Transfer plasmid, packaging system of lentivirus and application in preparation of lentivirus, CAR-T cell and kit

Abstract

The application provides a transfer plasmid of a lentivirus, a packaging system and application in preparation of the lentivirus, CAR-T cells and a kit, and particularly relates to a lentivirus packaging system for CAR-T, which comprises a transfer plasmid, the transfer plasmid comprises a virus promoter, a CAR promoter, a coding nucleotide sequence of a CAR expression region and a virus PolyA, wherein the virus promoter is selected from RSV, the CAR promoter is selected from SFFV and EF1 alpha promoter, the hinge and transmembrane region of the CAR expression region are from CD8a or CD8b, and the virus PolyA is selected from SV40 polyA. The lentivirus packaged by the packaging system has high packaging efficiency and transduction titer, so as to meet the demand of high production of the lentivirus for CAR-T cell treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a transfer plasmid for lentiviruses, a packaging system, and their applications in lentiviruses, CAR-T cells, and kits. Background Technology

[0002] Lentiviral vector systems (LV) are commonly used in cell gene therapy for the preparation of CAR-T cells, which involves delivering CARs into T cells to generate CAR-T cells. LV is typically obtained by co-transfecting host cells with transfer plasmids (containing the target gene and essential regulatory sequences), packaging plasmids (providing viral structural proteins), envelope plasmids (determining host range), and other helper plasmids in a specific ratio. The resulting virus-like particles (lentiviruses) selectively infect recipient cells.

[0003] CAR-T, short for Chimeric Antigen Receptor T-Cell Immunotherapy, is similar to other immunotherapies in that it utilizes the patient's own immune cells to eliminate cancer cells. A chimeric antigen receptor (CAR) consists of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. CAR-T cell therapy involves genetically recombining the CAR structure to obtain a recombinant lentivirus, which is then introduced into the patient's T cells. This results in CAR-T cells that express the chimeric antigen receptor and perform the function of killing tumor cells.

[0004] However, in actual production, the lentiviral packaging titers used for CAR-T are generally low, which restricts the large-scale preparation and clinical application of CAR-T cells. Current technologies typically improve viral titers by optimizing cell culture conditions, transfection methods, or packaging system composition, but these methods have limited effectiveness and are complex. Therefore, a more comprehensive approach is needed to improve viral titers.

[0005] Existing plasmid vectors used for lentiviral packaging still have design flaws in transcriptional regulatory elements, coding sequence structures, and target gene expression elements. These flaws may lead to insufficient expression efficiency of virus-related genes, low viral particle assembly efficiency, or low target gene expression efficiency in packaging cells, thus limiting further improvements in lentiviral yield and functional titer. Designing a high-titer CAR-T lentiviral packaging system remains a problem to be solved. Summary of the Invention

[0006] To improve the packaging efficiency and transduction titer of lentiviruses for CAR-T without changing the production process, this invention provides a lentiviral packaging system for CAR-T. This packaging system includes a transfer plasmid comprising a viral promoter, a CAR promoter, a coding nucleotide sequence for the CAR expression region, and a viral PolyA. The viral promoter is an RSV promoter, the CAR promoter is selected from SFFV and EF1α promoters, the hinge and transmembrane region of the CAR expression region are derived from CD8a or CD8b, and the viral PolyA is SV40 polyA.

[0007] In some embodiments, the present invention provides a lentiviral packaging system for CAR-T, the packaging system comprising a transfer plasmid, the transfer plasmid comprising a viral promoter, a CAR promoter, a coding nucleotide sequence of a CAR expression region, and a viral PolyA, wherein the viral promoter is an RSV promoter, the CAR promoter is selected from SFFV and EF1α promoters, the hinge and transmembrane region of the CAR expression region are derived from CD8a or CD8b, and the viral PolyA is SV40 polyA, and when the CAR promoter is EF1α, the hinge and transmembrane region of the CAR expression region are derived from CD8b. Preferably, the CAR promoter is an SFFV promoter, and the hinge and transmembrane region of the CAR expression region are derived from CD8b.

[0008] In some implementations, the viral promoter is the RSV promoter, the CAR promoter is the SFFV promoter, the hinge and transmembrane region of the CAR expression region are derived from CD8a, and the viral PolyA is SV40 polyA.

