the present application, the code for the fusion protein encoded by the nucleotide sequence set forth in SEQ ID NO:32 is S3.

[0076] The fusion protein having the nucleotide sequence set forth in SEQ ID NO:33 is a fusion protein comprising a single chain antibody capable of binding to CD3, and in the present application, the code for the fusion protein encoded by the nucleotide sequence set forth in SEQ ID NO:33 is S4.

[0077] The fusion protein having the nucleotide sequence set forth in SEQ ID NO:34 is a fusion protein comprising a single chain antibody capable of binding to CD28 and CD3, and in the present application, the code for the fusion protein encoded by the nucleotide sequence set forth in SEQ ID NO:34 is S34.

[0078] [ka]

[0079] [ka]

[0080] [ka]

[0081] [ka]

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[0088] According to an embodiment of the present invention, the second nucleic acid molecule further comprises a nucleic acid sequence encoding a signal peptide. According to a specific embodiment of the present invention, the signal peptide expressed by the nucleic acid sequence encoding a signal peptide is located at the amino terminus of the fusion protein precursor protein and is a membrane-localizing telopeptide of the envelope protein. The signal peptide serves to position the envelope protein in the endoplasmic reticulum and is hydrolyzed and removed after protein maturation. Therefore, the fusion protein of the virus particle does not contain this signal peptide.

[0089] According to an embodiment of the invention, the nucleic acid sequence encoding the signal peptide has the nucleotide sequence shown in SEQ ID NO:26.

[0090] ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGC (sequence number 26).

[0091] According to an embodiment of the present invention, the copy number ratio of the first nucleic acid molecule and the second nucleic acid molecule is 1:1 to 4:1. In this specification, the "copy number ratio of the first nucleic acid molecule and the second nucleic acid molecule" refers to the ratio of the number of the first nucleic acid molecule and the second nucleic acid molecule carried on the vector when the first viral vector and the second viral vector are the same vector, i.e., when the first nucleic acid molecule and the second nucleic acid molecule are the same vector, so as to ensure that the ratio of the protein expression amount of the first nucleic acid molecule and the second nucleic acid molecule is approximately the same. The present inventors have found that when the ratio of the number of the first nucleic acid molecule and the second nucleic acid molecule carried on the vector is 1:1 to 4:1, both the virus titer and the virus infection efficiency are higher.

[0092] According to an embodiment of the present invention, the copy number ratio of the first nucleic acid molecule and the second nucleic acid molecule is 2: 1 to 4: 1. According to a specific embodiment of the present invention, when the number ratio of the first nucleic acid molecule and the second nucleic acid molecule carried on the vector is 2: 1 to 4: 1, both the virus titer and the virus infection efficiency are further improved.

[0093] According to an embodiment of the present invention, the copy number ratio of the first nucleic acid molecule and the second nucleic acid molecule is 2: 1. The present inventors found that when the ratio of the number of the first nucleic acid molecule and the second nucleic acid molecule carried on the vector is 2: 1, the virus titer and the virus infection efficiency reach an optimal balance.

[0094] According to an embodiment of the present invention, the first viral vector and the second viral vector are the same vector.

[0095] According to an embodiment of the present invention, the first and second viral vectors are the same vector, and the vector further comprises an internal ribosome entry site (IRES) located between the first and second nucleic acid molecules. The expression of the two proteins before and after the internal ribosome entry site is usually proportional. The introduction of the internal ribosome entry site sequence allows the first and second nucleic acid molecules to be translated and expressed independently, and the resulting envelope protein and fusion protein are in a non-fusion form. The introduction of the internal ribosome entry site sequence effectively guarantees the biological effect of the envelope protein and the fusion protein, so that the specific binding and adsorption of the virus obtained by packaging is remarkable, and it can directly transduce non-preactivated T cells, resulting in high viral titer.

[0096] According to an embodiment of the present invention, the first and second viral vectors are the same vector, and the vector further comprises a third nucleic acid molecule located between the first and second nucleic acid molecules, the third nucleic acid molecule encoding a cleavable third binding peptide. The introduction of the third nucleic acid molecule makes the expression of the envelope protein and the fusion protein in a non-fused form, thereby ensuring the biological function of the envelope protein and the fusion protein, so that the virus obtained by packaging has remarkable specific binding and infection ability, can directly transduce non-preactivated T cells, and has high viral titer.

[0097] According to an embodiment of the present invention, the first and second viral vectors are pMD2.G, pCMV, pMD2.G mutant, or pCMV mutant. The types of the first and second viral vectors according to the embodiment of the present invention are not particularly limited, and a vector capable of expressing VSV-G, or a mutant of a vector capable of expressing VSV-G or a VSV-G mutant can be used.

[0098] According to an embodiment of the present invention, the viral vector further comprises a third viral vector and a fourth viral vector, the third viral vector carrying a gene of interest, and the fourth viral vector carrying a viral structural protein gene and a viral packaging enzyme gene and optionally a regulatory factor rev gene.

[0099] According to an embodiment of the present invention, the structural protein gene, the viral packaging enzyme gene and the regulatory factor rev gene are located on the same or different fourth viral vectors. For example, the expression product of lentiviral vector psPAX2 includes the structural protein gag, the packaging enzyme pol (including reverse transcriptase, protease and integrase) and the regulatory factor rev, where rev can increase the product titer to some extent but is not required for lentiviral packaging. Rev (pRSV-rev) and gag-pol (pMDLg-pRRE) can be divided into two expression plasmids, or rev (pRSV-rev), gag (pCMV-gag), pol (pCMV-gag) can be divided into three expression plasmids.

[0100] According to an embodiment of the invention, the viral packaging enzymes include at least one of a reverse transcriptase, a protease, and an integrase.

[0101] According to an embodiment of the invention, the third viral vector is a transfer vector.

[0102] According to an embodiment of the invention, the transfer vector comprises a lentiviral packaging signal.

[0103] According to an embodiment of the invention, the lentiviral packaging signal comprises Ψ.

[0104] According to an embodiment of the present invention, the transfer vector is a pLV vector.

[0105] According to an embodiment of the present invention, the fourth viral vector is psPAX2.

[0106] According to an embodiment of the present invention, the viral vector is a non-pathogenic virus.

[0107] According to an embodiment of the present invention, the third viral vector further carries a gene of interest which is a nucleic acid molecule encoding a chimeric antigen receptor.

[0108] In a second aspect of the present invention, a method for obtaining lentivirus is provided herein. According to an embodiment of the present invention, the method includes the steps of: introducing the viral vector provided herein into a first recipient cell; culturing the first recipient cell into which the viral vector is introduced to obtain the virus. The lentivirus obtained by the method has high titer and significantly improved ability to target and infect cells, and can directly transduce non-preactivated or preactivated T cells.

[0109] According to embodiments of the present invention, the method provided herein may further include at least one of the following additional technical features:

[0110] According to an embodiment of the present invention, the virus is a lentivirus, the first viral vector and the second viral vector are different vectors, and the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 1:1:1:0.25 to 2:1:1:1. According to the ratio of the viral vectors of the embodiment of the present invention, both the lentivirus titer and the lentivirus infection efficiency are high.

[0111] According to an embodiment of the present invention, the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 2:1:1:0.5.

[0112] According to an embodiment of the present invention, the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 1:1:1:0.5.

[0113] According to an embodiment of the present invention, the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 1:1:1:1.

[0114] The inventors have found that when the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 2:1:1:0.5, 1:1:1:0.5, 1 or 1:1:1:1, the lentivirus titer and infection efficiency reach an optimal balance.

[0115] According to an embodiment of the present invention, the first recipient cell is 293T.

[0116] In a third aspect of the present invention, a lentivirus is provided herein. According to an embodiment of the present invention, the lentivirus is obtained by packaging as described herein. The lentivirus according to an embodiment of the present invention has high titer and has the ability to target and infect immune cells, and can directly transduce non-preactivated or preactivated T cells.

[0117] In a fourth aspect of the present invention, a lentivirus is provided herein. According to an embodiment of the present invention, the lentivirus expresses an envelope protein and a fusion protein, and the fusion protein comprises at least one single-chain antibody and a C-terminal domain of the envelope protein. The single-chain antibody can bind to CD28 or CD3, the C-terminal domain of the envelope protein comprises the transmembrane and intracellular domains of the envelope protein, and the C-terminus of the at least one single-chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein. The lentivirus according to an embodiment of the present invention has high titer and the ability to target and infect immune cells, and can directly transduce non-preactivated or preactivated T cells.

[0118] According to an embodiment of the invention, the envelope protein is the envelope G glycoprotein of vesicular stomatitis virus or a mutant of the envelope G glycoprotein.

[0119] In a fifth aspect of the present invention, a lentivirus is provided herein. According to an embodiment of the present invention, the lentivirus expresses an envelope protein and a fusion protein, the fusion protein comprising a first single-chain antibody and a second single-chain antibody, and a C-terminal domain of the envelope protein. The first single-chain antibody can bind to CD28, the second single-chain antibody can bind to CD3, and the C-terminal domain of the envelope protein comprises the transmembrane and intracellular domains of the envelope protein. The C-terminus of the first single-chain antibody is linked to the N-terminus of the second single-chain antibody, and the C-terminus of the second single-chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein; or the C-terminus of the second single-chain antibody is linked to the N-terminus of the first single-chain antibody, and the C-terminus of the first single-chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein. Lentiviruses according to embodiments of the present invention have high titers and the ability to target and infect immune cells, and can directly transduce non-preactivated or preactivated T cells.

[0120] According to an embodiment of the invention, the envelope protein is the envelope G glycoprotein of vesicular stomatitis virus or a mutant of the envelope G glycoprotein.

