Use of cholesterol synthesis enzyme in preparation of products for enhancing the killing function of CAR-T cells and their derivatives

Abstract

The application discloses application of a cholesterol synthesis enzyme in preparation of a product for enhancing killing function of CAR-T cells and derivatives thereof, wherein the cholesterol synthesis enzyme is SQLE or LSS, and the product enhances the killing function by separately knocking out SQLE or separately overexpressing LSS in the CAR-T cells and the derivatives thereof. The application significantly enhances the killing ability of the CAR-T cells and the derivatives thereof by knocking out a key enzyme SQLE in cholesterol synthesis or overexpressing LSS.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of a cholesterol synthase in the preparation of products for enhancing the cytotoxic function of CAR-T cells and their derivatives. Background Technology

[0002] CAR-T cells (chimeric antigen receptor T cells) are autologous or allogeneic T cells modified through genetic engineering. Their core feature is the expression of a chimeric antigen receptor (CAR) on the cell surface. This receptor consists of an antigen recognition domain (single-chain antibody scFV), a transmembrane domain, and an intracellular signaling domain. It can bypass MHC molecule restrictions, directly recognize specific antigens on the surface of tumor cells, rapidly activate T cell killing function, and specifically eliminate target cells, making it an important means of treating malignant hematological malignancies. However, CAR-T cells suffer from problems such as insufficient infiltration in solid tumors, easy depletion, and strong toxic side effects. This has led to the development of various optimized products, including dual / multi-target CAR-T cells, armored CAR-T cells, universal CAR-T cells, and CAAR-T cells (chimeric autoantibody receptor T cells).

[0003] Among related technologies, CAR-T-related technologies have lower killing function due to factors such as tumor microenvironment inhibition.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing an application of cholesterol synthase in the preparation of products for enhancing the killing function of CAR-T cells and their derivatives. This application significantly enhances the killing ability of CAR-T cells by knocking out the key cholesterol synthesis enzyme SQLE or overexpressing LSS.

[0006] To achieve the above objectives, this application provides the following technical solution: According to one aspect of this application, there is provided the use of a cholesterol synthase in the preparation of a product for enhancing the killing function of CAR-T cells and their derivatives, said cholesterol synthase being SQLE or LSS, said product enhancing the killing function by knocking out SQLE alone or overexpressing LSS alone in said CAR-T cells and their derivatives.

[0007] In some embodiments, the CAR-T cells and their derivatives are CAR-T cells or CAAR-T cells.

[0008] In some embodiments, the CAR-T cells include, but are not limited to, CD19 CAR-T, BCMA CAR-T, FcRL5 CAR-T, BAFF-R CAR-T, GPRC5D CAR-T, HER2 CAR-T, EGFR CAR-T, CD38 CAR-T, CD22 CAR-T, and CD20 CAR-T cells.

[0009] In some embodiments, the CAR-T cells are CD19 CAR-T cells.

[0010] In some embodiments, the CAAR-T cells include, but are not limited to, AQP4 CAAR-T, MOG CAAR-T, NMDARCAAR-T, AchR CAAR-T, Musk CAAR-T, Dsg3 CAAR-T, LGI1 CAAR-T, GFAP CAAR-T, GAD65CAAR-T, and MBP CAAR-T cells.

[0011] In some embodiments, the CAAR-T cells are AQP4 CAAR-T or MOG CAAR-T cells.

[0012] In some embodiments, the AQP4 CAAR-T cells are AQP4 M1 CAAR-T cells or AQP4 M23 CAAR-T cells.

[0013] Compared with the prior art, the advantages and positive effects of this application are as follows: The killing ability of CAR-T cells was significantly enhanced by knocking out SQLE, a key enzyme in cholesterol synthesis, or by overexpressing LSS.

[0014] Furthermore, this application can maintain or even improve the killing efficiency of CAR-T cells under conditions of a low effector cell to target cell ratio (E:T ratio), reducing dependence on high-dose CAR-T cell infusion. By reducing the amount of CAR-T cells used in clinical treatment, the risk of excessive cytokine release is reduced, mitigating clinical side effects such as cytokine storms, thereby improving treatment safety and patient tolerability.

[0015] Furthermore, this application achieves intrinsic enhancement of CAR-T cell function without increasing the structural complexity of the CAR or introducing additional exogenous immune stimulation, thereby improving the overall efficacy and safety of CAR-T cell therapy and possessing high clinical application value. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results of the detection of the killing efficiency of CD19 CAR-T and Nalm6 co-incubation under different treatment conditions are shown in Example 1 of this application.

[0018] Figure 2 The results of co-incubation of CD19 CAR-T and Nalm6 under different treatment conditions are shown in Example 1 of this application.

[0019] Figure 3 The results of co-incubation of M1 CAAR-T and Nalm6 rAb53 under different treatment conditions are shown in Example 2 of this application.

[0020] Figure 4 The results of co-incubation of M23 CAAR-T and Nalm6 rAb53 under different treatment conditions are shown in Example 2 of this application.