[0009] In some implementations, the viral promoter is the RSV promoter, the CAR promoter is the SFFV promoter, the hinge and transmembrane region of the CAR expression region are derived from CD8b, and the viral PolyA is SV40 polyA.

[0010] In some implementations, the viral promoter is the RSV promoter, the CAR promoter is the EF1α promoter, the hinge and transmembrane region of the CAR expression region are derived from CD8b, and the viral PolyA is SV40 polyA.

[0011] In addition to the regions described above, the transfer plasmid provided by this invention also includes other common and necessary elements required to maintain the normal operation of the transfer plasmid, such as lentiviral long terminal repeat (LTR) sequences and spacer sequences between the elements.

[0012] Transfer plasmids, also known as gene transfer vectors or expression vectors, are the core component of lentiviral vector systems. Essentially, they are modified plasmid DNA whose sole and most important function is to carry the target gene (or foreign sequence) to be delivered to the target cell and integrate it into the host cell's genome, achieving long-term, stable expression.

[0013] Viral promoters typically refer to the 5' long terminal repeat promoter of the viral vector itself. In self-inactivating vectors, it is usually inactivated after integration. Therefore, modern CAR vectors also include potent CAR promoters to drive CAR gene transcription, such as: EF1α (elongation factor-1α) promoter: provides potent and durable constitutive expression in most mammalian cells (especially T cells). It is one of the most commonly used promoters in CAR-T vectors, ensuring stable and high expression of the CAR protein. CMV (cytomegalovirus) promoter: has extremely high initiation strength, but may exhibit expression silencing in vivo (especially in T cells), i.e., expression levels decrease over time. EF1α generally has more stable long-term expression characteristics. The promoter determines the transcriptional efficiency of the CAR gene, directly affecting the expression level of CAR on the surface of T cells. Insufficient expression levels lead to poor therapeutic effects, while excessively high levels may cause T cell exhaustion. Therefore, the CAR promoter should be selected in conjunction with other components. In some embodiments, the viral promoter used in this invention is RSV. In some embodiments, the CAR promoter used in this invention is the EF1α promoter.

[0014] The CAR expression region provided by this invention includes a signal peptide domain, an scFv target cell recognition domain, a hinge and transmembrane region, a co-stimulatory domain, and an activation domain.

[0015] The signal peptide domain, a short amino acid sequence located at the N-terminus of a protein, guides newly synthesized CAR proteins into the secretory pathway. It directs the CAR being synthesized on the ribosome to the endoplasmic reticulum, where it is subsequently modified and processed by the Golgi apparatus, and finally transported and anchored to the cell membrane of T cells. Without the correct signal peptide, the CAR protein may fail to reach the membrane surface and instead be degraded intracellularly. Commonly used signal peptides are derived from natural membrane proteins (such as CD8α or the heavy chain of IgG). In some embodiments, the signal peptide domain used in this invention is derived from CD8a.

[0016] The scFv target cell recognition domain is formed by the light chain variable region and heavy chain variable region of an antibody (usually a monoclonal antibody) linked by a short, artificially designed peptide chain. It specifically recognizes and binds to antigens on the surface of tumor cells (such as CD19, BCMA, etc.), and is a determining element of CAR targeting. Different scFvs target different tumor antigens, and their binding affinity needs careful optimization; too weak an affinity will be ineffective, while too strong an affinity may lead to off-target toxicity or T cell overactivation. In some embodiments, the scFv target cell recognition domain used in this invention is FMC63, through which CAR-T cells target the CD19 antigen. In some embodiments, the lentiviral packaging system for CAR-T provided by this invention is a CD19-targeting CAR-T lentiviral packaging system. In some embodiments, the scFv target cell recognition domain can also target other antigens.

[0017] The hinge region is a structural domain connecting the scFv (cell fungus) and the transmembrane region, allowing the scFv to extend and orient more freely to contact the antigen and overcome the steric hindrance of the cell membrane surface. Common sources include the hinge regions of CD8α or IgG.

[0018] The transmembrane region is a hydrophobic amino acid sequence, typically consisting of 20-25 amino acids. In the CAR coding sequence, it is located between the extracellular hinge region and the intracellular signaling domain. In the post-translational protein structure, it spans the lipid bilayer of the T cell membrane, firmly anchoring the entire CAR molecule to the cell membrane.