[0121] In the sixth aspect of the present invention, a method for introducing lentivirus into non-activated T lymphocytes is provided herein. According to an embodiment of the present invention, the lentivirus vector is used to electroporate or transfect non-activated T lymphocytes, or the lentivirus is used to infect non-activated T lymphocytes. As described above, after the viral vector is introduced into recipient cells, a virus with high viral titer can be packaged, and the single chain antibody expressed by the virus achieves specific target binding of the virus to immune cells via binding to CD28 or CD3, and then realizes infection of immune T cells with the virus; according to the method of the embodiment of the present invention, the lentivirus can be directly introduced into pre-activated T cells in vivo or ex vivo.

[0122] In a seventh aspect, a method for expressing a gene of interest is provided herein. According to an embodiment of the present invention, the method includes the steps of: introducing a viral vector or lentivirus incorporating a gene of interest into a second recipient cell; culturing the second recipient cell into which the viral vector or lentivirus has been introduced to express the gene of interest. According to an embodiment of the present invention, the expression of the gene of interest in the recipient cell is effectively achieved. For example, in an embodiment of the present invention, mCherry is a gene of interest that can be carried to verify the feasibility of the targeting vector platform, and the inventors used the mCherry gene as a tag gene for expressing a fluorescent protein, and characterized the positive transduction rate of the virus in the recipient cell.

[0123] According to embodiments of the present invention, the method provided herein may further include at least one of the following additional technical features:

[0124] According to an embodiment of the present invention, the introduction into the second recipient cell is performed by electrotransfection, transfection, or infection. It is specified that "electrotransfection" or "transfection" refers to a method of introducing a viral vector into a recipient cell, and "infection" refers to a process in which a virus actively binds and fuses with the cell membrane to enter the cell. However, "electrotransfection" refers to a method of introducing a viral packaging vector into a recipient cell by electrical stimulation, and "transfection" refers to a method of introducing a viral packaging vector into a recipient cell via a chemical mediator, such as a liposome.

[0125] According to an embodiment of the invention, the second recipient cell is an immune cell.

[0126] According to an embodiment of the invention, the second recipient cell is a T cell. Expression of a gene of interest in the T cell can achieve a direct or indirect therapeutic effect. The method according to an embodiment of the invention enhances the immune response by achieving activation of immune cells.

[0127] In the eighth aspect of the present invention, the present invention provides a method for obtaining CAR-T cells. According to an embodiment of the present invention, the method includes the steps of: introducing the viral vector or lentivirus into T lymphocytes, in which the nucleic acid encoding the chimeric antigen receptor is incorporated; and culturing the T lymphocytes into which the viral vector or lentivirus is introduced to express the chimeric antigen receptor. As described above, after the viral vector is introduced into the first recipient cell, a virus with high viral titer can be packaged, and the virus can express a single-chain antibody. Thus, the viral vector is directly introduced into T lymphocytes, or the packaged virus with high titer is introduced into T lymphocytes, and the resulting single-chain antibody binds to CD28 or CD3, thereby achieving specific targeting of the virus to T lymphocytes, and thereby achieving infection of immune T cells. According to the method of the present invention, CAR-T cells can be obtained ex vivo or in vivo from non-preactivated or preactivated T lymphocytes, and the CAR-T cells can effectively inhibit the proliferation of tumor cells.

[0128] According to an embodiment of the present invention, the introduction into T lymphocytes is performed by electrotransfection, transfection, or infection. It is specified that "electrotransfection" or "transfection" refers to a method of introducing a viral vector into a recipient cell, and "infection" refers to a process in which a virus actively binds and fuses with the cell membrane to enter the cell. However, "electrotransfection" refers to a method of introducing a viral packaging vector into a recipient cell by electrical stimulation, and "transfection" refers to a method of introducing a viral packaging vector into a recipient cell via a chemical mediator, such as a liposome.

[0129] According to an embodiment of the present invention, both "pMD2.G mutant" or "pMD2.G-Mut" refer to a plasmid expressing a lentiviral envelope protein containing a K47Q\R354Q mutation site (VSV-G-K47Q\R354Q).

[0130] In a ninth aspect of the present invention, a CAR-T cell is provided herein. According to an embodiment of the present invention, the CAR-T cell is prepared by the method described in the eighth aspect of the present invention. The CAR-T cell prepared according to an embodiment of the present invention has low cost, high cell activity, high purity, and exhibits excellent killing activity against tumor cells.

[0131] In a tenth aspect of the present invention, a pharmaceutical composition is provided herein. According to an embodiment of the present invention, the pharmaceutical composition comprises a viral vector as described in the first aspect of the present invention, a lentivirus as described in the fourth aspect of the present invention, or a CAR-T cell as described in the eighth aspect of the present invention. As mentioned above, the CAR-T cell has the advantages of high cell activity, strong cell killing ability, and excellent immune activation effect. The delivery of the CAR-T cell into the body encourages the CAR-T cell to exert its killing ability against tumor cells.

[0132] In an eleventh aspect of the present invention, the present invention provides the use of the viral vector, lentivirus, CAR-T cell, and pharmaceutical composition for activating immunity or treating or preventing disease. The present inventors have found that the drug comprising the viral vector, lentivirus, or CAR-T cell has a better effect on activating immunity, treating or preventing disease.

[0133] In a twelfth aspect of the present invention, the present invention provides the use of the viral vector, lentivirus, CAR-T cell and pharmaceutical composition for treating or preventing tumors.The present inventors have found that the drug comprising the viral vector, lentivirus or CAR-T cell has excellent efficacy in treating or preventing malignant tumors.

[0134] In a thirteenth aspect of the present invention, there is provided herein a method for activating immunity or treating or preventing a disease in a body. According to an embodiment of the present invention, the method comprises administering to a subject a pharma- ceutically acceptable amount of said viral vector, lentivirus, CAR-T cell, or pharmaceutical composition. According to an embodiment of the present invention, the viral vector, lentivirus, CAR-T cell, or pharmaceutical composition may be used to activate immunity, treat, or prevent a disease after administration to a subject in an effective amount.

[0135] According to an embodiment of the present invention, the disease is a tumor.

[0136] The above and / or further aspects and advantages of the present invention will become apparent and will be readily understood from the following detailed description of the embodiments taken in conjunction with the drawings, in which: [Brief description of the drawings]

[0137] [Figure 1] 1A and 1B are structural model diagrams of envelope fusion proteins targeting CD28, CD3, and CD3 antigens (anti-CD28 / 3-G, anti-CD3-G), respectively, according to an embodiment of the present invention, where anti-CD28scFv refers to the variable region sequence of CD28 single chain antibody, anti-CD3scFv refers to the variable region sequence of CD3 single chain antibody, and VSV-G C-terminus refers to the C-terminal domain of lentiviral envelope protein. [Diagram 2] 1 is a map of pMD2.G mutant (pMD2.G-Mut) according to an embodiment of the present invention. [Diagram 3] FIG. 1 shows the titers of lentiviruses with CD28 and CD3 targeting ability obtained by vector packaging under different conditions according to an embodiment of the present invention, where the abscissa represents the type of lentivirus, the type and ratio of vectors contained in each lentivirus are shown in Table 1, and the ordinate represents the lentivirus titer measured by lentivirus infecting HEK-293T cells. [Figure 4]FIG. 1 shows the results of detecting the positivity of HEK-293T cells infected with lentiviruses targeting CD28 and CD3 obtained by different vector packaging according to an embodiment of the present invention, where the abscissa represents the type of lentivirus, the types and ratios of vectors contained in each lentivirus are shown in Table 1, and the ordinate represents the mCherry positivity of HEK-293T cells infected with the lentivirus. [Diagram 5] FIG. 1 is an analysis diagram of the positive rate detection results of T cells and Nalm-6 cells transfected with different lentiviruses according to an embodiment of the present invention, in which the abscissa represents the type of lentivirus, the type and ratio of vectors contained in each lentivirus are shown in Table 1, and the ordinate represents the mCherry positive rate of HEK-293T cells infected with lentivirus. [Figure 6] 6A shows the results of analyzing the titer of different lentiviruses obtained by packaging according to an embodiment of the present invention, and the ability to infect 293T cells, T cells, and Nalm-6 cells. FIG. 6A shows the results of analyzing the titer of different lentiviruses, where the abscissa represents the type of lentivirus, the type and ratio of vectors contained in each lentivirus are shown in Table 2, and the ordinate represents the titer of HEK-293T cells infected with lentivirus. FIG. 6B shows the results of testing the positive rate of HEK-293T cells transduced with different lentiviruses, where the abscissa represents the type of lentivirus, the type and ratio of vectors contained in each lentivirus are shown in Table 2, and the ordinate represents the mCherry positive rate of HEK-293T cells infected with lentivirus. FIG. 6C shows the results of detecting the mCherry positivity rate of T cells and Nalm-6 cells after a mixed system of T cells and Nalm-6 cells was infected with different lentiviruses, in which the abscissa represents the type of lentivirus, the type and ratio of vectors contained in each lentivirus are shown in Table 2, and the ordinate represents the mCherry positivity rate of T cells and Nalm-6 cells infected with the lentivirus. [Figure 7]7A and 7B respectively show the CAR positive rates of Nalm-6 cells and T cells after infecting a mixed system of T cells and Nalm-6 cells or K562 cells with different lentiviruses according to an embodiment of the present invention. The abscissa represents the type of lentivirus, and the type and ratio of vectors contained in each lentivirus are shown in Table 3. The ordinate represents the CAR positive rates of T cells, K562 cells, and Nalm-6 cells infected with lentiviruses. 7A and 7B respectively show the CAR positive rates of Nalm-6 cells and T cells after infecting a mixed system of T cells and Nalm-6 cells with different lentiviruses. 7C represents the CAR positive rates of K562 cells after infecting a mixed system of T cells and K562 cells with different lentiviruses. [Figure 8] 8A shows the results of detecting the total number of live cells of T cells, K562 cells, and Nalm-6 cells after infecting a mixture of T cells and Nalm-6 cells or K562 cells with different lentiviruses according to an embodiment of the present invention. The abscissa represents the number of days after infecting a mixture of T cells and Nalm-6 cells or K562 cells with lentiviruses, and the types and ratios of vectors contained in each lentivirus are shown in Table 3, and the ordinate represents the total number of live cells. FIG. 8A shows the total number of live cells of T cells and Nalm-6 cells after infecting a mixture of T cells and Nalm-6 cells with lentiviruses. FIG. 8B shows the total number of live cells of T cells and K562 cells after infecting a mixture of T cells and K562 cells with lentiviruses. [Figure 9] 1 is a flow chart showing the operation of a validation experiment in which T cells and Nalm-6 cells are transplanted into mice according to an embodiment of the present invention. [Figure 10] FIG. 1 shows flow cytometry detection of mouse peripheral blood leukocytes after engraftment of T cells and Nalm-6 according to an embodiment of the present invention. [Figure 11] FIG. 1 is an analysis diagram of the detection results of the positive rates of T cells transfected with different lentiviruses according to an embodiment of the present invention. [Figure 12]FIG. 1 shows titer results of CAR-T prepared using lentiviral vectors according to an embodiment of the present invention. [Figure 13] FIG. 1 compares the efficiency of transducing non-activated T cells with LV-CAR-2 and LV-S2-CAR vectors according to an embodiment of the invention. [Figure 14] FIG. 1 compares the efficiency of transducing activated T cells with LV-CAR-2 and LV-S2-CAR vectors according to an embodiment of the invention. [Figure 15] FIG. 1 compares ex vivo CAR-T killing results according to an embodiment of the invention. [Figure 16] Comparison of the in vivo potency of CAR-T products prepared based on different lentiviral vector processes. [Figure 17] Comparison of transduction efficiency of CD3+ Jurkat cells by lentiviral vectors packaged with CD3-targeting fusion proteins containing different lengths of the VSV-G C-terminal domain. [Figure 18] Comparison of the titers of a lentiviral vector packaged with a fusion protein having an scFv located at the N-terminus of intact VSV-G (LV-2) and a lentiviral vector packaged with VSV-G and a fusion protein having an scFv in the C-terminal domain of VSV-G (LV-S2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0138] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the invention are described in detail below, examples of which are illustrated in the drawings.

