A viral envelope protein, lentiviral vector, lentivirus and application thereof

Abstract

The application discloses a viral envelope protein, a lentivirus vector, a lentivirus and application thereof. The viral envelope protein is a mutant VSVG envelope protein, and the mutant VSVG envelope protein does not bind to a low-density lipoprotein receptor. The lentivirus vector comprises a nucleotide or a fragment thereof coding the envelope protein. The application specifically modifies the lentivirus envelope to adapt to the surface biological characteristics of NK cells and T cells, which is a core technical link for improving the transduction efficiency, reducing the activation threshold, and finally guaranteeing the safety and effectiveness of treatment. The application effectively enhances the efficiency and specificity of the lentivirus in NK cell and gamma-delta T cell infection by modifying the lentivirus envelope, has a wide application prospect in the field of adoptive immunotherapy, and provides practical basis and technical accumulation for developing the next generation of efficient and precise immune cell gene therapy tools.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a viral envelope protein, a lentiviral vector, a lentivirus, and its applications. Background Technology

[0002] Lentivirals, as a highly efficient and stable gene delivery tool, play an increasingly important role in basic research, gene therapy, and cell engineering. Their core advantage lies in their ability to infect both dividing and non-dividing cells and stably integrate the target gene into the host genome, achieving long-term expression. However, the host range and infection efficiency of natural lentiviruses are often limited by their envelope proteins (i.e., envelope glycoproteins), which restricts their application in specific cell types, especially in the promising field of immune cell modification. Summary of the Invention

[0003] This invention is based on the following discoveries of the inventors: In adoptive cellular immunotherapy, particularly therapies based on natural killer (NK) cells and T cells, efficient gene delivery is a prerequisite for conferring novel functions on these cells (such as chimeric antigen receptor expression, gene editing, and functional enhancement). However, NK cells and T cells, due to their unique physiological state and surface receptor composition, often exhibit low susceptibility to conventional lentiviral vectors. Based on markers specifically expressed on the surface of NK and T cells, such as CD2 and CD7 molecules, and CD58, also known as LFA-3, a glycoprotein widely expressed on the surface of human nucleated cells (such as antigen-presenting cells, endothelial cells, and epithelial cells), the binding of CD2 and CD58 is an important adhesion molecule interaction between immune cells. Utilizing this property, introducing CD58 membrane chimeric molecules or CD2 and CD7 single-chain antibodies into the lentiviral envelope can specifically guide the lentivirus to target NK cells, T cells, or CIK cells, thereby enhancing the lentiviral infection efficiency.

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. To this end, this invention proposes a viral envelope protein, a lentiviral vector, a lentivirus, and its applications.

[0005] In a first aspect, the present invention provides a viral envelope protein, said envelope protein being a mutant VSVG envelope protein, said mutant VSVG envelope protein not binding to low-density lipoprotein receptors.

[0006] In a second aspect, the present invention provides a lentiviral vector comprising nucleotides or fragments thereof encoding the envelope protein as described above.

[0007] In a third aspect, the present invention provides a lentivirus comprising the aforementioned envelope protein, or the lentivirus being obtained by viral packaging via the aforementioned lentivirus vector system.

[0008] In a fourth aspect, the present invention provides a cell in which the genome of the cell is integrated with exogenous nucleotides or fragments thereof encoding envelope proteins as described above; or the cell is prepared by cell transduction from the lentiviral vector described above, or the cell is prepared by cell transduction from the lentivirus described above.

[0009] In a fifth aspect of the invention, the invention proposes the use of the above-mentioned envelope protein, lentiviral vector, or lentivirus in packaging viruses, increasing viral titers, reducing toxicity to packaging cells, and / or improving cell transduction efficiency.

[0010] In a sixth aspect, the present invention provides a medicament or medicament composition, said medicament being made from any of the following: the envelope protein described above, the lentiviral vector described above, the lentivirus described above, or the cell described above. And pharmaceutically acceptable excipients.

[0011] This invention specifically modifies the lentiviral envelope to adapt it to the surface biological characteristics of NK cells and T cells, which is a core technical aspect for improving transduction efficiency, lowering the activation threshold, and ultimately ensuring the safety and efficacy of treatment. This invention will effectively enhance the efficacy and specificity of lentiviral envelope modification in NK cell and γδT cell infection, showing broad application prospects in the field of adoptive immunotherapy, and also providing practical evidence and technical accumulation for the development of next-generation, highly efficient, and precise immune cell gene therapy tools.

[0012] The present invention has the following beneficial effects: Lentiviral viruses packaged with the chimeric membrane protein constructed in this invention, compared to wild-type lentivirus VSVG or lentiviruses packaged with the existing lentivirus modified BaEVRless, showed the strongest infectivity in NK and T cell infections after infection with these viruses. The modified lentivirus envelope of this invention exhibits stronger cellular fluorescence, allowing for clear and intuitive observation. Flow cytometry analysis revealed the highest percentage of positive GFP cells, followed by lentivirus NK2 containing the exCD58-TM chimeric membrane protein molecule antiCD2 scFv-TM, while lentivirus NT containing the antiCD7 scFv-TM chimeric membrane protein molecule showed the weakest infectivity. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the exCD58-TM vector structure of the chimeric membrane protein molecule in Example 1 of the present invention; Figure 2 This is a schematic diagram of the structure of the chimeric membrane protein molecule antiCD7 scFv-TM carrier in Example 1 of the present invention; Figure 3 This is a schematic diagram of the antiCD2 scFv-TM vector structure of the chimeric membrane protein molecule in Example 1 of the present invention; Figure 4 This is a schematic diagram of the Trans-Vector structure in Embodiment 2 of the present invention; Figure 5 This is a statistical chart showing the percentage of GFP-positive cells after infecting NK cells with negative control BLANK, VSVG lentivirus, VSVG-m lentivirus, and NT lentivirus, respectively, in Example 3 of the present invention. Figure 6 This is a statistical chart showing the percentage of GFP-positive cells after infecting NK cells with negative control BLANK, VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, NK lentivirus, and NK2 lentivirus in Example 3 of the present invention. Figure 7 This is a statistical chart showing the percentage of GFP-positive cells after infecting αβT cells with negative control BLANK, VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, NK lentivirus, and NK2 lentivirus, respectively, in Example 3 of the present invention. Figure 8 This is a statistical chart showing the percentage of GFP-positive cells after infecting γδT cells with negative control BLANK, VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, NK lentivirus, and NK2 lentivirus, respectively, in Example 3 of the present invention. Figure 9 This is a statistical chart showing the percentage of GFP-positive cells after infecting CIK cells with NK and T cell characteristics with negative control BLANK, VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, and NK lentivirus respectively in Example 3 of the present invention. Detailed Implementation