[0019] In some embodiments, the hinge region and the transmembrane region used in this invention are connected together, i.e., the hinge and the transmembrane region are derived from CD8a or CD8b.

[0020] The co-stimulatory domain is an enhancement module of the CAR molecule. When CAR binds to the antigen, in addition to the activation signal, the co-stimulatory signal provides a "second signal," which is essential for the complete activation, proliferation, survival, memory formation, and powerful killing function of T cells. Without the co-stimulatory signal, T cells are prone to becoming incompetent or undergoing apoptosis.

[0021] The activation domain is typically the intracellular segment of the CD3ζ chain. When a CAR binds to an antigen and undergoes dimerization / oligomerization, the immune receptor tyrosine activation motif on CD3ζ is phosphorylated, initiating downstream signaling cascade reactions (such as activation of kinases like ZAP70), ultimately leading to T cell activation and the exercise of killing functions (releasing perforin, granzymes, etc.). It is a "basic activation module" that all functional CARs must possess.

[0022] The lentiviral packaging system provided by this invention includes, in addition to the transfer plasmid for delivering the target gene, auxiliary plasmids necessary for packaging lentiviruses, such as packaging plasmids providing Gag and Pol proteins, REV expression plasmids providing Rev proteins to assist viral RNA exiting the nucleus, and envelope plasmids providing envelope proteins.

[0023] In some embodiments, the coding nucleotide sequence of the CAR expression region of the transfer plasmid provided by the present invention is shown in SEQ ID NO: 12. In some embodiments, the CAR expression region can be further optimized; specifically, the optimized CAR expression region is shown in SEQ ID NO: 13.

[0024] In some embodiments, the nucleotide sequence of the viral promoter is as shown in SEQ ID NO: 2 or a mutant sequence thereof. The mutant sequence can be obtained by making single-point or multi-point mutations based on the sequence shown in SEQ ID NO: 2, such as replacing, inserting or deleting specific bases in the core region of the promoter to enhance its transcription initiation efficiency or binding ability to transcription factors, and the mutated sequence can still maintain the basic function of the viral promoter.

[0025] In some implementations, the nucleotide sequence of the CAR promoter is as shown in SEQ ID NO:3 or SEQ ID NO:4, or a mutant sequence thereof.

[0026] In some embodiments, the nucleotide sequences encoding the hinge and transmembrane regions of the CAR expression region encode amino acid sequences as shown in SEQ ID NO: 7 or SEQ ID NO: 8, or mutant sequences thereof. These mutant sequences can be obtained by single-point or multi-point mutations based on the amino acid sequences shown in SEQ ID NO: 7 or SEQ ID NO: 8. For example, the flexible amino acid residues in the hinge region can be replaced to enhance its structural flexibility, or the hydrophobic amino acid residues in the transmembrane region can be adjusted to optimize its binding ability to the cell membrane. The mutated sequences retain the basic functions of the hinge and transmembrane regions, such as connecting the scFv to the intracellular signaling domain, anchoring the CAR molecule to the cell membrane, and supporting the effective binding of the CAR to the antigen.

[0027] In some embodiments, the nucleotide sequences encoding the hinge and transmembrane region of the CAR expression region encode an amino acid sequence as shown in SEQ ID NO: 8, or a mutant of SEQ ID NO: 8, having at least 70%, at least 80%, preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the original SEQ ID NO: 8 sequence.

[0028] In some embodiments, the nucleotide sequence of the virus PolyA is as shown in SEQ ID NO: 11 or a mutated sequence thereof. In some embodiments, the nucleotide sequence of the virus PolyA is as shown in SEQ ID NO: 11 or a mutated sequence thereof, which can be obtained by single-point or multi-point mutation based on the sequence shown in SEQ ID NO: 11, for example by adjusting the length of the PolyA tail or specific base sequences to enhance its transcription termination efficiency or improve mRNA stability, while the mutated sequence still maintains the basic function of the virus PolyA.

[0029] In some embodiments, the packaging system includes a transfer plasmid comprising a viral promoter, a CAR promoter, a nucleotide sequence encoding the CAR expression region, and a viral PolyA, wherein the viral promoter is shown in SEQ ID NO: 2, the CAR promoter is shown in SEQ ID NO: 4, the nucleotide sequence encoding the hinge and transmembrane region of the CAR expression region is shown in SEQ ID NO: 8, and the viral PolyA is shown in SEQ ID NO: 11.