[0139] According to a specific embodiment of the present invention, a group of novel lentiviral vectors capable of targeted transfection are provided herein, which vectors comprise a coding region carrying the envelope protein VSV-G or a VSV-G mutant, and a coding region carrying a fusion protein obtained by linking a single-chain antibody scFv and the C-terminal domain of VSV-G via a linking peptide.

[0140] The lentiviral vectors provided herein have the following features: the VSV-G mutants are mutants that attenuate the ability of VSV-G to attach to target cells but retain the ability of cell membrane fusion; the scFvs can be one or more tandem; the C-terminal domain of VSV-G includes at least the intracellular and transmembrane domains of VSV-G; the viral vectors can contain one or more fusion proteins.

[0141] The advantage of this study is that the fusion protein formed by scFv and the C-terminal domain of VSV-G has the same transmembrane and intracellular regions as VSV-G, so the fusion protein maintains the interaction with the matrix protein and efficiently assembles the matrix protein on the lentiviral particle envelope without interfering with the budding of the virus particle, without significant effect on the virus titer. The lentiviral particle envelope still contains the complete VSV-G or its mutants that can maintain the stability of the virus particle. Through the binding of scFv to the corresponding antigen, the lentiviral particle can actively infect the target cells that express the corresponding antigen, increasing the infection efficiency of the target cells under the same multiplicity of infection conditions. The scFv has a clear functional background and guaranteed safety, and any antigen that specifically binds to the scFv can be screened, making the virus packaged by the lentiviral vector universally applicable to the targeted infection of various types of cells. VSV-G is replaced with a mutant with reduced ability to adsorb target cells, so that the specific binding force between scFv and corresponding antigen governs the process of viral particle attachment, further improving the targeting of recombinant lentivirus.The lentivirus vector constructed in the present invention can directly transduce unactivated blood cells, as the CAR-T product prepared by directly transducing T cells, which facilitates the manufacturing process, reduces costs, and avoids the loss of drug potency caused by cell activation; the ex vivo killing ability is extremely strong under the condition of low effective target ratio.

[0142] According to a specific embodiment of the present invention, there is provided herein a method for constructing and using a novel lentiviral vector capable of transfecting a gene of interest, the method comprising the steps of: + One example is lentiviral vectors targeted for cell transfection).

[0143] 1. Designing a fusion protein (CD3scFv-VSV-G-CT (for short anti-CD3-G)) formed by a CD3-targeting single chain antibody and the C-terminal domain of VSV-G. The structural pattern diagram is shown in Figure 1. However, the VSV-G signal peptide is located at the N-terminus of the fusion protein precursor protein. The original VSV-G signal peptide is located at the amino terminus of the VSV-G precursor protein, is a membrane-localized telopeptide of the VSV-G protein, serves to place the VSV-G protein in the endoplasmic reticulum, and is hydrolyzed and removed after protein maturation, so the mature VSV-G protein of the virus particle and the fusion protein does not contain this signal peptide.

[0144] The signal peptide has the amino acid sequence shown below.

[0145] MKCLLYLAFLFIGVNC (SEQ ID NO:27)

[0146] The gene encoding the signal peptide has the nucleotide sequence shown below.

[0147] ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGC (SEQ ID NO: 26)

[0148] The amino acid sequence of the anti-CD3 scFv is shown in SEQ ID NO:5.

[0149] [ka]

[0150] The amino acid sequence of the second connecting peptide (linker) is shown in SEQ ID NO:12.

[0151] AAATT (SEQ ID NO: 12)

[0152] The VSV-G C-terminus (VSV-G-CT) contains amino acids 405 to 495 (a total of 91) of the VSV-G protein, and the amino acid sequence is shown in SEQ ID NO:13.

[0153] [ka]

[0154] 2. Construction of CG fusion membrane protein expression plasmid A DNA sequence was designed using the VSV-G signal peptide-scFv-linker (VSV-G-CT) pattern and commissioned to a gene synthesis company (General Biosystems (Anhui) Co., Ltd.), and the pMD2.G plasmid (VSV-G protein expression plasmid) was used as a vector to construct the fusion protein expression plasmid pMD2.anti-CD28 / 3-G, where the scFv contains CD28 and / or CD3.

[0155] 3. The process of constructing a mutant with reduced VSV-G attachment (Reference: Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein. Nature Communications. 2018) The DNA sequence of the double point mutant of K47Q and R354Q was designed by a gene synthesis company. VSV-G expression plasmid pMD2.G was used as a vector to construct VSV-G-K47Q\R354Q expression plasmid pMD2.G-Mut. The study found that the mutations of K47Q and R354Q could only reduce the membrane attachment ability of VSV-G, but did not affect the membrane fusion ability of VSV-G.

[0156] 4. Packaging and recovery of lentivirus (1) Lentiviral vector packaging plasmids were co-transfected into 293T cells.

[0157] (2) Virus-containing culture supernatants were harvested 48 to 72 h after transfection, filtered through a 0.45 μm filter, and either directly PEG-concentrated or purified and concentrated, then aliquoted and stored in an ultra-low temperature freezer.

[0158] 5. Detection and analysis of targeted transfection ability of lentiviral vectors (1) The gene of interest for transduction was mCherry. LVm-CD3ta-mCherry was titrated together with its corresponding control lentiviral vector, transduced into 293T cells, transduced into tumor cells and T cells, and then evaluated for its ability to specifically transduce T cells.

[0159] (2) The gene of interest for transduction was anti-hCD19-CAR. Following the previous step, the LVm-CD3ta vector was selected to add the gene of interest, anti-CD19-CAR, to obtain the lentiviral vector LVm-CD3ta-CAR. Together with the control vector, after titration, the mixed T cells and tumor cells were transduced at MOI=1 (simulating the coexistence of T cells and tumor cells in the body). Four, seven, and eleven days after transduction, the cell concentration, ratio of T cells to tumor cells, and CAR-positive ratio in each group were detected, respectively, and then a line graph of the change in the CAR-positive rate of each cell and the growth curve of each cell were drawn. Finally, the ability of LVm-CD3ta-CAR to target and transduce T cells to produce CAR-T, and the ability of the produced CAR-T to kill tumor cells were evaluated.

[0160] 6. Evaluating the ability of the lentiviral vector LVm-CD3ta-CAR to target and transduce T cells to produce CAR-T in mice, and the ability of the produced CAR-T to kill tumor cells Tumor cells and human T cells were mixed and injected into mice via the tail vein, and the lentiviral vector was injected into the mice via the tail vein the following day. Five weeks later, the contents of tumor cells and T cells in the peripheral blood of the mice, as well as the CAR-positive rate of each cell, were detected.

[0161] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The embodiments of the present invention are described in detail below, examples of which are illustrated in the drawings. The embodiments described below in conjunction with the drawings are exemplary and are intended to illustrate the present invention and should not be construed as limiting the present invention.

[0162] It is specified that "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

[0163] Example 1 Lentiviral vector packaging and yield assessment 1.1 Packaging a series of lentiviruses to target T cells via CD28 and CD3 to transduce the gene of interest mCherry 1.1.1 Lentiviral Packaging Information 293T cells were co-transfected and packaged with lentivirus according to the plasmids and mass ratios shown in Table 1 (the quality of pLV-mCherry and psPAX2 plasmids was consistently maintained between groups), except that the pMD2.Anti-CD28 / 3-G plasmid expresses a fusion protein targeting CD3 and CD28 (anti-CD28 / 3-G), and the pMD2.G-Mut plasmid expresses a mutant with reduced VSV-G attachment ability.