[0014] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as would be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual”; “Lewin, “Genes VIII”; and Roitt et al., “Immunology” (8th edition), *Medical Immunology*, *Clinical Cell Therapy*, *Tumor Biotherapy*, and general prior art cited herein; furthermore, unless otherwise stated, all methods, procedures, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as would be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0015] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0016] In a first aspect, the present invention provides a viral envelope protein, said envelope protein being a mutant VSVG envelope protein, said mutant VSVG envelope protein not binding to low-density lipoprotein receptors.

[0017] The lentiviral envelope is the outermost lipid bilayer structure of a lentivirus, an essential structure for lentiviruses. Its surface is embedded with envelope glycoproteins, which are primarily responsible for recognizing host cell receptors and mediating membrane fusion, thus assisting the viral core in entering target cells. Specifically, the viral envelope protein of this invention is a mutant VSVG envelope protein, that is, a mutant VSVG envelope protein obtained from the wild-type VSVG envelope protein that does not bind to the low-density lipoprotein receptor.

[0018] The viral envelope protein of this invention is obtained by modifying the VSVG envelope protein (or wild-type VSVG envelope protein, Vesicular Stomatitis Virus Glycoprotein, abbreviated as VSVG) to obtain a mutant VSVG envelope protein (abbreviated as VSVG-m). This mutant VSVG envelope protein does not bind to the low-density lipoprotein receptor (LDLR), thus eliminating the specificity of the viral envelope protein for cells expressing low-density lipoprotein and eliminating the broad-spectrum non-specificity mediated by LDLR. The mutant VSVG envelope protein's non-binding to the low-density lipoprotein receptor can be achieved through various methods, such as modifying the wild-type VSVG envelope protein, including mutating, adding, or deleting amino acid sequences. Compared to the wild-type VSVG envelope protein, the mutant VSVG envelope protein has at least one different amino acid site.

[0019] As one embodiment, the amino acid sequence of the mutant VSVG envelope protein is based on the amino acid sequence shown in SEQ ID NO.1, with at least specific mutations at the K63 and R370 sites; as a preferred embodiment, the specific mutations of the mutant VSVG envelope protein are K63Q and R370A.

[0020] The amino acid sequence of the wild-type VSVG envelope protein is shown in SEQ ID NO.1, and the nucleotide sequence of the wild-type VSVG envelope protein is shown in SEQ ID NO.2. The amino acid sequence of the mutant VSVG envelope protein, based on the amino acid sequence shown in SEQ ID NO.1, contains specific mutations at least at positions K63 and R370. Preferably, the specific mutations in the mutant VSVG envelope protein are K63Q and R370A. The amino acid sequence of the mutant VSVG envelope protein is shown in SEQ ID NO.3, encoding... In a second aspect, the present invention provides a lentiviral vector comprising nucleotides or fragments thereof encoding the envelope protein as described above.

[0021] In a third aspect, the present invention provides a lentivirus comprising the aforementioned envelope protein, or the lentivirus being obtained by viral packaging via the aforementioned lentivirus vector system.

[0022] In one embodiment, the lentivirus includes a chimeric membrane protein selected from at least one of exCD58-TM, antiCD7 scFv-TM, or antiCD2 scFv-TM; the chimeric membrane protein is expressed on the surface of the lentivirus and specifically binds to targeting markers on the surface of NK cells, T cells, or CIK cells.

[0023] CD2 and CD7 molecules are markers specifically expressed on the surface of NK cells and T cells; CD58 (LFA-3) is an adhesion glycoprotein widely expressed on human nucleated cells (including antigen-presenting cells, endothelial cells, epithelial cells, etc.). The binding of CD2 and CD58 is an important adhesion molecule interaction between immune cells. Based on the above molecular expression characteristics, this invention constructs exCD58-TM (NK), anti-CD7 scFv-TM (NT), and anti-CD2scFv-TM (NK2) as chimeric membrane proteins, enabling lentiviruses to specifically target NK cells and T cells, thereby improving the infection efficiency and targeting specificity of these immune cells.

[0024] exCD58-TM (NK for short) is a chimeric membrane protein that includes a transmembrane domain and an extracellular domain. Its transmembrane domain is anchored in the lipid bilayer of the lentiviral envelope, while the CD58 extracellular domain is exposed on the surface of the lentiviral envelope, thereby enabling it to recognize and bind to CD2 molecules on the surface of target cells.

[0025] antiCD7 scFv-TM (NT) is a chimeric membrane protein that includes a transmembrane domain and an extracellular domain. Its transmembrane domain is anchored in the lipid bilayer of the lentiviral envelope, while the antiCD7 scFv extracellular domain is exposed on the lentiviral envelope surface, thereby enabling it to recognize and bind to CD7 molecules on the surface of target cells.

[0026] antiCD2 scFv-TM (NK2 for short) is a chimeric membrane protein that includes a transmembrane domain and an extracellular domain. Its transmembrane domain is anchored in the lipid bilayer of the lentiviral envelope, while the antiCD2 scFv extracellular domain is exposed on the surface of the lentiviral envelope, thereby enabling it to recognize and bind to CD2 molecules on the surface of target cells.