[0030] The present invention also provides a transfer plasmid for packaging lentiviruses, the transfer plasmid comprising a viral promoter, a viral PolyA, a CAR promoter, and a nucleotide sequence encoding a CAR expression region, wherein the viral promoter is shown in SEQ ID NO: 2, the CAR promoter is shown in SEQ ID NO: 4, the nucleotide sequence encoding the hinge and transmembrane region of the CAR expression region is shown in SEQ ID NO: 8, and the viral PolyA is shown in SEQ ID NO: 11.

[0031] This invention also provides a method for pseudo-packaging lentiviruses, using any of the lentivirus packaging systems described above. The method includes co-transfecting a CAR transfer plasmid with helper plasmids such as viral structural protein expression plasmids into host cells, achieving efficient packaging of lentivirus particles through transient or stable transfection.

[0032] The present invention also provides a pseudomorphic lentivirus, including a lentivirus packaged using the above-described lentivirus pseudomorphic packaging method.

[0033] In some embodiments, the lentiviruses provided by this invention can be used for in vivo or in vitro CAR-T therapy or for the preparation of CAR-T cells. CAR-T cells refer to T cells (CAR-T) capable of expressing chimeric antigen receptors (CARs). CARs consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. The advantages of CAR-T therapy are: First, its binding to tumor antigens does not depend on HLA presentation, effectively avoiding the immune escape mechanism of HLA downregulation in tumor cells; Second, since the basic principle of CAR-T therapy is the specific binding of antibodies to antigens or a specific target, and the CAR structure includes a tumor-associated extracellular antigen-binding domain, CAR-T therapy is a specific targeted therapy. In comparison, traditional cell immunotherapies such as cytokine-induced killer cells (CIKs) and natural killer cells (NKs) are mostly non-targeted; Third, CAR-T therapy can generate a large number of T cells with tumor-killing efficacy in a short period of time. Therefore, CAR-T therapy has significant clinical application prospects for the cure of tumors. In some embodiments, the CAR-T cells prepared by this invention target cells with CD19 antigen on their surface.

[0034] This invention also provides a kit comprising a transfer plasmid for packaging lentivirus as defined above, or a pseudotyped lentivirus as defined above, or CAR-T cells as defined above. In some embodiments, the kit comprises a transfer plasmid for packaging lentivirus as described above, and optionally also comprises helper plasmids required for virus packaging, transfection reagents, and instructions for use, suitable for research and preclinical studies. The components are optimized in proportion to ensure efficient virus packaging and stable production. In some embodiments, the kit comprises a pseudotyped lentivirus as described above, and optionally also comprises instructions for use, suitable for research and preclinical studies. In some embodiments, the kit comprises CAR-T cells as described above, and optionally also comprises instructions for use, suitable for research and preclinical studies.

[0035] This invention also relates to pharmaceutical compositions comprising pseudotyped lentivirus or CAR-T cells as defined above and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be administered via various routes, including but not limited to intravenous injection, local injection, or oral administration, and can be formulated and dose-optimized according to disease type and treatment needs to ensure effective drug concentration and safety at the target site. This invention also relates to a method for treating a subject in need of the drug, comprising administering a therapeutically effective amount of pseudotyped lentivirus as defined above to the subject in need of the drug.

[0036] In the context of this invention, "subject" refers to a human or non-human mammal, such as rodents (rats, mice, rabbits), primates (chimpanzees), felines (cats), and canines (dogs). Preferably, the subject is a human.

[0037] This invention provides a lentiviral packaging system for CAR-T therapy. The system includes a transfer plasmid comprising a viral promoter, a CAR promoter, a coding nucleotide sequence for the CAR expression region, and a viral polyA. The viral promoter is an RSV promoter, and the CAR promoter is selected from SFFV and EF1α promoters. The hinge and transmembrane region of the CAR expression region are derived from CD8a or CD8b, and the viral polyA is SV40 polyA. Lentiviral viruses packaged using this system improve the packaging efficiency and transduction titer of lentiviruses for CAR-T therapy without altering the production process, thereby meeting the high-yield demand for lentiviruses in CAR-T cell therapy. Attached Figure Description

[0038] To better understand the present invention and more clearly demonstrate how to implement it, features of embodiments according to the present invention are now illustrated by way of example and with reference to the accompanying drawings, wherein:

[0039] Figure 1 Schematic diagram of the YM-CARLi transfer plasmid structure.