[0164] The gene encoding anti-CD28 / 3-G has the nucleotide sequence shown below (SEQ ID NO:24).

[0165] The gene encoding anti-CD28 / 3-G encodes a fusion protein having the amino acid sequence shown below (SEQ ID NO: 16).

[0166] [Table 1]

[0167] where pLV-mCherry is a transfer plasmid carrying an mCherry sequence, and the gene of interest is the mCherry sequence; psPAX2 is a plasmid expressing the lentiviral structural protein gag, and the nonstructural proteins pol and rev; pMD2.G is a plasmid expressing a lentiviral membrane protein (VSV-G); pMD2.G-Mut is a plasmid expressing a lentiviral membrane protein mutant (VSV-G-K47Q\R354Q); and pMD2.Anti-CD28 / 3-G is a plasmid expressing a fusion protein (anti-CD28 / 3-G) containing an scFv targeting CD28 and CD3 and the C-terminal domain of the VSV-G protein, where the C-terminal domain of the VSV-G protein has the amino acid sequence shown in SEQ ID NO: 13. A map of the pMD2.G mutant used in this example is shown in FIG. 2.

[0168] 1.1.2 Assessment of lentiviral vector yield 1) Lentivirus packaging, recovery, titer determination, and T cell transfection Lentiviral vectors obtained according to Table 1 were concentrated with PEG, aliquoted, frozen in a deep freezer (<-75°C) and titered using 293T cells.

[0169] As a positive control, 293T cells were transduced at MOI=0.05 according to the experimental groups in Table 1, viral vehicle was used as a negative control, and Nalm-6 (CD28 - CD3 - ) and T cells (CD28 + CD3 + ) and 72 h post-transfection, flow cytometry was used to detect the percentage of mCherry+ cells in the transduced cells.

[0170] 2) Results and Analysis According to the results shown in FIG. 3, there is no significant difference in the titers of the four lentiviruses. The results in FIG. 4 show that at MOI=0.05, the transduction positivity rates of the four vectors in 293T cells were similar, indicating that all four vectors have the ability to transduce cells. The results in FIG. 5 show that in Nalm-6 cells and T cells, both LV and LV-CD28CD3ta have high transduction positivity rates and no transduction targeting, but the latter has a higher transduction ability for T cells than the former. LVm cannot transduce Nalm-6 cells and can only transduce a small amount of T cells. LVm-CD28CD3ta cannot transduce Nalm-6 cells but can transduce T cells, and its transduction ability is equivalent to that of LV, indicating that LVm-CD28CD3ta can target and transduce T cells.

[0171] Example 2 CD3 + Evaluation of targeting efficiency of lentiviral vectors targeting T cells 2.1 Packaging a series of lentiviruses to transduce the gene of interest, mCherry, through CD3-targeted T cells To co-transfect HEK 293T cells to package lentivirus, follow the plasmids and ratios in Table 2.

[0172] [Table 2]

[0173] where pLV-mCherry is a transfer plasmid carrying an mCherry sequence, and the gene of interest is the mCherry sequence; psPAX2 is a plasmid expressing the lentiviral structural protein gag, and the nonstructural proteins pol and rev; pMD2.G is a plasmid expressing a lentiviral membrane protein (VSV-G); pMD2.G-Mut is a plasmid expressing a lentiviral membrane protein mutant (VSV-G-K47Q\R354Q); and pMD2.anti-CD3-G is a plasmid expressing a fusion protein (anti-CD3-G) containing an scFv targeting CD3 and the C-terminal domain of the VSV-G protein, with the proviso that the C-terminal domain of the VSV-G protein has the amino acid sequence shown in SEQ ID NO: 13. A map of the pMD2.G mutant used in this example is shown in FIG. 2.

[0174] The gene encoding anti-CD3-G has the nucleotide sequence shown below (SEQ ID NO:23).

[0175] The gene encoding anti-CD3-G has the amino acid sequence shown below (SEQ ID NO:15).

[0176] 2.2 CD3 + Targeting evaluation of lentiviral vectors targeting T cells 1) Lentivirus packaging, recovery, titer determination, and target cell transfection 293T cells were co-transfected according to the experimental groups in Table 2, and viruses were harvested from each group 48-72 hours after transfection, then aliquoted and frozen in a deep freezer (<-75°C). Titer determination was performed using 293T cells.

[0177] The two lentiviral vectors were used to transduce 293T cells at MOI=0.1 as a positive control, the viral vehicle was used as a negative control, and Nalm-6 (CD3 - ) and T cells (CD3 +) were transduced at MOI=2, 10, and 50, respectively, for 3-4 days, and then the percentage of mCherry+ cells in the infected cells was detected by flow cytometry.

[0178] 2) Results and Analysis Specific experimental results are shown in Figure 6. 6A shows that there is no significant difference in the titers of the two lentiviruses. Figure 6B shows that at MOI=0.1, the transduction positive rates of the two vectors in 293T cells are similar, indicating that both vectors have the ability to transduce cells. Figure 6C shows that LV can transduce both Nalm-6 and T cells, but LVm-CD3ta alone cannot transduce Nalm-6 cells but can transduce T cells, and its transduction ability is equivalent to that of LV, indicating that LVm-CD3ta can target transduce T cells.

[0179] Example 3 CD3 + Detection of transduction efficiency and tumor-killing effect of lentiviral vectors targeting T cells 3.1 Packaging a series of lentiviruses to transduce the gene of interest anti-hCD19 scFv-CTM4O (CAR) through CD3-targeted T cells To co-transfect HEK 293T cells to package lentivirus, follow the plasmids and ratios in Table 3.

[0180] [Table 3]

[0181] where pLV-CAR is a transfer plasmid carrying the CAR sequence, the gene of interest is anti-hCD19 scFv-CTM4O (CAR), pMDLg-pRRE is a plasmid expressing the lentiviral structural protein gag and the nonstructural protein pol, pRSV-rev is a plasmid expressing the regulatory protein rev, and pMD2.G is a plasmid expressing the lentiviral envelope protein (VSV- pMD2.G-Mut is a plasmid expressing a lentiviral membrane protein mutant (VSV-G-K47Q\R354Q); pMD2.anti-CD3-G is a plasmid expressing a fusion protein containing an scFv targeting CD3 and the C-terminal domain of the VSV-G protein (anti-CD3-G, the nucleotide sequence is shown in SEQ ID NO:23 and the amino acid sequence is shown in SEQ ID NO:15), whereby the C-terminal domain of the VSV-G protein has the amino acid sequence shown in SEQ ID NO:13; a map of the pMD2.G mutant used in this example is shown in FIG. 1.

[0182] The gene encoding anti-hCD19 scFv-CTM4O (CAR) has the nucleotide sequence shown below (the bolded portion is the nucleotide sequence encoding the anti-hCD19 scFv).

[0183] [ka]

[0184] Anti-hCD19 scFv-CTM4O (CAR) has the amino acid sequence shown below (the bold part is the sequence of anti-hCD19 scFv).

[0185] [ka]

[0186] 3.2 Lentiviral vector transduction T cells were co-cultured with tumor cells Nalm-6 or K562 (cell number, T cells:tumor cells = 1:2) and transduced with lentiviral vectors (MOI = 1), and an equal volume of vehicle was used as a negative control. On days 4, 7, and 11 after transduction, the cell number of each group was measured to draw a growth curve, and the CAR-positive rate of each cell was measured to evaluate the transduction function of the vector targeting T cells. Tumor cells Nalm-6 express CD19, which can be effectively killed by anti-CD19 CAR-T. + K562 is a CD19 cell that promotes CAR-T cell proliferation and cannot be killed by anti-CD19 CAR-T. - Control cells, which cannot promote CAR-T cell proliferation. The number of cells used to draw cell proliferation curves was calculated as follows:

[0187] Cell number = cell density x volume x cell ratio corresponding to flow cytometry

[0188] 3.3 Results analysis Figure 7 presents the aggregated data of the positivity across different cell populations. LV-CAR shows robust transduction potency for T cells and tumor cell lines Nalm-6 or K562, while it is lacking in terms of targeted transduction specificity. On the other hand, LVm-CD3ta excels in precisely targeting T cells for transduction, with a CAR-T positivity rate of about 10% by day 11. Before day 7, CAR-T cells continue to eliminate tumor cells. Although the CAR-T cells can specifically proliferate, they also undergo significant cell death. Thus, no CAR positive signal can be detected on days 4 and 7. After day 7, when all tumor cells are eradicated, the CAR-T cells persist in specific proliferation. This leads to an increase in the positivity rate, which reaches approximately 10% by day 11.

[0189] The proliferation curves of each cell are shown in Figure 8. Under co-culture conditions of T cells and K562 (shown in Figure 8B), transduction of lentiviral vectors does not affect the proliferation rate of K562 and T cells. Under co-culture conditions of T cells and Nalm-6 (shown in Figure 8A), the proliferation rate of Nalm-6 in the LVm-CD3ta-CAR transduction group was the slowest until day 4, and the number of Nalm-6 cells began to decrease rapidly after day 4, demonstrating the strongest tumor killing effect for the first time. The number of Nalm-6 cells in the LV transduction group was still increasing until day 7, but the proliferation rate was greater than that of the LVm-CD3ta group and smaller than that of the NC group, indicating that although the proliferation of tumor cells was inhibited, the effect was not as strong as that of LVm-CD3ta. The number of Nalm-6 cells in the LV-transduced group began to rapidly decrease toward 0 after day 7, whereas the number of Nalm-6 cells in the NC group also began to rapidly decrease during this period, indicating that most of the decrease in tumor cell number during this period was due to the effect of non-CAR-T-dependent tumor killing.

[0190] In summary, under co-culture conditions of T cells and tumor cells, which to some extent mimic the conditions in which T cells and tumor cells coexist in animals, LVm-CD3ta can achieve targeted transduction of T cells to generate CAR-T cells, which efficiently and specifically kill tumor cells.