[0027] As one implementation, the transmembrane domains of exCD58-TM, anti-CD7 scFv-TM, and anti-CD2 scFv-TM may be the same or different. As an optional approach, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO.5, and the nucleotide sequence encoding the transmembrane domain is shown in SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8.

[0028] As one embodiment, the amino acid sequence of the exCD58-TM is shown in SEQ ID NO.9, the amino acid sequence of the antiCD7 scFv-TM is shown in SEQ ID NO.11, and the amino acid sequence of the antiCD2 scFv-TM is shown in SEQ ID NO.13.

[0029] As one implementation, the nucleotide sequence encoding the exCD58-TM is shown in SEQ ID NO.10, the nucleotide sequence encoding the antiCD7 scFv-TM is shown in SEQ ID NO.12, and the nucleotide sequence encoding the antiCD2 scFv-TM is shown in SEQ ID NO.14.

[0030] In a fourth aspect, the present invention provides a cell in which the genome of the cell is integrated with exogenous nucleotides or fragments thereof encoding envelope proteins as described above; or the cell is prepared by cell transduction from the lentiviral vector described above, or the cell is prepared by cell transduction from the lentivirus described above.

[0031] Those skilled in the art can transfect lentiviral vectors into host cells (such as 293T cells) to prepare lentiviral particles using methods well-known in the art. For example, lentiviral vectors can be introduced into eukaryotic packaging cells via calcium phosphate co-precipitation, electroporation, microinjection, liposome transfection, or transfection using polyamine transfection reagents. The obtained lentiviral particles can be further transduced into target cells (such as NK cells, γδT cells, T cells, and other immune cells) to achieve efficient delivery of the target gene.

[0032] In a fifth aspect of the invention, the invention proposes the use of the above-mentioned envelope protein, lentiviral vector, or lentivirus in packaging viruses, increasing viral titers, reducing toxicity to packaging cells, and / or improving cell transduction efficiency.

[0033] In a sixth aspect, the present invention provides a medicament or medicament composition, said medicament being made from any of the following: the envelope protein described above, the lentiviral vector described above, the lentivirus described above, or the cell described above. And pharmaceutically acceptable excipients.

[0034] The excipients include various excipients and diluents, which are not essential active ingredients and do not cause excessive toxicity after application. The excipients contain sterile water or physiological saline, stabilizers, excipients, etc. When the excipients are used in an aqueous solution for injection, they are selected from physiological saline, isotonic glucose solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG) and / or nonionic surfactants (Tween 80, HCO-50, etc.).

[0035] In the pharmaceutical composition provided by this invention, the aforementioned lentiviral particles or cells can be a single active ingredient, or they can be combined with one or more other active components useful for disease treatment to form a combined formulation. The active components are various other drugs used for treating diseases.

[0036] The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient in the pharmaceutical composition depends on the patient's weight, the type of application, and the condition and severity of the disease.

[0037] The relevant sequences in this invention are shown in Table 1.

[0038] Table 1 Sequence List

[0039] Some of the reagents and consumables used in Examples 1-3 of this invention are as follows: Phanta Max Super-Fidelity DNA Polymerase, Novizan Bio (P505-d1); GBclonart Seamless Assembly Kit, Suzhou Shenzhou Gene Co., Ltd. (GB2002-48). Gel and PCR Clean-up, MACHEREY-NAGEL (740609.250); EcoRI, NEB (R3101V); XbaI, NEB (R0145V); Plasmid small-dose preparation kit, Uelandy (UE-MN-P-250G). PrimeSTAR enzyme, Novizan, (P101-03) pMDLg / pRRE plasmid, addgene(#12251). pRSV-Rev plasmid, addgene (#12253); Complete culture medium (DMEM, Gibco, C11995500BT).

[0040] Example 1: Vector Construction I. Construction of exCD58-TM(NK) vector 1. Based on the VSVG gene information of the pHCMV-VSVG type packaging plasmid (i.e., VSVG plasmid) from Shanghai Shengbo Biotechnology Co., Ltd., design the pHCMV-exCD58-TM gene DNA fragment (i.e., replace the VSVG gene in the VSVG plasmid with the exCD58-TM gene (SEQ ID NO.10)) and synthesize a double-stranded DNA molecule. 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, PCR amplification reactions were performed using the synthesized PCR primers (forward primer F (SEQ ID NO. 17: AGCACTGAGATCTGAATTC) and reverse primer R (SEQ ID NO. 18: CTGCACTGGTGGGGTTCT) to obtain the PCR product of the target DNA fragment.

[0041] The PCR reaction system is as follows: 32.5 μL H2O, 10 μL 5× Buffer (containing Mg2+), 4 μL dNTPs (2.5 mM each), 1 μL forward primer Primer1 (+) (10 μM), 1 μL reverse primer Primer2 (-) (10 μM), 1 μL target gene template DNA (15 ng / μL), and 0.5 μL PrimeSTAR enzyme (concentration: 1 U / μL).

[0042] The PCR procedure is as follows: denaturation at 98°C for 3 minutes; annealing at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1 minute, repeated for 30 cycles; extension at 72°C for 10 minutes.

[0043] 3. Take the pHCMV-VSVG type packaging plasmid, digest it with restriction endonucleases EcoRI and XbaI, and then recover the vector backbone; The enzyme digestion system was as follows: BamHI: 1 μL, buffer: 3 μL, pHCMV-VSVG plasmid: 1 μg, water added to 30 μL; digestion at 37℃ for 4 hours.

[0044] 4. The PCR product obtained in step 2 and the vector backbone recovered in step 3 were recombined, transformed into E. coli, positive bacteria were screened, and the plasmids were extracted using the Uelandy plasmid small-dose preparation kit to obtain the recombinant vector.

[0045] The recombinant system consisted of: 15 μL of recombinase (GBclonart Seamless Assembly Kit), 40 ng of recovered PCR product DNA, and 20 ng of recovered plasmid; after incubating in a water bath at 42°C for 30 min, the cells were transformed into E. coli.