[0040] Figure 2 Results of CAR lentiviral transduction titer analysis of three transfer plasmids: YM-CARLi, YM-CARLi_1.1, and YM-CARLi_2.0.

[0041] Figure 3 Results of CAR lentiviral transduction titer analysis of four transfer plasmids: YM-CARLi_2.0, YM-CARLi_2.1, YM-CARLi_2.2, and YM-CARLi_2.3.

[0042] Figure 4 Results of CAR lentiviral transduction titer analysis before and after CAR expression region optimization in YM-CARLi_2.0. Detailed Implementation

[0043] Definitions: To provide a clear and consistent understanding of the terminology used in this specification, some definitions are provided below. Furthermore, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] When used in conjunction with the term "comprising" in the claims and / or description, the word "a" can mean "one," but it also aligns with the meanings of "one or more," "at least one," and "one or more." Similarly, the word "another" can mean at least a second or more.

[0045] The terms “comprising” (and any form of inclusion, such as “comprising” and “including”), “having” (and any form of having, “having”, “including”, and “containing”) as used in this specification and claims are inclusive and open-ended and do not exclude additional unlisted elements or processing steps.

[0046] As used in this article, "pseudovirus" or "pseudotyped lentivirus" are interchangeable terms. The process of introducing a heterologous envelope glycoprotein into the core of a lentiviral vector is called "pseudotypening." Pseudotyped lentiviruses are typically produced by co-transfecting a plasmid encoding a heterologous envelope glycoprotein into packaging cells along with a lentiviral vector system, resulting in recombinant lentiviral particles carrying the heterologous envelope glycoprotein on their surface. These particles have a core structure similar to the original lentivirus, but their surface envelope glycoprotein determines the virus's targeting and invasion efficiency in infecting host cells. Through pseudotypening technology, envelope glycoproteins from different sources can be flexibly replaced to regulate the tissue specificity or cell tropism of the viral vector, thereby expanding its application potential in gene therapy, vaccine delivery, and functional genomics research.

[0047] As used herein, the term "variant" or "mutant" means a mutant that has at least 70%, at least 80%, preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the initial sequence, provided that the variant sequence retains the ability of the initial sequence.

[0048] Unless otherwise specified, the experimental methods described below are standard methods, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.

[0049] Examples: The present invention will be more readily understood by referring to the following examples, which are used to illustrate the invention and should not be construed as limiting the scope of the invention in any way.

[0050] Unless otherwise defined or the context clearly requires, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention.

[0051] Although the invention has been described in detail with reference to embodiments thereof, these embodiments are provided for illustration and not limitation. Other embodiments that can be obtained according to the principles of the invention fall within the scope defined by the claims of the invention.

[0052] Experimental methods not specifically described in this invention are performed according to the methods described in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (4th Edition) or according to the relevant product instructions. When used herein, unless otherwise stated, all terms in this invention should be understood in their ordinary meaning as known in the art. Unless otherwise specified, all biological reagents used in this invention are commercially available.

[0053] Example 1: Construction of CAR transfer plasmid and lentivirus packaging

[0054] 1.1 Design of transfer plasmid vectors

[0055] Three different transfer plasmids were designed, denoted as YM-CARLi, YM-CARLi_1.1, and YM-CARLi_2.0, respectively. The structure of YM-CARLi is as follows: Figure 1 As shown. Using YM-CARLi as the starting plasmid, the CAR hinge-transmembrane region CD8a of YM-CARLi was... Figure 1 The CD8a Hinge TM portion in the plasmid map shown is replaced with CD8b to obtain the YM-CARLi_1.1 plasmid. Using YM-CARLi as the starting plasmid, the following modifications are made: (1) The viral promoter CMV ( Figure 1(1) Replace the CMV enhancer and its CMV promoter portion in the diagram shown with the promoter RSV; (2) Add SV40 polyA downstream of 3'LTR; (3) Replace the CAR-expressing promoter EF1a with SFFV to obtain YM-CARLi_2.0. Partial regions of YM-CARLi, YM-CARLi_1.1, and YM-CARLi_2.0 are shown in Table 1. The nucleotide sequences of the CMV promoter are shown in SEQ ID NO: 1, the RSV promoter in SEQ ID NO: 2, the EF1α promoter in SEQ ID NO: 3, the SFFV promoter in SEQ ID NO: 4, the CD8a signal peptide in SEQ ID NO: 5, the FMC63 scFv in SEQ ID NO: 6, the CD8a hinge transmembrane region in SEQ ID NO: 7, the CD8b hinge transmembrane region in SEQ ID NO: 8, the 4-1BB co-stimulatory domain in SEQ ID NO: 9, the CD3ζ activation domain in SEQ ID NO: 10, and the viral polyA nucleotide sequence are shown in SEQ ID NO: 1. As shown in NO:11, the nucleic acid sequences of the three groups of transfer plasmids were synthesized separately. After obtaining the correct plasmids, they were extracted and used for lentivirus packaging experiments.