[0191] Example 4 Evaluating the tumor-killing function of LVm-CD3ta-CAR in mouse models of hematological malignancies 4.1 Functional evaluation of lentiviral vector LVm-CD3ta-CAR in the NCG mouse Nalm-6 hematoma model Functional evaluation was performed based on the lentiviral vector LVm-CD3ta-CAR obtained in Example 3. The specific experimental steps are as follows:

[0192] 1) After the NCG mice had completed the isolation period, a vaccination plan was planned, and the detailed experimental method is shown in Figure 9. The specific implementation method for T cells and tumor cells is as follows.

[0193] Nalm-6 cells were subjected to cell counting and viability testing. If the viability was greater than 95%, the cells were ready for seeding, and the culture medium was removed by centrifugation and the cell density was adjusted to 2.0E+06 cells / mL by adding PBS. The T cells frozen in liquid nitrogen were resuscitated, followed by cell counting and viability testing. If the viability was greater than 80%, the cells were ready for injection, and the cell density was adjusted to 1.0E+07 cells / mL by adding PBS, and the cell density adjusted Nalm-6 and T cell suspensions were mixed uniformly in a 1:1 volume and 200 μL / mouse were injected via the tail vein of the mice. The injection time point was recorded as day -1.

[0194] 2) Lentiviral vector administration: Mice engrafted with cells the day before were randomly divided into three groups. LV-CAR and LVm-CD3ta-CAR were each diluted to 6.0E+07TU / mL in lentiviral vector vehicle. Mice were administered via the tail vein, with the NC group receiving 200μL / mouse of vehicle, the LV-CAR group receiving 200μL / mouse of LV-CAR dilution, and the LVm-CD3ta-CAR group receiving 200μL / mouse of LVm-CD3ta-CAR dilution, and the injection time was recorded as day 0.

[0195] 3) Detection of tumor cells and CAR-T cells in the peripheral blood of mice On day 35, blood was collected from the orbit of each group of mice, and the proportion of erythroblast lysed red blood cells was detected using cell death dye-antibody incubation-flow cytometry.

[0196] 4.2 Results analysis The experimental results are shown in Figure 10. The Q1 gate is Nalm-6 tumor cells, hCD19 + hCD3 - The Q1 gate contained human T cells, the Q2 gate contained unrelated cells, and the Q3 gate contained engrafted human T cells, hCD19 - hCD3 +The Q1 gate contains hCD19 cells, and the Q2 gate contains hCD19 cells, which are other white blood cells in mouse blood. - hCD3 - Contains cells.

[0197] In the NC group, 74.7% of the leukocytes in the peripheral blood of the mice were Nalm-6 cells and 3.3% were T cells, and it was found that Nalm-6 proliferated rapidly and had considerable tumorigenicity.

[0198] In the LV-CAR group, 71.1% of the white blood cells in the peripheral blood of the mice were Nalm-6 cells and 2.38% were T cells, which was not significantly different from the NC group. CAR-positive cells were detected in all cell groups, indicating that LV-CAR nonspecifically transduced mouse cells and that the generated CAR-T cells could not effectively kill tumor cells.

[0199] In the LVm-CD3ta-CAR group, no Nalm-6 cells were detected in the peripheral blood leukocytes of the mice, and 16.9% were T cells. CAR-positive cells were only detected in T cells, indicating that LVm-CD3ta-CAR can target the transduction of T cells in mice, and the generated CAR-T cells can efficiently kill Nalm-6 tumor cells in this process and promote the specific proliferation of CAR-T cells.

[0200] In summary, in the Nalm-6 hematological malignancy NCG mouse model, intravenous administration of LVm-CD3ta-CAR can achieve targeted transduction of T cells to generate CAR-T cells with normal function, reflecting antitumor potency.

[0201] Example 5 Construction of a series of mCherry-loaded lentiviral vectors for transducing T cells 5.1 Lentiviral Packaging Information The plasmids and mass ratios shown in Table 4 of the present invention were used to co-transfect 293T cells to package lentivirus.

[0202] [Table 4]

[0203] Among them, pLV-mCherry is a transfer plasmid carrying the mCherry sequence, pMDLg-pRRE is a packaging plasmid expressing the lentiviral structural protein gag and the nonstructural protein pol, pRSV-rev is a regulatory plasmid expressing the regulatory protein rev, pMD2.G is an envelope plasmid expressing the envelope protein VSV-G, and pMD2.S2 / pMD2.S12 / pMD2.S3 / pMD2.S4 / pMD2.S34 are plasmids targeting CD3, CD28, or CD3 and CD28, respectively. and fusion proteins S2 / S12 / S3 / S4 / S34 containing the scFv of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0204] 5.2 Recovery and storage of lentiviral vectors Virus-containing culture supernatants were collected 48-72 h after transfection, then filtered through a 0.45 μm filter, concentrated with PEG, aliquoted, and stored in a deep freezer (<-75°C).

[0205] 5.3 Evaluation of T cells transfected with lentiviral vectors The lentiviral vectors were used to transduce T cells, respectively. Seven days after transduction, the positive rate of mCherry expression in each group, i.e., the positive rate of transduction, was detected by flow cytometry.

[0206] 5.4 Results and Analysis The results are shown in FIG. 11, where NC is a negative control group that used a vehicle without lentiviral vector during transduction.

[0207] The results in Figure 11 show that a series of fusion proteins containing scFvs that specifically bind to T cell surface antigens (CD3\CD28), which are designed to improve the transduction efficiency of T cells by lentiviral vectors carrying the mCherry gene, are packaged as membrane proteins on the surface of lentiviral vectors. However, the transduction positivity rates of LV-S2-mCherry, LV-S12-mCherry, LV-S3-mCherry, LV-S4-mCherry, and LV-S34-mCherry are significantly increased, more than three times that of LV-mCherry.

[0208] Example 6 Construction of lentiviral vector carrying anti-CD19-CAR (referred to as CAR) for transduction of T cells 6.1 Based on the results from Example 5, the lentiviral vectors that demonstrated the most substantial increase in transduction positivity rate were selected for further use in carrying the CAR gene to evaluate transduction efficiency. The plasmids and their corresponding mass ratios detailed in Table 5 were utilized to co-transfect 293T cells to package the indicated lentiviral vectors.

[0209] [Table 5]

[0210] However, pLV-CAR2 is a transfer plasmid carrying the CAR sequence and carries the gene of interest, anti-hCD19 scFv-CAR.

[0211] The gene encoding anti-hCD19scFv-CAR2 has the nucleotide sequence shown in SEQ ID NO:30.

[0212] [ka]

[0213] Anti-hCD19 scFv-CAR2 has the amino acid sequence shown in SEQ ID NO:31.

[0214] [ka]

[0215] 6.2 Lentiviral Vector Recovery, Purification, and Storage 48-72 h after transfection, the virus-containing culture supernatant was collected and then filtered using a 0.45 μm filter; a portion of the sample was used as a stock solution sample for titer determination, and the remaining sample was purified, concentrated, and then aliquoted and stored in an ultra-low temperature freezer (<-75°C).

[0216] 6.3 Lentiviral vector titration a. 293T cells are seeded into 24-well plates at appropriate confluence and cultured overnight to allow cells to adhere.

[0217] b. Samples were taken and the virus samples were diluted using a two-fold gradient dilution method, and 8 μg / mL polybrene was added to the diluted virus solution.

[0218] The culture medium was removed from the 293T culture plate, and the cells in any two wells were counted after trypsinization to calculate the average cell number in each well (A). The initial culture medium in the remaining wells was discarded, and then 0.4 mL of virus dilution was added, and the mixture was centrifuged at 1000×g for 40 min at room temperature, then incubated at 37° C., 5% CO2 for 16 h, and then the culture medium was replaced with fresh DMEM containing 8% FBS.

[0219] d. After 72 h of culture, the positive rate of CAR expression was detected by flow cytometry (B).

[0220] e. Titer calculation: The values ​​of wells with a positive rate of CAR expression between 2% and 20% were selected for calculation: Titer (PFU / mL) = BxA / (0.4 / corresponding dilution).

[0221] 6.4 Evaluation of positivity rate after transduction of T cells transfected with lentiviral vectors The lentiviral vectors were used to transduce non-activated or activated T cells, respectively, at an MOI of 2. Two, four, and seven days after transduction, the positive rate of CAR expression in cells in each group, i.e., the positive transduction rate, was detected by flow cytometry.

[0222] 6.5 Comparison of killing ability of CAR-T cells transduced with different lentiviral vectors a. Cells were seeded as shown in Table 6 and cultured statically at 37° C. and 5% CO2.

[0223] [Table 6]

[0224] Note: K562 tumor cells in Table 6 are plated as above; Target: target cells; E, effector cells, CAR-T cells; T, target cells, Nalm6 or K562 cells; "Target+lysis" and "Target+NC" plate only target cells; T(NegCtrl): non-transduced T cells.

[0225] b. After incubation for 18-20 h, 20 μl of lysis buffer was added to each well of the [target+lysis] group, and the mixture was mixed thoroughly and lysed in a cell incubator at 37° C. for 30 min.

[0226] c. The whole culture medium was gently transferred to a new round-bottom 96-well plate and centrifuged at 400×g for 5 min at room temperature. For LDH cell killing detection, 100 μL of supernatant was taken from each well into a flat-bottom 96-well plate and a cytotoxic LDH assay kit (DOJINDO MOLECULAR, CK12) was used.

[0227] d. 100 μL of standard solution was added to each well, mixed thoroughly, and incubated at 37° C. for 30-60 min. Then, 50 μL of stop solution was added to each well, mixed thoroughly, and absorbance values ​​were measured at 490 nm.