[0046] II. Construction of antiCD7 scFv-TM(NT) vectors 1. Based on the VSVG gene information of the pHCMV-VSVG type packaging plasmid (i.e., VSVG plasmid) from Shanghai Shengbo Biotechnology Co., Ltd., design the pHCMV-antiCD7-TM gene DNA fragment (i.e., replace the VSVG gene in the VSVG plasmid with the antiCD7-TM gene (SEQ ID NO.12)) and synthesize a double-stranded DNA molecule.

[0047] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, PCR amplification reactions were performed using the synthesized PCR primers (forward primer F (SEQ ID NO. 17: AGCACTGAGATCTGAATTC) and reverse primer R (SEQ ID NO. 18: CTGCACTGGTGGGGTTCT) to obtain the PCR product of the target DNA fragment.

[0048] The PCR reaction system is as follows: 32.5 μL H2O, 10 μL 5× Buffer (containing Mg2+), 4 μL dNTPs (2.5 mM each), 1 μL forward primer Primer1 (+) (10 uM), 1 μL reverse primer Primer2 (-) (10 uM), 1 μL target gene template DNA (20 ng / μL), and 0.5 μL PrimeSTAR enzyme (1 U / μL).

[0049] The PCR procedure is as follows: denaturation at 98°C for 3 minutes; annealing at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1 minute, repeated for 30 cycles; extension at 72°C for 10 minutes.

[0050] 3. Take the pHCMV-VSVG type packaging plasmid, digest it with restriction endonucleases EcoRI and XbaI, and then recover the vector backbone.

[0051] The enzyme digestion system was as follows: BamHI: 1 μL, buffer: 3 μL, pHCMV-VSVG plasmid: 1 μg, water added to 30 μL; digestion at 37℃ for 4 hours.

[0052] 4. The PCR product obtained in step 2 and the vector backbone recovered in step 3 are recombined, transformed into E. coli, positive bacteria are screened and their plasmids are extracted to obtain the recombinant vector.

[0053] The recombination system consisted of: 15 μL of recombinase (GBclonart Seamless Assembly Kit), 40 ng of recovered PCR product DNA, and 20 ng of recovered plasmid; after incubating in a water bath at 42°C for 30 min, the cells were transformed into E. coli.

[0054] III. Construction of antiCD2 scFv-TM (NK2) vectors 1. Based on the VSVG gene information of the pHCMV-VSVG type packaging plasmid (i.e., VSVG plasmid) from Shanghai Shengbo Biotechnology Co., Ltd., design the pHCMV-antiCD2-TM gene DNA fragment (i.e., replace the VSVG gene in the VSVG plasmid with the antiCD2scFv-TM gene (SEQ ID NO.14)) and synthesize a double-stranded DNA molecule.

[0055] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, PCR amplification reactions were performed using the synthesized PCR primers (forward primer F (SEQ ID NO. 17: AGCACTGAGATCTGAATTC) and reverse primer R (SEQ ID NO. 18: CTGCACTGGTGGGGTTCT) to obtain the PCR product of the target DNA fragment.

[0056] The PCR reaction system is as follows: 32.5 μL H2O, 10 μL 5× Buffer (containing Mg2+), 4 μL dNTPs (2.5 mM each), 1 μL forward primer Primer1 (+) (10 μM), 1 μL reverse primer Primer2 (-) (10 μM), 1 μL target gene template DNA (20 ng / μL), and 0.5 μL PrimeSTAR enzyme (1 U / μL).

[0057] The PCR procedure is as follows: denaturation at 98°C for 3 minutes; annealing at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1 minute, repeated for 30 cycles; extension at 72°C for 10 minutes.

[0058] 3. Take the pHCMV-VSVG-m type packaging plasmid, digest it with restriction endonucleases EcoRI and XbaI, and then recover the vector backbone.

[0059] The enzyme digestion system was as follows: BamHI: 1 μL, buffer: 3 μL, pHCMV-VSVG-m plasmid: 1 μg, water added to 30 μL; digestion at 37℃ for 4 hours.

[0060] 4. The PCR product obtained in step 2 and the vector backbone recovered in step 3 are recombined, transformed into E. coli, positive bacteria are screened and their plasmids are extracted to obtain the recombinant vector.

[0061] The recombinant system consisted of: 15 μL of recombinase (GBclonart Seamless Assembly Kit), 40 ng of recovered PCR product DNA, and 20 ng of recovered plasmid; after incubating in a water bath at 42°C for 30 min, the cells were transformed into E. coli.

[0062] After successful construction and identification, NK plasmids (structures as shown) were obtained. Figure 1 As shown), NT plasmid (structure as shown) Figure 2 As shown), NK2 plasmid (structure as shown) Figure 3 (As shown).

[0063] The same method was used to construct VSVG-m plasmid (K63Q, R370A double-point mutation envelope plasmid, SEQ ID NO.4) and BV plasmid (BaEVRless plasmid, the amino acid sequence of BV is shown in SEQ ID NO.15, and the nucleotide sequence encoding BV is shown in SEQ ID NO.16), which were used for lentivirus packaging in Example 2.

[0064] Example 2 Lentiviral Packaging I. Cell passage Select healthy 293T cells, discard the old culture medium, wash the cells with sterile PBS, and discard the PBS. Add trypsin digestion solution and digest for 1-2 minutes until the cells become rounded and begin to detach. Add complete culture medium (DMEM, Gibco, C11995500BT) to stop digestion, and pipette the cells from the bottom of the culture dish into a single-cell suspension. Centrifuge to collect the cells. Seed the cells into 10cm culture dishes, maintaining a pre-transfection cell density of 70%–80%.

[0065] II. Virus Packaging (Transfection) 1. Discard the original culture medium 1 hour before transfection and replace it with fresh complete culture medium.