[0056] Table 1

[0057]

[0058] 1.2 Packaging lentiviruses using HEK293TH suspension cells

[0059] HEK293TH seeding: In T125mL cell culture flasks, at a concentration of 1-1.5 × 10⁻⁶ cells / mL. 6 Cells were seeded at 30 mL of BalanCD HEK293 (FUJIFILM) + 4 mM L-Glutamine + 1% penicillin / streptomycin culture system and cultured at 37°C, 5% CO2 incubator, and 180 rpm for 18-20 h.

[0060] Virus packaging: Shuttle plasmids (YM-CARLi, YM-CARLi_1.1, YM-CARLi_2.0, etc.) were added sequentially at a mass ratio of 48:28:16:5, along with helper plasmids 1 (expressing Gag-pol protein), 2 (expressing Rev protein), and 3 (expressing envelope protein VSVG). After mixing, 300 μL of BalanCD HEK293 medium was added and thoroughly mixed to prepare a DNA solution. 300 μL of BalanCD HEK293 medium and 100 μL of PEI pro (polyplustransfection) were added to prepare a PEI solution. The PEI solution was added dropwise to the DNA solution, vortexed thoroughly, and incubated for 15–20 min. The mixture was then added dropwise to cultured cells for transfection. HEK293TH culture flasks were incubated at 37°C and 180 rpm in a 5% CO2 incubator.

[0061] Twenty-four hours after cell transfection, BalanCD HEK293 Viral Feed (FUJIFILM) was added at 12% of the system volume.

[0062] Forty-eight hours after cell transfection, the viral supernatant was collected, which was the crude lentivirus. Sucrose was added, and the mixture was aliquoted and stored at -80°C.

[0063] 1.3. Lentiviral transduction titer test

[0064] Jurkat-T cells were counted, and the viable cell density was adjusted to 5E5 cells / ml. The cells were seeded in 96-well cell culture plates at a volume of 100 μl per well.

[0065] The crude lentivirus was serially diluted 5-fold. The stock solution was labeled as dilution gradient 1, the 5-fold diluted sample as dilution gradient 2, and so on. Each dilution consisted of 30 μl of the previous dilution of stock virus solution + 120 μl of 1640 complete culture medium.

[0066] Add 100 μL of diluted lentivirus to each well and mix thoroughly with the cells by pipetting. Add an equal volume of virus dilution to the negative control wells and mix by pipetting. Incubate in a CO2 incubator at 37°C for 3 days.

[0067] Cells were harvested and stained with PE anti-CAR antibody to determine transduction titers. The transduction titers (TU / ml) of the virus in each group were calculated retrospectively using the positive rate and dilution factor. The calculated results are as follows: Figure 2 As shown. (Through) Figure 2 It can be seen that the group with the highest titer of CARLi_2.0 is 2.25 times higher than that of CARLi.

[0068] Example 2: Optimization of CAR transfer plasmids

[0069] 2.1 Plasmid Vector Design

[0070] Using the transfer plasmid CARLi_2.0 from Example 1 as a template, three sets of transfer plasmids were set up, denoted as YM-CARLi_2.1, YM-CARLi_2.2, and YM-CARLi_2.3, respectively. Some regions are shown in Table 2.

[0071] Table 2

[0072]

[0073] 2.2 Packaging lentiviruses using HEK293TH suspension cells

[0074] HEK293TH seeding: In T125mL cell culture flasks, at a concentration of 1-1.5 × 10⁻⁶ cells / mL. 6 Cells were seeded at 30 mL of BalanCD HEK293 (FUJIFILM) + 4 mM L-Glutamine + 1% penicillin / streptomycin culture system and cultured at 37°C, 5% CO2 incubator, and 180 rpm for 18-20 h.