[0228] e. Data processing: Positive control (PS) = (target + lysis) - (target + NC) Experimental group extinction (EFF) = (E / T=n) - (T only, E / T=n) Cytotoxicity=100%x(EFF) / (PS)

[0229] 6.6 Results and Analysis 1) CAR-T titer detection The comparison results of the stock solution titers are shown in Figure 12. From the results in Figure 12, it can be seen that there is no significant difference between the production titers of the LV-CAR2 vector and the LV-S2-CAR2 vector.

[0230] 2) Transduction efficiency of CAR-encoding lentiviral vectors into unactivated T cells LV-CAR2 and LV-S2-CAR2 lentiviruses were used at MOI=2 to transduce non-activated T cells, and NC was a negative control group (negative control) that added vehicle without lentivirus vector during transduction. At 2 days (day 2), 4 days (day 4), and 7 days (day 7) after transduction, the positive rate of CAR expression in each group was detected by flow cytometry. The comparison of the positive rate of CAR expression in each group is shown in Figure 13.

[0231] FIG. 13 illustrates a comparative analysis of transduction efficiency between non-activated T cells using LV-CAR2 and LV-S2-CAR2 vectors. Initially, the LV-CAR2 vector demonstrates substantial presence of CAR-positive cells, but the majority show weak CAR signals. As the culture time progresses, the proportion of CAR-positive cells declines significantly (day 2: 58.5%, day 4: 38.7%, day 7: 9.73%), suggesting that the weak CAR signal is not derived from stable integration of the gene into the host cell genome for sustained expression. Thus, the use of the positivity rate on day 7 provides a more accurate assessment of the transduction potency of LV-CAR2 for non-activated T cells. When transduced with the LV-S2-CAR2 vector, the percentage of CAR positivity increases and stabilizes after day 4 (day 2: 8.33%, day 4: 42.8%, day 7: 41.6%). This pattern may be due to delayed kinetics of reverse transcription and integration processes with suppressed protein expression in non-activated T cells. Therefore, CAR expression levels on day 2 may be insufficient for effective detection. The CAR-positive cell rate on day 7 of LV-S2-CAR2 was 4.28-fold higher than that of LV-CAR2.

[0232] The above results clearly demonstrate that conventional lentiviral vectors, such as LV, exhibit significantly lower efficacy in delivering the CAR gene to non-activated T cells, whereas the LV-S2 lentiviral vector exhibits a significant ability to transduce non-activated T cells with high efficiency, exhibiting stable and sustained expression of the CAR molecule.

[0233] 3) Evaluation of CAR transduction efficiency in activated T cells After 3 days of activation with CD3 / CD28 magnetic beads, both LV-CAR2 and LV-S2-CAR2 vectors were introduced into activated T cells at MOI 2. A negative control (NC) group was established at the same time, characterized by the addition of vehicle alone, lacking any lentiviral vector, during the transduction step. The expression positivity of chimeric antigen receptor (CAR) in each experimental cohort was then quantitatively evaluated by flow cytometry at different time intervals after transduction, namely, 2 days (day 2), 4 days (day 4), and 7 days (day 7). A comparative analysis of the CAR expression positivity across all groups is illustrated in FIG. 14.

[0234] Figure 14 shows a comparative analysis depicting the transduction efficiency of LV-CAR2 and LV-S2-CAR2 vectors in activated T cells. Both vectors can effectively transduce activated T cells, leading to the generation of CAR-T cells characterized by stable CAR expression. However, the transduction potency of the LV-S2 lentiviral vector is significantly superior to that of conventional lentiviral vectors (LV), approximately two-fold.

[0235] 4) Detection of in vitro CAR-T killing activity A lactate dehydrogenase (LDH) assay was utilized to assess the cytotoxic potential of CAR-T cells harvested on day 4. Effector cells were quantified according to the proportion of CAR-positive cells within each CAR-T cell population. Target cells included Nalm6 cells, which stand out for their CD19 positivity. Conversely, K562 cells, which lack CD19 expression, served as a negative control for target cells. The CAR-T cell negative control group was referred to as the T cell group. The resulting data are shown in Figure 15.

[0236] As evidenced by the data in Figure 15, a comparison of CAR-T cell in vitro cytotoxicity reveals that at comparable effector-to-target ratios, CAR-T cells generated using the LV-S2-CAR2 vector exhibit superior killing ability compared to CAR-T cells produced using the LV-CAR2 vector. This observation suggests that in addition to increasing the transduction positivity rate, the LV-S2 vector exerts an additional, as yet unknown, effect of increasing the tumor-killing potential of CAR-T cells promoted by the LV-S2 vector. Particularly noteworthy is the enhanced tumor-killing potency of CAR-T cells derived from unactivated T cells transduced with LV-S2-CAR2 at a low effector-to-target ratio (0.1). Considering that in clinical settings, CAR-T cells and tumor cells frequently encounter each other at similarly low ratios, this finding suggests that CAR-T cells generated from non-activated T cells using the LV-S2-CAR2 vector may exhibit superior clinical outcomes versus conventional methods (i.e., CAR-T cells generated from activated T cells using the LV vector).

[0237] Example 7 Comparative potency analysis of CAR-T cell products derived from various lentiviral vectors in mouse models a. Preparation of CAR-T samples by conventional protocol: Upon thawing of cryopreserved T cells, activation was initiated using magnetic beads conjugated with CD3 / CD28 antibodies. After activation, typically 72 hours later, the beads were carefully removed. The transduction enhancer Lentiboost was then incorporated before the LV-CAR2 lentiviral vector was introduced to transduce the now activated T cells. Approximately 2 to 7 days after transduction, CAR-T cells were cryopreserved. The percentage of CAR-positive cells was quantified at various time points throughout this time series.

[0238] b. Novel method 1 for CAR-T sample preparation (LV-S2-CAR2): Upon recovery of cryopreserved T cells, the T cells were cultured for 48 hours under standard conditions. The transduction enhancer Lentiboost was then introduced and the LV-S2-CAR2 lentiviral vector was administered to transduce the T cells in a non-activated state. Following an interval of approximately 24 hours after transduction, the CAR-T cells were appropriately cryopreserved to ensure optimal preservation of cell health and function.

[0239] c. New protocol 2 for CAR-T sample preparation (LV-S2-CAR2): After recovery of cryopreserved T cells, the T cells were subjected to a 48-hour culture period for adaptation and recovery. The transduction enhancer Lentiboost was then introduced and the LV-S2-CAR2 lentiviral vector was administered to transduce the T cells in a non-activated state. Approximately 24 hours after transduction, the resulting CAR-T cells were cryopreserved.

[0240] d. After resuscitating the cryopreserved CAR-T cells prepared by novel method 1 and novel method 2, the CAR-T cells were cultured for 3 to 7 days, and the CAR positivity rate was evaluated for each.

[0241] e. To establish the tumor model, 1E+06 Nalm6-luciferase cells per mouse were intravenously injected into NCG female mice. After 5-7 days, the tumor burden was detected by in vivo imaging, and based on the tumor burden, the mice were divided into different groups and administered drugs on the same day.

[0242] f. To align CAR-T positive cell rates across samples, samples prepared in a, b, and c were recovered, suspended in PBS, and diluted with untransduced T cells. The experiment consisted of the following groups: G1 used PBS only, G2 included untransduced T cells, G3 reflected the conventional method (LV-CAR2) from sample a, and G4 and G5 represented the two new methods (LV-S2-CAR2) from samples b and c, respectively. G6 also served as a blank control without tumor cell engraftment. To ensure consistency, groups G2 to G5 had equal T cell numbers, and G3 to G5 had the same number of CAR positive cells (1E+06 cells / mouse). This setup allowed a fair evaluation of the different CAR-T cell generation technologies.

[0243] g. To detect the tumor burden in mice in each group, in vivo imaging was performed 5, 8, 12, and 15 days after administration, and tumor burden curves were drawn. The results are shown in Figure 16.

[0244] According to the findings shown in Figure 16, a comparison of the potency of CAR-T products prepared by different lentiviral vector processes in mice reveals that under uniform experimental conditions, the potency of CAR-T samples generated by the two novel methods utilizing the targeted LV-S2-CAR2 lentiviral vector is significantly higher than that of samples prepared by conventional lentiviral methods known from the prior art. This suggests that targeted lentiviral vectors, such as LV-S2-CAR2, provide unexpected utility in the preparation of CAR-T products.

[0245] Example 8 Comparative analysis of the functionality of lentiviral vectors encapsulating CD3-targeting fusion proteins incorporating different lengths of the VSV-G CT domain a. A series of lentiviruses encoding mCherry were packaged and used to transduce T cells via CD3 targeting.

[0246] Plasmids were co-transfected into HEK-293T cells to package lentivirus according to Table 7.

[0247] [Table 7]

[0248] In Table 7, pLV-mCherry refers to the transfer plasmid containing the mCherry sequence, psPAX2 represents the packaging plasmid expressing the lentivirus structural protein gag, the nonstructural protein pol, and used as the plasmid for the regulatory protein rev, pMD2.G-Mut represents the envelope plasmid expressing the mutant envelope protein VSV-G (VSV-G-K47Q\R354Q), and pMD2.anti-CD3-G-1-11 are plasmids encoding the fusion proteins anti-CD3-G-1-11 containing the scFv targeting CD3 and the C-terminal domain of the VSV-G protein of different lengths, where anti-CD3 The scFv has the amino acid sequence shown in SEQ ID NO:5, and the C-terminal domain of the VSV-G protein embedded in the fusion proteins anti-CD3-G-1 to -11 corresponds to the amino acid sequence ranging from positions 455 to 495, 445 to 495, 435 to 495, 425 to 495, 415 to 495, 405 to 495, 395 to 495, 385 to 495, 375 to 495, 365 to 495, and 355 to 495, respectively.

[0249] The C-terminal domain of the VSV-G protein contains amino acids 455 to 495 (a total of 41) of the VSV-G protein, and the amino acid sequence is shown in SEQ ID NO:36.