[0066] 2. Prepare the DNA / CaCl2 solution. For each 293T cell culture dish, use the following ratio for plasmid transfection: pMDLg / pRRE plasmid (10 μg), pRSV-Rev plasmid (8 μg), VSVG-m plasmid (6 μg), one of the following plasmids (VSVG, VSVG-m, NK, NK2, NT, or BV) (6 μg), and Trans-Vector (16 μg). (Trans-Vector is a lentiviral transfer plasmid expressing the target gene (EGFP reporter gene), with the structure shown below.) Figure 4As shown, the nucleotide sequence between LTRs is shown in SEQ ID NO. 19. Take a new 5 mL or 15 mL centrifuge tube and add 0.5 M CaCl2 (0.25 mL), pMDLg / pRRE plasmid (10 μg), pRSV-Rev plasmid (8 μg), VSVG-m plasmid (6 μg), one of the following plasmids (VSVG, VSVG-m, NK, NK2, NT, or BV) (6 μg), and Trans-Vector (16 μg). Add ultrapure water to 0.5 mL. Mix thoroughly.

[0067] The experimental groups are shown in Table 2.

[0068] Table 2 Lentiviral Packaging Plasmid Experimental Groups

[0069] 3. After mixing thoroughly, slowly add 2×HBS solution (2×HBS solution is: in 500ml system, NaCl: 4.09g, Na2HPO4: 0.269g, HEPES: 5.96g, pH: 7.05-7.10), and incubate at room temperature for 2-5min.

[0070] 4. Add the calcium transfer mixture dropwise to the cell culture dish and incubate in a CO2 incubator.

[0071] 5. Replace with fresh 5% FBS complete medium 6-8 hours after transfection, and continue culturing in a CO2 incubator. Take photos to observe the transfection status 24 hours after transfection.

[0072] III. Receiving Poison 1. Harvest the virus for the first time 36 hours after transfection, and collect the supernatant. Add 9 mL of fresh 5% FBS complete culture medium and incubate in a CO2 incubator.

[0073] 2. Collect the virus a second time 72 hours after transfection and collect the supernatant.

[0074] IV. Virus Concentration and Purification 1. Combine the two viral supernatants, centrifuge to remove cell precipitate, filter the supernatant through a 0.2 μm filter membrane into an ultrafiltration tube, and centrifuge at 100,000 g and 4 °C for 2 hours.

[0075] 2. After centrifugation, discard the supernatant and add Opti to the virus pellet. Dissolve the virus in MEM medium at 4°C overnight. The next day, disperse the virus precipitate to ensure complete dissolution.

[0076] 3. Collect all identical virus concentrates together, filter through a 0.22 μm filter membrane, and take 15 μL of the virus solution for titer detection. Aliquot the remaining virus and... Store at 80℃ for long-term preservation.

[0077] Using the methods described above, VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, NK lentivirus, NK2 lentivirus, and NT lentivirus were respectively packaged and obtained.

[0078] Example 3 Cell Culture and Infection I. NK Cell Culture and Infection 1. Sample Requirements 1) Cell viability: The viability of mononuclear cells is greater than 90%.

[0079] 2) Initial cell count: The total number of mononuclear cells from fresh peripheral blood is 2-2.5 × 10⁻⁶. 7 The total number of mononuclear cells from fresh umbilical cord blood is 2.5-3.0 × 10⁻⁶. 7 The total number of mononuclear cells derived from cryopreserved peripheral blood was 2.5-3.0 × 10⁻⁶. 7 .

[0080] 3) Initial culture medium: The initial culture medium used is XR NK basal medium (XR NK basal medium is a serum-free lymphocyte culture medium, sourced from Corning, Corning 88-581-CM). 2. Preparation - Preparation of Culture Medium 1) Basic culture medium: Add IL-2 (final concentration 200 U / mL) and gentamicin (final concentration 80 U / mL) to the initial culture medium to prepare the basic culture medium. After preparation, store at 4℃.

[0081] 2) Autologous plasma separation: Fresh whole blood samples were centrifuged at 700g for 10 min. The upper pale yellow plasma layer was collected, inactivated by water bath at 56℃ for 30 min, and then centrifuged at 900g for 10 min. The supernatant was collected, frozen at -20℃ for 15 min, and centrifuged again at 900g for 10 min. The upper plasma layer was stored at 4℃ for later use. The lower blood cell layer was used to extract mononuclear cells.

[0082] 3) Mononuclear cell extraction: Different lymphocyte separation solutions can be used according to the relevant instructions.

[0083] 4) NK autologous plasma culture medium: Based on the initial NK culture volume, add 5% autologous plasma to the T basal culture medium prepared in step 1). (If plasma is insufficient or the anticoagulant ratio in the umbilical cord blood sample is greater than 30%, HPL or AB serum can be mixed with autologous plasma at a 1:1 ratio before addition to avoid excessive anticoagulant ratio affecting the use of autologous plasma). Add 5% before bagging and 1% after bagging.

[0084] 3. Cultivation process 1) Cultivation focuses on controlling cell density. The total volume may fluctuate due to individual sample differences and adjustments to the cultivation method. Cell density is calculated based on the density of viable mononuclear cells.

[0085] 2) The initial vaccination dose should be 1.0 × 10⁻⁶. 6 Mononuclear cell density per mL.

[0086] 3) If plasma is insufficient or the anticoagulant ratio in the umbilical cord blood sample is greater than 30%, add HPL or AB serum after mixing it with plasma at a 1:1 ratio to avoid the anticoagulant ratio being too high and affecting the use of autologous plasma. Add 5% before bagging and 1% after bagging.

[0087] 4) First, add the autologous plasma to the prepared basal culture medium and mix well before using it for fluid replenishment and fluid replacement.

[0088] 4. Daily training operation details: Following the prescribed procedure, fluid was administered to maintain cell density, and lentiviral infection was performed on day 14, specifically as follows: 1) On day 0, NK cells were initially cultured in T75 square flasks treated with TC (T75 adherent cell culture flasks), with a mononuclear cell viable density >1.0 × 10⁻⁶ cells / year. 6 (Incubate at 37°C in a 5% CO2 incubator at a concentration of 1 mL / mL).