[0075] Virus packaging: Shuttle plasmids (YM-CARLi_2.0, YM-CARLi_2.1, YM-CARLi_2.2, YM-CARLi_2.3, etc.) and helper plasmids 1 (expressing Gag-pol protein), 2 (expressing Rev protein), and 3 (expressing envelope protein VSVG) were added sequentially at a mass ratio of 50 μg total plasmid (48:28:16:5). After mixing, 300 μl of BalanCD HEK293 medium was added and thoroughly mixed to prepare a DNA solution. 300 μl of BalanCD HEK293 medium and 100 μl of PEI pro (polyplus transfection) were added to prepare a PEI solution. The PEI solution was added dropwise to the DNA solution, vortexed thoroughly, and incubated for 15–20 min. The mixture was then added dropwise to cultured cells for transfection. HEK293TH culture flasks were incubated at 37°C and 180 rpm in a 5% CO2 incubator.

[0076] Twenty-four hours after cell transfection, BalanCD HEK293 Viral Feed (FUJIFILM) was added at 12% of the system volume.

[0077] Forty-eight hours after cell transfection, the viral supernatant was collected, which was the crude lentivirus. Sucrose was added, and the mixture was aliquoted and stored at -80°C.

[0078] 2.3 Lentiviral transduction titer assay

[0079] Jurkat-T cells were counted, and the viable cell density was adjusted to 5E5 cells / ml. The cells were seeded in 96-well cell culture plates at a volume of 100 μl per well.

[0080] The crude lentivirus was serially diluted 5-fold. The stock solution was labeled as dilution gradient 1, the 5-fold diluted sample as dilution gradient 2, and so on. Each dilution consisted of 30 μl of the previous dilution of stock virus solution + 120 μl of 1640 complete culture medium.

[0081] Add 100 μL of diluted lentivirus to each well and mix thoroughly with the cells by pipetting. Add an equal volume of virus dilution to the negative control wells and mix by pipetting. Incubate in a CO2 incubator at 37°C for 3 days.

[0082] Cells were harvested and stained with PE anti-CAR antibody to determine transduction titers. The transduction titers (TU / ml) of each group were calculated retrospectively using the positive rate and dilution factor. The results are shown below. Figure 3 As shown. (Through) Figure 3 It can be seen that both CARLi_2.1 and CARLi_2.3 have high transduction titers, and CARLi_2.1 is significantly superior to other transfer plasmids.

[0083] Example 3: Codon Optimization of CAR Expression Region Sequence

[0084] 3.1. Plasmid Vector Design

[0085] The CAR expression region in the aforementioned CARLi_2.0 was codon optimized. The nucleotide sequence of the CAR expression region before optimization is shown in SEQ ID NO: 12, and the nucleotide sequence of the CAR expression region after optimization is shown in SEQ ID NO: 13. The optimized plasmid is denoted as YM-optiCARLi_2.0.

[0086] 3.2 Packaging lentiviruses using HEK293TH suspension cells

[0087] HEK293TH seeding: In T125mL cell culture flasks, at a concentration of 1-1.5 × 10⁻⁶ cells / mL. 6 Cells were seeded at 30 mL of BalanCD HEK293 (FUJIFILM) + 4 mM L-Glutamine + 1% penicillin / streptomycin culture system and cultured at 37°C, 5% CO2 incubator, and 180 rpm for 18-20 h.

[0088] Virus packaging: Shuttle plasmids (YM-CARLi_2.0 and YM-opCARLi_2.0, respectively), helper plasmid 1 (expressing Gag-pol protein), helper plasmid 2 (expressing Rev protein), and helper plasmid 3 (expressing envelope protein VSVG) were added sequentially at a mass ratio of 50 μg total plasmid (48:28:16:5). After mixing, 300 μl of BalanCD HEK293 medium was added and thoroughly mixed to prepare a DNA solution. 300 μL of BalanCD HEK293 medium and 100 μL of PEI pro (polyplustransfection) were added to prepare a PEI solution. The PEI solution was added dropwise to the DNA solution, vortexed thoroughly, and incubated for 15–20 min. The mixture was then added dropwise to cultured cells for transfection. HEK293TH culture flasks were incubated at 37°C and 180 rpm in a 5% CO2 incubator.