[0250] IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (sequence number 36).

[0251] The C-terminal domain of the VSV-G protein contains amino acids 445 to 495 (a total of 51) of the VSV-G protein, and the amino acid sequence is shown in SEQ ID NO:37.

[0252] WKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (sequence number 37).

[0253] The C-terminal domain of the VSV-G protein contains amino acids 435 to 495 (a total of 61) of the VSV-G protein, and the amino acid sequence is shown in SEQ ID NO:38.

[0254] [ka]

[0255] The C-terminal domain of the VSV-G protein contains amino acids 425 to 495 (a total of 71) of the VSV-G protein, and the amino acid sequence is shown in SEQ ID NO:39.

[0256] [ka]

[0257] The C-terminal domain of the VSV-G protein contains amino acids 415 to 495 of the VSV-G protein (a total of 81 amino acids), and the amino acid sequence is shown in SEQ ID NO:40.

[0258] [ka]

[0259] The C-terminal domain of the VSV-G protein contains amino acids 405 to 495 of the VSV-G protein (a total of 91 amino acids), and the amino acid sequence is shown in SEQ ID NO:13.

[0260] [ka]

[0261] The C-terminal domain of the VSV-G protein contains amino acids 395 to 495 of the VSV-G protein (a total of 101 amino acids), and the amino acid sequence is shown in SEQ ID NO:41.

[0262] [ka]

[0263] The C-terminal domain of the VSV-G protein contains amino acids 385 to 495 of the VSV-G protein (a total of 111 amino acids), and the amino acid sequence is shown in SEQ ID NO:42.

[0264] [ka]

[0265] The C-terminal domain of the VSV-G protein contains amino acids 375 to 495 of the VSV-G protein (a total of 121 amino acids), and the amino acid sequence is shown in SEQ ID NO:43.

[0266] [ka]

[0267] The C-terminal domain of the VSV-G protein contains amino acids 365 to 495 of the VSV-G protein (a total of 131 amino acids), and the amino acid sequence is shown in SEQ ID NO:44.

[0268] [ka]

[0269] The C-terminal domain of the VSV-G protein contains amino acids 355 to 495 of the VSV-G protein (a total of 141 amino acids), and the amino acid sequence is shown in SEQ ID NO:45.

[0270] [ka]

[0271] The amino acid sequence of the fusion protein anti-CD3-G-1 is shown in SEQ ID NO:46.

[0272] [ka]

[0273] The amino acid sequence of the fusion protein anti-CD3-G-2 is shown in SEQ ID NO:47.

[0274] [ka]

[0275] The amino acid sequence of the fusion protein anti-CD3-G-3 is shown in SEQ ID NO:48.

[0276] [ka]

[0277] The amino acid sequence of the fusion protein anti-CD3-G-4 is shown in SEQ ID NO:49.

[0278] [ka]

[0279] The amino acid sequence of the fusion protein anti-CD3-G-5 is shown in SEQ ID NO:50.

[0280] [ka]

[0281] However, anti-CD3-G-6 is the fusion protein S2 and contains the amino acid sequence shown in SEQ ID NO:15.

[0282] The amino acid sequence of the fusion protein anti-CD3-G-7 is shown in SEQ ID NO:51.

[0283] [ka]

[0284] The amino acid sequence of the fusion protein anti-CD3-G-8 is shown in SEQ ID NO:52.

[0285] [ka]

[0286] The amino acid sequence of the fusion protein anti-CD3-G-9 is shown in SEQ ID NO:53.

[0287] [ka]

[0288] The amino acid sequence of the fusion protein anti-CD3-G-10 is shown in SEQ ID NO:54.

[0289] [ka]

[0290] The amino acid sequence of the fusion protein anti-CD3-G-11 is shown in SEQ ID NO:55.

[0291] [ka]

[0292] b. Lentiviral vector recovery, purification, and storage 48-72 hours after transfection, the culture supernatants, enriched in viral particles, were harvested. They were subsequently filtered through a 0.45 μm filter to remove cellular debris. The filtrate was then purified and concentrated. Finally, the processed viral preparations were aliquoted and stored in a deep freezer (<-75°C) for future use.

[0293] c. Lentivirus titer determination After removing and thawing the frozen samples, the titer is measured, and the titer measurement and calculation refer to step 6.3 of Example 6.

[0294] d. All vectors were used to transduce CD3+ Jurkat cells at a multiplicity of infection (MOI) of 5. After a period of 3-7 days, flow cytometry was used to determine the percentage of mCherry expressing cells. The percentage of positive cells in each experimental group was normalized by dividing it by the percentage of positive cells in the LVm-CD3ta-6 group. A histogram representing this relative percentage was constructed and is illustrated in FIG. 17.

[0295] Based on the findings presented in FIG. 17, lentiviral vectors packaged with CD3-targeting fusion proteins incorporating different lengths of VSV-G C-terminal domain were used to evaluate the transduction efficiency of CD3+ Jurkat cells. Specifically, vectors LVm-CD3ta-4, LVm-CD3ta-5, LVm-CD3ta-6, and LVm-CD3ta-7 showed the highest transduction efficiency. In contrast, the transduction efficiency of other lentiviral vectors was significantly lower. This observation indicates that the optimal length of the VSV-G C-terminal domain in the fusion protein is when the VSV-G C-terminal domain spans between amino acids 386 and 434 of the envelope protein, as well as up to amino acid 495. In other words, the ideal length of the VSV-G C-terminal domain is longer than 61 amino acids, but shorter than 111 amino acids. Under this condition, the lentiviral vector achieves the highest transduction efficiency.

[0296] Example 9 Assessment of titer variation among various CD3-targeted lentiviral vectors a. A series of lentiviruses encoding mCherry were packaged and used to transduce T cells via CD3 targeting.

[0297] Plasmids were co-transfected into HEK-293T cells to package lentivirus according to Table 8.

[0298] [Table 8]

[0299] pLV-mCherry denotes a transfer plasmid containing the mCherry sequence, psPAX2 denotes a packaging plasmid expressing the lentiviral structural protein gag, the nonstructural protein pol, and used as a plasmid for the regulatory protein rev, pMD2.S2 is a plasmid expressing a fusion protein comprising a single chain variable fragment (scFv) targeting CD3 and the C-terminal domain of the VSV-G protein. In particular, the C-terminal domain of the VSV-G protein is characterized by the amino acid sequence shown in SEQ ID NO: 13, and the fusion protein itself contains the amino acid sequence shown in SEQ ID NO: 15, and pMD2.VSV-G-anti-CD3 is a plasmid expressing anti-CD3 scFv-VSV-G, a fusion protein incorporating a CD3-targeting scFv at the N-terminus of the full-length VSV-G protein, the fusion protein anti-CD3 scFv-VSV-G having the amino acid sequence shown in SEQ ID NO: 56.

[0300] [ka]

[0301] b. Lentivirus packaging and titer determination For lentivirus packaging methods, see Example 1. 48-72 hours after transfection, the culture supernatant loaded with viral particles was harvested. It was then passed through a 0.45 μm filter to remove any cell debris. The viral titer was then determined and calculated according to the procedure outlined in step 6.3 of Example 6.

[0302] The titer results are illustrated in Figure 18. As the findings in Figure 18 show, there is a significant enhancement in the titer of the lentiviral vector (LV-S2) compared to the lentiviral vector (LV-2) packaged using a fusion protein in which the scFv is located at the N-terminus of the complete VSV-G protein. This improvement was observed in LV-S2 packaged with VSV-G combined with a fusion protein containing the scFv within the C-terminal domain of the VSV-G protein. Specifically, the titer of LV-2 is only 7.8E+03 TU / mL, whereas the titer of LV-S2 increases significantly to 5.8E+07 TU / mL.

[0303] Moreover, the use of terms such as "first" and "second" merely serves a descriptive role and should not be misconstrued as implying or suggesting any preference or quantity in terms of importance with respect to the technical features referred to. Thus, an element designated by "first" or "second" may inherently include one or more instances of those features. The term "plurality" within the context of describing the present invention is utilized to mean a minimum of two, unless expressly stated otherwise, and may represent two, three, or any higher number.

[0304] Throughout this specification, reference to "an embodiment," "some embodiments," "one embodiment," "another example," "one example," "particular example," or "some examples" indicates that a particular feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of the disclosure. Thus, the presence of such expressions throughout this specification does not necessarily refer to the same embodiment or example of the disclosure. Furthermore, as will be apparent to one of ordinary skill in the art, while particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples, various embodiments and examples may be presented separately, and it is within the ability of one of ordinary skill in the art to combine elements from different embodiments or examples if there is no contradiction or inconsistency in doing so.

[0305] Although exemplary embodiments have been shown and described, those skilled in the art will recognize that the above embodiments are not to be construed as limiting the present disclosure, and that changes, substitutions, and alterations may be made in the embodiments without departing from the spirit, principles, and scope of the present disclosure.

Claims

1. a first viral vector carrying a first nucleic acid molecule encoding an envelope protein; at least one second viral vector carrying a second nucleic acid molecule encoding at least one fusion protein, the fusion protein comprising at least one single chain antibody capable of binding to CD28 or CD3, and a C-terminal domain of an envelope protein comprising the transmembrane and intracellular domains of the envelope protein, the C-terminus of the at least one single chain antibody being linked to the N-terminus of the C-terminal domain of the envelope protein; A group of viral vectors comprising: A group of viral vectors in which the first and second nucleic acid molecules are configured to express an envelope protein and a fusion protein, the envelope protein and the fusion protein being in a non-fused form.

2. 2. The viral vector of claim 1, which is a retroviral vector, a lentiviral vector, or another enveloped viral vector.