[0089] Fresh peripheral blood: approximately 20 mL XR NK basal culture medium + 5% autologous plasma (v / v, 1-1.25 mL) + 2.0-2.5 × 10⁻⁶ mg / L. 7 One live mononuclear cell derived from fresh peripheral blood + the first NK cell activation solution (Shouning Biotechnology, NK amplification kit, RP03030).

[0090] Fresh umbilical cord blood: Approximately 25 mL XR NK basal culture medium + 5% autologous plasma (v / v, 1.25-1.5 mL) + 2.5-3.0 × 10⁻⁶ mg / L 7 One live mononuclear cell derived from fresh umbilical cord blood + the first XR NK cell activation solution.

[0091] Cryopreservation of peripheral blood: Approximately 25 mL XR NK basal culture medium + 5% autologous plasma (v / v, 1.25-1.5 mL) + 2.5-3.0 × 10⁻⁶ mg / L. 7 One cryopreserved live mononuclear cells derived from peripheral blood + the first XR NK cell activation solution.

[0092] 2) On days 1 and 2, carefully observe whether the cells have clumped. If cells have clumped, handle the culture flask gently to avoid breaking the cell clusters. If the culture medium does not turn significantly yellow and the cells are in good condition, no further treatment is necessary.

[0093] 3) On day 3, after cell sedimentation, centrifuge part of the cells and change the medium (without changing the bottle): Transfer all cell suspension from the culture flask to a 50mL centrifuge tube. Place the centrifuge tube vertically and allow the cell suspension to settle naturally for 3 minutes. Then, collect the top 2 / 3 of the culture medium supernatant and centrifuge (300g, 6min). Transfer the remaining 1 / 3 of the cell suspension at the bottom of the centrifuge tube back into the culture flask (avoid disrupting the cell clusters). After centrifugation, resuspend the cell pellet in 10mL of NK autologous plasma culture medium and transfer it back to the culture flask. Replenish the total culture medium volume in the culture flask to the original volume (maintaining a viable cell density of 1.0-1.5 x 10⁻⁶ cells / mL). 6 / mL), with an autologous plasma addition of 5% (v / v).

[0094] 4) On day 4, if the culture medium turns yellow, NK autologous plasma culture medium can be added as needed. The amount of autologous plasma added is 5% (v / v) (it needs to be mixed well beforehand), and the viable cell density is controlled at 0.8-1.0×10⁻⁶. 6 Between / mL.

[0095] 5) On day 5, if the culture medium turns yellow, NK autologous plasma culture medium can be added as needed. The amount of autologous plasma added is 5% (v / v) (it needs to be mixed well beforehand), and the viable cell density is controlled at 0.8-1.0×10⁻⁶. 6 Between / mL.

[0096] 6) On day 6, if the culture medium turns yellow, NK autologous plasma culture medium can be added as needed. The amount of autologous plasma added is 5% (v / v) (it needs to be mixed well beforehand), and the viable cell density is controlled at 0.8-1.0×10⁻⁶. 6 Between / mL.

[0097] 7) On day 7, supplement with NK autologous plasma culture medium (live cell density controlled at 0.8-1.0 × 10⁶ cells / day). 6Add two vials of XR NK cell activation solution (between / mL) for the second activation. After replenishing the solution, the volume should reach more than 250mL. Transfer the cells to a bag and add autologous plasma at 1% (v / v) to replenish the culture medium. If the volume is less than 250mL after replenishing the solution, continue culturing at T175 or T225, delaying the transfer to a bag, and add autologous plasma at 5% (v / v) to replenish the culture medium.

[0098] 8) Observe daily on days 8-9. Estimate the amount of solution to add based on experience and the color of the culture medium. Maintain a viable cell density of 1.0-1.5 × 10⁻⁶ cells / day. 6 The autologous plasma addition is 1% (v / v) between / mL (must be premixed). The XR NK cell activator single product supports large-scale culture systems; 4L culture systems can be divided into smaller bags after the total cell suspension volume is greater than 1L.

[0099] 9) On days 10-11, observe daily, estimating the amount of solution to be added based on experience and the color of the culture medium. Maintain the viable cell density at 1.5-2.0 × 10⁶ cells / day. 6 The amount of autologous plasma added is 1% (v / v) between / mL (must be mixed beforehand).

[0100] 10) From day 12 to 15, administer fluid replacement daily or every two days, maintaining a viable cell density of 2.0-3.5 × 10⁻⁶. 6 The cell count can be between [value] / mL. The decision to terminate the culture, collect the cells for use, or freeze them can be made based on the total cell count.

[0101] 11) On day 14, NK cells were infected with lentivirus. 48 hours after infection, cells were collected and transduction efficiency was detected by flow cytometry. The percentage of GFP-positive cells was analyzed by detecting GFP expression. Each group had three replicates.

[0102] Lentiviral infection of NK cells included two sets of experiments: First set of experiments and results: NK cells were infected with VSVG lentivirus, VSVG-m lentivirus, and NT lentivirus obtained in Example 2, respectively, while BLANK cells without lentivirus were used as a negative control group; the results are as follows. Figure 5 As shown, from Figure 5 It can be seen that the transduction efficiency of the NT lentivirus group is higher than that of the VSVG-m lentivirus group, but lower than that of the VSVG lentivirus group.

[0103] Second set of experiments and results: NK cells were infected with VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, NK lentivirus, and NK2 lentivirus obtained in Example 2, respectively, while BLANK cells without lentivirus were used as a negative control group; the results are as follows. Figure 6 As shown, from Figure 6It can be seen that the NK lentivirus group has the highest transduction efficiency, followed by the NK2 lentivirus group, both of which are superior to the VSVG lentivirus group, VSVG-m lentivirus group and BV lentivirus group.