[0089] Twenty-four hours after cell transfection, BalanCD HEK293 Viral Feed (FUJIFILM) was added at 12% of the system volume.

[0090] Forty-eight hours after cell transfection, the viral supernatant was collected, which was the crude lentivirus. Sucrose was added, and the mixture was aliquoted and stored at -80°C.

[0091] 3.3. Lentiviral transduction titer assay

[0092] Jurkat-T cells were counted, and the viable cell density was adjusted to 5E5 cells / ml. The cells were seeded in 96-well cell culture plates at a volume of 100 μl per well.

[0093] The crude lentivirus was serially diluted 5-fold. The stock solution was labeled as dilution gradient 1, the 5-fold dilution as dilution gradient 2, and so on. Each dilution consisted of 30 μl of the previous dilution of stock virus solution + 120 μl of 1640 complete culture medium.

[0094] Add 100 μL of diluted lentivirus to each well and mix thoroughly with the cells by pipetting. Add an equal volume of virus dilution to the negative control wells and mix by pipetting. Incubate in a CO2 incubator at 37°C for 3 days.

[0095] Cells were harvested and stained with PE anti-CAR antibody to determine transduction titers. The transduction titers (TU / ml) of each group were calculated retrospectively using the positive rate and dilution factor. The results are shown below. Figure 4 As shown. By Figure 4 It can be seen that after codon optimization of the CAR expression region, the packaging titer increased by more than three times compared with that before optimization.

[0096] Although the invention has been described in detail with reference to embodiments thereof, these embodiments are provided for illustration and not limitation. Other embodiments that can be obtained according to the principles of the invention fall within the scope defined by the claims of the invention.

Claims

1. A lentiviral packaging system for CAR-T, characterized in that, The packaging system includes a transfer plasmid comprising a viral promoter, a CAR promoter, a nucleotide sequence encoding a CAR expression region, and a viral PolyA, wherein the nucleotide sequence of the viral promoter is shown in SEQ ID NO: 2, the nucleotide sequence of the CAR promoter is shown in SEQ ID NO: 4, the nucleotide sequence of the hinge and transmembrane region of the CAR expression region encodes an amino acid sequence as shown in SEQ ID NO: 8, and the nucleotide sequence of the viral PolyA is shown in SEQ ID NO:

11.

2. The lentiviral packaging system for CAR-T as described in claim 1, characterized in that, The packaging system also includes an auxiliary plasmid for packaging lentiviruses.

3. The lentiviral packaging system for CAR-T as described in claim 1 or 2, characterized in that, The nucleotide sequence encoding the CAR expression region of the transfer plasmid is shown in SEQ ID NO:

12.

4. The lentiviral packaging system for CAR-T as described in claim 3, characterized in that, The coding nucleotide sequence of the CAR expression region has been codon optimized, and the optimized sequence is shown in SEQ ID NO:

13.

5. A transfer plasmid for packaging lentiviruses, characterized in that, The transfer plasmid comprises a viral promoter, a CAR promoter, a nucleotide sequence encoding the CAR expression region, and a viral PolyA, wherein the nucleotide sequence of the viral promoter is shown in SEQ ID NO: 2, the nucleotide sequence of the CAR promoter is shown in SEQ ID NO: 4, the nucleotide sequence of the hinge and transmembrane region of the CAR expression region encodes the amino acid sequence shown in SEQ ID NO: 8, and the nucleotide sequence of the viral PolyA is shown in SEQ ID NO:

11.

6. A method for pseudo-packaging lentiviruses, characterized in that, The lentiviral packaging system according to any one of claims 1 to 4 or the transfer plasmid according to claim 5 is used for pseudo-packaging.

7. A pseudomorphic lentivirus, characterized in that, The pseudomorphic lentivirus is packaged using the pseudomorphic packaging method for lentivirus as described in claim 6.

8. A method for preparing CAR-T cells, characterized in that, This includes transfecting T cells with the pseudotyped lentivirus as described in claim 7.

9. A reagent kit, characterized in that, The kit contains the transfer plasmid for packaging lentivirus as described in claim 5 or the pseudotyped lentivirus as described in claim 7.

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