3. the enveloped virus comprises at least one selected from the group consisting of Bornaviridae, Nyamaviridae, Arenaviridae, Filoviridae, Hantaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Fenuiviridae, Rhabdoviridae, Arteriviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepadnaviridae, Spumavirus, Iridoviridae, Herpesviridae, Poxviridae, and Deltavirus; Optionally, the envelope protein is the envelope G glycoprotein from Vesicular Stomatitis virus, which belongs to the Rhabdoviridae family, or a mutant variant thereof; 3. The viral vector of claim 2, wherein optionally the envelope G glycoprotein has the amino acid sequence shown in SEQ ID NO:

1.

4. The envelope protein mutant has a mutation that reduces adhesion ability, Optionally, the mutant envelope G glycoprotein has a K47Q and R354Q mutation; 4. The viral vector of claim 3, wherein optionally the mutant envelope G glycoprotein has the amino acid sequence shown in SEQ ID NO:

2.

5. the single chain antibody can bind to CD28, optionally wherein the single chain antibody has the amino acid sequence shown in SEQ ID NO: 3 or 4; Optionally, the single chain antibody is capable of binding to CD3, and optionally the single chain antibody has the amino acid sequence shown in SEQ ID NO: 5 or 6; Optionally, the fusion protein comprises a first single chain antibody, a second single chain antibody, and a C-terminal domain of an envelope protein, wherein the first single chain antibody is capable of binding to CD28 and the second single chain antibody is capable of binding to CD3, the C-terminus of the first single chain antibody is linked to the N-terminus of the second single chain antibody and the C-terminus of the second single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein; or the C-terminus of the second single chain antibody is linked to the N-terminus of the first single chain antibody and the C-terminus of the first single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein, Optionally, the first single chain antibody has the amino acid sequence set forth in SEQ ID NO: 3 or 4; Optionally, the second single chain antibody has the amino acid sequence set forth in SEQ ID NO: 5 or 6; Optionally, the C-terminal domain of the envelope protein further comprises at least a portion of the extracellular region of the envelope protein.

6. the fusion protein further comprises a first binding peptide, the N-terminus of the first binding peptide being linked to the C-terminus of the first single chain antibody and the C-terminus of the first binding peptide being linked to the N-terminus of the second single chain antibody; or the N-terminus of the first binding peptide being linked to the C-terminus of the second single chain antibody and the C-terminus of the first binding peptide being linked to the N-terminus of the first single chain antibody; Optionally, the first binding peptide has an amino acid sequence as set forth in SEQ ID NO: 7, 8, 9, 10 or 11; Optionally, the fusion protein further comprises a second binding peptide, the N-terminus of the second binding peptide being linked to the C-terminus of the at least one single chain antibody, and the C-terminus of the second binding peptide being linked to the N-terminus of the C-terminal domain of the envelope protein; 6. The viral vector of claim 1, wherein the second binding peptide has the amino acid sequence shown in SEQ ID NO:

12.

7. the C-terminal domain of the envelope protein comprises a peptide chain beginning at an amino acid between amino acids 386 and 434 of the envelope protein and ending at amino acid 495 of the envelope protein; Preferably, the C-terminal domain of the envelope protein comprises a peptide chain starting at an amino acid between amino acids 395 and 425 of the envelope protein and ending at amino acid 495, Optionally, the C-terminal domain of the envelope protein comprises amino acids 425-495, 415-495, 405-495, or 395-495 of the VSV-G protein; Optionally, the C-terminal domain of the envelope protein has the amino acid sequence set forth in SEQ ID NO: 13, 39, 40 or 41; 7. A viral vector as defined in any one of claims 1 to 6, optionally wherein the fusion protein has the amino acid sequence as set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19 or 20.

8. a first promoter operably linked to the first nucleic acid molecule; and 2. The viral vector of claim 1, further comprising a second promoter operably linked to the second nucleic acid molecule.

9. The viral vector of claim 8, wherein each of the first promoter and the second promoter is independently selected from a CMV, EF-1, or RSV promoter.

10. the first nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID NO: 21 or 35; Optionally, the second nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID NO: 22, 23, 24, 25, 32, 33 or 34; Optionally, the second nucleic acid molecule further comprises a nucleic acid sequence encoding a signal peptide, Optionally, the nucleic acid sequence encoding the signal peptide has the nucleotide sequence set forth in SEQ ID NO:26, 2. The viral vector as defined in claim 1, optionally wherein the copy number ratio of the first nucleic acid molecule and the second nucleic acid molecule is between 1:1 and 4:

1.

11. 2. The viral vector of claim 1, wherein the first viral vector and the second viral vector are the same vector.

12. The viral vector of claim 11, further comprising an internal ribosome entry site sequence disposed between the first nucleic acid molecule and the second nucleic acid molecule.

13. The viral vector of claim 11, further comprising a third nucleic acid molecule encoding a third cleavable binding peptide disposed between the first and second nucleic acid molecules.

14. 2. The viral vector of claim 1, wherein the first viral vector and the second viral vector are pMD2.G, pCMV, a pMD2.G mutant, or a pCMV mutant.

15. a third viral vector carrying a gene of interest, and a fourth viral vector carrying a viral structural protein gene and a viral packaging enzyme gene and optionally a regulatory factor rev gene; Optionally, the structural protein genes, the viral packaging enzyme gene and the regulatory rev gene are located on the same or different fourth viral vectors; Optionally, the viral packaging enzymes include at least one of a reverse transcriptase, a protease, and an integrase.

16. the third viral vector is a transfer vector containing a lentiviral packaging signal; Optionally, the lentiviral packaging signal comprises Ψ, Optionally, the transfer vector is pLV; 16. The viral vector of claim 15, optionally wherein the fourth viral vector is psPAX2.

17. 17. The viral vector of claim 15 or 16, wherein the gene of interest is a nucleic acid molecule encoding a chimeric antigen receptor.

18. 20. A method for obtaining a lentivirus, comprising the steps of: introducing into a first recipient cell a viral vector defined in any one of claims 1 to 17; and culturing the first recipient cell to obtain the virus.

19. the virus is a lentivirus, the first viral vector and the second viral vector are different vectors, and the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 1:1:1:0.25 to 2:1:1:1; Preferably, the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 2:1:1:0.5; Preferably, the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 1:1:1:0.5; Preferably, the mass ratio of the third viral vector, the fourth viral vector, the first viral vector, and the second viral vector is 1:1:1:1; 20. The method of claim 18, optionally wherein the first recipient cell is 293T.

20. A lentivirus obtained by packaging using the method of claim 18 or 19.

21. expressing an envelope protein and a fusion protein, the fusion protein comprising at least one single chain antibody capable of binding to CD28 or CD3, and a C-terminal domain of the envelope protein comprising the transmembrane and intracellular regions of the envelope protein, the C-terminus of the at least one single chain antibody being linked to the N-terminus of the C-terminal domain of the envelope protein; Optionally, the envelope protein is the envelope G glycoprotein of vesicular stomatitis virus or a mutant of the envelope G glycoprotein.

22. expressing an envelope protein and a fusion protein, the fusion protein comprising a first single chain antibody capable of binding to CD28, a second single chain antibody capable of binding to CD3, and a C-terminal domain of the envelope protein comprising the transmembrane and intracellular regions of the envelope protein, wherein the C-terminus of the first single chain antibody is linked to the N-terminus of the second single chain antibody and the C-terminus of the second single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein; or wherein the C-terminus of the second single chain antibody is linked to the N-terminus of the first single chain antibody and the C-terminus of the first single chain antibody is linked to the N-terminus of the C-terminal domain of the envelope protein. Optionally, the envelope protein is the envelope G glycoprotein of vesicular stomatitis virus or a mutant of the envelope G glycoprotein.

23. A method for introducing a lentivirus into non-activated T lymphocytes, comprising using a lentiviral vector as defined in any one of claims 1 to 17 to electroporate or transfect non-activated T lymphocytes, or using a lentivirus as defined in any one of claims 20 to 22 to transduce non-activated T lymphocytes.

24. Introduction of a viral vector as defined in any one of claims 1 to 17 or a lentivirus as defined in any one of claims 20 to 22, comprising a gene of interest, into a second recipient cell, A step of culturing the second recipient cell into which the viral vector or lentivirus has been introduced to express the gene of interest. A method for expressing a gene of interest, comprising: Optionally, introducing into the second recipient cell is by electrotransfection, transfection, or infection; Optionally, the method, wherein the second recipient cell is a T cell.

25. Introducing into a T lymphocyte a viral vector as defined in any one of claims 1 to 17 or a lentivirus as defined in any one of claims 20 to 22, the vector incorporating a nucleic acid encoding a chimeric antigen receptor; A step of culturing T lymphocytes into which a viral vector or lentivirus has been introduced to express a chimeric antigen receptor. A method for obtaining CAR-T cells, comprising: Optionally, the method wherein introduction into the T lymphocyte is by electrotransfection, transfection or infection.

26. 26. A CAR-T cell prepared by the method of claim 25.

27. 27. A pharmaceutical composition comprising a viral vector as defined in any one of claims 1 to 17, a lentivirus as defined in any one of claims 20 to 22, or a CAR-T cell as defined in claim 26.

28. Use of a viral vector as defined in any one of claims 1 to 17, a lentivirus as defined in any one of claims 20 to 22, a CAR-T cell as defined in claim 26, or a pharmaceutical composition as defined in claim 27 for activating immunity or for treating or preventing a disease.

29. 28. Use of a viral vector as defined in any one of claims 1 to 17, a lentivirus as defined in any one of claims 20 to 22, a CAR-T cell as defined in claim 26, or a pharmaceutical composition as defined in claim 27 for treating or preventing a tumor.

30. 27. A method for activating immunity or treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of a viral vector as defined in any one of claims 1 to 17, a lentivirus as defined in any one of claims 20 to 22, a CAR-T cell as defined in claim 26, or a pharmaceutical composition as defined in claim 27.

31. 32. The method of claim 31 , wherein the disease is a tumor.

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