[0104] II. αβT cell culture and infection 1. Culture medium composition 1) T cell culture medium: serum-free medium (TAKARA, WK593S) supplemented with 1000 IU / mL IL-2 and 5% FBS.

[0105] 2. Cultivation process 1) Collect peripheral blood.

[0106] 2) Obtaining mononuclear cells from peripheral blood. Dilute peripheral blood containing EDTA anticoagulant with an equal volume of sterile PBS and add it to Ficoll separation solution (approximately 1:2 ratio). Centrifuge at 800g for 30 minutes at room temperature. After separation, aspirate the middle white membrane layer, i.e., peripheral blood mononuclear cells (PBMCs), wash twice with 0.9% sodium chloride (1450rpm × 5 minutes), resuspend, stain with trypan blue, and count. Finally, collect the PBMCs for later use.

[0107] 3) CD3 + T cell sorting The PBMC suspension was prepared at 1×10 7 Add CD3 magnetic beads at a cell / 10 μl ratio, incubate at 4°C in the dark for 15 minutes, then wash and centrifuge. Collect CD3 cells using a magnetic separation column via positive selection. + T lymphocytes. 4) CD3 + T cell activation CD3 + Cells were centrifuged at 1450 rpm for 5 minutes and resuspended in T cell culture medium. A culture flask pre-coated with anti-CD3 / CD28 antibody was added, along with IL-2 (final concentration 1000 IU / ml). The cell volume was adjusted to 2 × 10⁶ cells / ml. 6 / ml density, incubated and activated in a 37℃, 5% CO2 incubator.

[0108] 5) Lentiviral transduction of cells After 24 hours of activation, remove the T cell culture flask, collect a small number of T cells, and stain with trypan blue. Based on the cell count results, adjust the cell concentration to 1×10⁻⁶. 6 / ml. Add an appropriate amount of virus to the cell suspension at an MOI of 100, mix well, and transduce in a 37°C, 5% CO2 incubator.

[0109] 6) Expansion of αβ T cells Observe cell status daily, including density, aggregation, and changes in culture medium, and count cells periodically. Use a cell concentration of 5 × 10⁶ cells / day. 5 Adjust the fluid level by adding fluid at a rate of / ml, and continue culturing for 10 to 12 days.

[0110] 7) On day 14, αβT cells were infected with lentivirus. 48 hours after infection, cells were collected and transduction efficiency was detected by flow cytometry. The percentage of GFP-positive cells was analyzed by detecting GFP expression. Each group had three replicates.

[0111] The experiment and results of lentiviral infection of αβT cells are as follows: αβT cells were infected with VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, NK lentivirus, and NK2 lentivirus obtained in Example 2, respectively, while BLANK without lentivirus was used as a negative control group; the results are as follows. Figure 7 As shown, from Figure 7 It can be seen that the VSVG lentivirus group has the highest transduction efficiency, followed by the NK lentivirus group, and then the NK2 lentivirus group, BV lentivirus group, and VSVG-m lentivirus group.

[0112] III. γδT cell culture and infection 1. Culture medium composition 1) T cell culture medium: serum-free medium (TAKARA, WK593S) supplemented with 1000 IU / mL IL-2 and 5% FBS.

[0113] 2) γδT cell stimulation medium: T009 medium (Bioengine, FG0103801) supplemented with 1000 IU / mL IL-2 and 5% FBS.

[0114] 2. T cell stimulation 1) After counting T cells, the total cell count was adjusted to 1×10⁻⁶. 7 According to the manufacturer's instructions, add T Cell TransAct™ reagent to the culture system at a ratio of 1 × 10⁻⁶. 6 Add 10 μL of the solution to each cell. Incubate the cells under standard culture conditions for 48 h.

[0115] 3. γδT cell stimulation 1) Day 0 (Activation): Peripheral blood mononuclear cells (PBMCs) were introduced at a rate of 2 × 10⁻⁶. 6 Cells were resuspended at a density of 10 cells / mL in γδT cell stimulation medium, and zoledronic acid was added to a final concentration of 5 μM to induce specific activation of γδT cells.

[0116] 2) Day 2 (Transduction): After counting and adjusting the cell concentration, the cells were seeded into 24-well plates for lentiviral transduction. Polybrene was added to each transduction group at a final concentration of 8 μg / mL to improve viral transduction efficiency. The specific cell count, virus dosage, and experimental group settings were all performed according to the experimental design table.

[0117] 4. Culture medium replenishment 1) Day 4: Add 400 μL of fresh culture medium to each well of the αβT cell and γδT cell culture systems.

[0118] 5. Transduction efficiency detection 1) Day 5: Cells were collected and transduction efficiency was detected by flow cytometry. The percentage of GFP-positive cells was analyzed by detecting GFP expression. Each group had three replicates.

[0119] The experiment and results of lentiviral infection of γδT cells are as follows: γδT cells were infected with VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, NK lentivirus, and NK2 lentivirus obtained in Example 2, respectively, while BLANK without lentivirus was used as a negative control group; the results are as follows. Figure 8 As shown, from Figure 8 It can be seen that the NK lentivirus group has the highest transduction efficiency, followed by the VSVG lentivirus group, then the VSVG-m lentivirus group, the BV lentivirus group, and the NK2 lentivirus group.

[0120] IV. CIK Cell Culture and Infection 1. Sample Requirements 1) Cell viability: The viability of mononuclear cells is greater than 90%.

[0121] 2) Starting cell count: Fresh peripheral blood was collected from volunteers, and the initial PBMC count was 2-2.5 × 10⁻⁶. 7 .

[0122] 3) Initial culture medium: Corning 88-581-CM was selected as the initial culture medium.

[0123] 2. Preparation - Preparation of basal culture medium 1) Basic culture medium: Add IL-2 (final concentration 200 U / mL) and gentamicin (final concentration 80 U / mL) to the initial culture medium to prepare the basic culture medium, and store it at 4℃.

[0124] 2) Autologous plasma separation: Fresh whole blood samples were centrifuged at 700g for 10 min. The upper pale yellow plasma layer was collected, inactivated by water bath at 56℃ for 30 min, and then centrifuged at 900g for 10 min. The supernatant was collected, frozen at -20℃ for 15 min, and centrifuged again at 900g for 10 min. The upper plasma layer was stored at 4℃ for later use. The lower blood cell layer was used to extract mononuclear cells.

[0125] 3) Mononuclear cell extraction: Different lymphocyte separation solutions can be used according to the relevant instructions.

[0126] 3. Cultivation process 1) Cultivation focuses on controlling density. The total volume may fluctuate due to individual sample differences and adjustments to the cultivation method. Cell density is calculated based on the density of viable mononuclear cells. In the later stages of cell culture, the degree of yellowing of the culture medium may not be proportional to the cell proliferation rate; one cannot simply judge whether to add more medium based on the color of the culture medium.

[0127] 2) The initial vaccination dose should be 1.0 × 10⁻⁶. 6 Mononuclear cell density per mL.

[0128] 3) First, add the autologous plasma to the prepared basal culture medium and mix well before using it for replenishment and replacement of fluids.

[0129] 4. Daily training operation details: Following the prescribed procedure, fluid was administered to maintain cell density, and lentiviral infection was performed on day 14, specifically as follows: 1) On day 1, in T75 square flasks (T75 adherent cell culture flasks) with TC treatment for CIK initiation, the viable mononuclear cell density was >1.0 × 10⁻⁶ cells / year. 6 (Incubate at 37°C in a 5% CO2 incubator at a concentration of 1 mL / mL).

[0130] Fresh peripheral blood: approximately 40 mL CIK basal culture medium + 5% autologous plasma (v / v, 1-1.25 mL).

[0131] 2) On days 2-3, carefully observe whether the cells have clumped. If cells have clumped, handle the culture flask gently to avoid breaking the cell clusters. If the culture medium does not turn significantly yellow and the cells are in good condition, no further treatment is necessary.

[0132] 3) From day 4 to 8, supplement CIK autologous plasma culture medium as needed based on cell density. The amount of autologous plasma added is 5% (v / v) (must be pre-mixed) to control the viable cell density at 0.8-1.0 × 10⁻⁶. 6 Between / mL.

[0133] 4) Observe daily from day 9 to 13. Estimate the amount of culture to add based on experience and the color of the culture medium. Once the culture medium volume exceeds 200 ml, transfer it to a cell culture bag for continued culture.

[0134] 5) On day 14, CIK cells were infected with lentivirus. 48 hours after infection, cells were collected and transduction efficiency was detected by flow cytometry. The percentage of positive GFP cells was analyzed by detecting GFP expression. Each group had three replicates.

[0135] The experiment and results of lentiviral infection of CIK cells are as follows: CIK cells were infected with VSVG lentivirus, VSVG-m lentivirus, BV lentivirus, and NK lentivirus obtained in Example 2, respectively, while BLANK cells without lentivirus were used as a negative control group; the results are as follows. Figure 9 As shown, from Figure 9 It can be seen that the NK lentivirus group has the highest transduction efficiency, followed by the VSVG lentivirus group, then the BV lentivirus group and the VSVG-m lentivirus group.

[0136] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0137] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A viral envelope protein, characterized in that, The envelope protein is a mutant VSVG envelope protein, which does not bind to the low-density lipoprotein receptor.

2. The envelope protein according to claim 1, characterized in that, The amino acid sequence of the mutant VSVG envelope protein is based on the amino acid sequence shown in SEQ ID NO.1, with at least specific mutations at the K63 and R370 sites; as a preferred embodiment, the specific mutations of the mutant VSVG envelope protein are K63Q and R370A.

3. A lentiviral vector, characterized in that, The lentiviral vector comprises a nucleotide or fragment thereof encoding an envelope protein as described in any one of claims 1-2.

4. A lentivirus, characterized in that, The lentivirus contains the envelope protein as described in any one of claims 1-2, or the lentivirus is obtained by viral packaging using the lentivirus vector system as described in claim 3.

5. The lentivirus according to claim 4, characterized in that, The lentivirus includes a chimeric membrane protein selected from at least one of exCD58-TM, antiCD7 scFv-TM, or antiCD2 scFv-TM; the chimeric membrane protein can be expressed on the surface of the lentivirus and specifically bind to targeting markers on the surface of NK cells, T cells, or CIK cells.

6. The lentivirus according to claim 5, characterized in that, The amino acid sequence of the exCD58-TM is shown in SEQ ID NO.9, the amino acid sequence of the antiCD7 scFv-TM is shown in SEQ ID NO.11, and the amino acid sequence of the antiCD2 scFv-TM is shown in SEQ ID NO.

13.

7. The lentivirus according to claim 5, characterized in that, The nucleotide sequence encoding the exCD58-TM is shown in SEQ ID NO.10, the nucleotide sequence encoding the antiCD7 scFv-TM is shown in SEQ ID NO.12, and the nucleotide sequence encoding the antiCD2 scFv-TM is shown in SEQ ID NO.

14.

8. A cell, characterized in that, The cell genome integrates exogenous nucleotides or fragments thereof encoding the envelope protein as described in any one of claims 1-2; or the cell is prepared by cell transduction from the lentiviral vector as described in claim 3; or the cell is prepared by cell transduction from the lentivirus as described in any one of claims 4-7.

9. The application of the envelope protein according to any one of claims 1-2, the lentiviral vector according to claim 3, or the lentivirus according to claims 4-7 in packaging viruses, increasing viral titers, reducing toxicity to packaging cells, and / or improving cell transduction efficiency.

10. A drug or drug composition, said drug being made from any one of the following: the envelope protein of any one of claims 1-2, the lentiviral vector of claim 3, the lentivirus of any one of claims 4-7, or the cell of claim 9; And pharmaceutically acceptable excipients.

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