[0021] Figure 5 The results of co-incubation of MOG CAAR-T and Nalm6 8-18C5 under different treatment conditions are shown in Example 3 of this application. Detailed Implementation

[0022] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0023] Terminology Explanation: Squalene epoxidase (SQLE) is a key rate-limiting enzyme in the endogenous cholesterol synthesis pathway. It is located in the endoplasmic reticulum and catalyzes the core reaction in the midstream of cholesterol synthesis. Lanosterol synthase (LSS) is the core rate-limiting enzyme in the endogenous cholesterol synthesis pathway. It is located in the endoplasmic reticulum, and its catalytic reaction is a key branch point in cholesterol synthesis. CD19 scFV: The extracellular antigen-binding domain of CART-19 is a single-chain antibody sequence (scFV) of human CD19. Hinge region: a structural domain that can enhance the binding ability between antigens and antibodies; Transmembrane region: A domain used to immobilize CARs displayed on the surface of T cells; Co-stimulatory domain: the second signal for T cell activation, such as CD28 / 4-1BB; Activation domain: the first signal for T cell activation, such as CD3ξ; CD19 CAR-T: Chimeric T cells that combine the CD19 antigen receptor, including secretory peptides, CD19 scFV, hinge region, transmembrane region, co-stimulatory domain, and activation domain, can target and specifically kill cells expressing CD19; NMOSD: Neuromyelitis optica spectrum disorder; MOGAD: MOG antibody-related disease; rAb53: A patented and recognized positive antibody for AQP4, which can specifically bind to AQP4. It is used to verify whether CAAR-T cells can be successfully killed and to construct the Nalm6 cell line expressing rAb53 as a target cell line to verify the killing effect of AQP4 on CAAR-T cells. 8-18C5: A patented and recognized positive antibody for MOG, which can specifically bind to MOG and is used to verify whether CAAR-T cells can successfully kill it. It is also used to construct the Nalm6 cell line expressing 8-18C5 as a target cell line to verify the killing effect of MOG CAAR-T cells. AQP4 CAAR-T: Chimeric T cells that combine the AQP4 antibody receptor, including the full-length AQP4 protein, transmembrane region, co-stimulatory domain, and activation domain, can target and specifically kill cells expressing AQP4 antibodies; MOG CAAR-T: Chimeric T cells that integrate MOG antibody receptors, including the extracellular domain, transmembrane region, co-stimulatory domain, and activation domain of the MOG protein; E:T ratio: The ratio of effector cells to target cells, where effector cells are CART-19, AQP4 CAAR-T, or MOGCAAR-T. FDA: U.S. Food and Drug Administration; Luciferase killing assay: This is a mature scientific method for indicating the degree of cell killing. Luciferase is pre-transferred into cells of interest, and after the killing experiment, firefly luciferase substrate is added. The chemiluminescence value is then detected. A higher number of surviving cells results in a higher chemiluminescence value, and vice versa.

[0024] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1: Knockout of squalene monooxygenase (SQLE) and overexpression of lanosterol synthase (LSS) can significantly enhance the killing ability of CD19 CAR-T cells.

[0026] 1. Construct CD19 CAR-T lentiviral plasmid vector.

[0027] The sequence required for constructing CD19 CAR-T was obtained from the patent (patent number: WO 2019 / 159193 A1). After the sequence was designed, it was synthesized by Genewiz Biotechnology Co., Ltd. and ligated with the pCDH GFP plasmid vector digested with XbaI and BamHI to obtain the CD19 CAR-T lentiviral plasmid vector, which is the plasmid vector used in this invention.

[0028] 2. Construct SQLE overexpression lentiviral plasmid vectors and LSS overexpression lentiviral plasmid vectors.

[0029] The full-length human SQLE sequence (protein ID Q14534, amino acid sequence M1-H574) and LSS sequence (protein ID P48449, amino acid sequence M1-P732) were selected from the Uniprot website. The full-length SQLE sequence is shown in SEQ ID NO:1, and the full-length LSS sequence is shown in SEQ ID NO:2. After sequence design, the sequences were synthesized by Genewiz Biotechnology Co., Ltd., and ligated into the pCDH GFP plasmid vector digested with XbaI and BamHI to obtain the SQLE overexpression lentiviral plasmid vector and the LSS overexpression lentiviral plasmid vector, which are the plasmid vectors used in this invention.

[0030] 3. Construct SQLE knockout lentivirus plasmid vectors and LSS knockout lentivirus plasmid vectors.

[0031] Based on CRISPR / Cas9 gene editing technology, specific sgRNA sequences targeting the SQLE or LSS genes were designed using the CHOPCHOP online tool to knock out SQLE or LSS in T cells. The SQLE sgRNA sequence is shown in SEQ ID NO:3, and the LSS sgRNA sequence is shown in SEQ ID NO:4. Primers were synthesized by Genewiz Biotechnology Co., Ltd., and then annealed and ligated. The ligation products were ligated with CRISPR v2 plasmids to construct lentiviral plasmids for SQLE gene knockout or LSS gene knockout. The constructed recombinant plasmids were sequenced for verification, and plasmid vectors with correct sequences were screened. Subsequently, the verified plasmids were transfected into packaging cells for amplification and identification. Finally, sgRNA sequences and corresponding lentiviral plasmid vectors that can effectively mediate SQLE or LSS gene knockout were screened.

[0032] 4. Lentiviral packaging of CD19 CAR, SQLE and LSS gene overexpression and knockout.

[0033] Lentivirals expressing target genes were packaged using the CaCl2 method.

[0034] ① Cell Culture: The 293T cell line was routinely cultured in our laboratory. The 293T cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number CL0005. Cells were placed in high-glucose DMEM medium containing 10% inactivated fetal bovine serum and incubated at 37°C in a 5% CO2 incubator according to standard methods. Cells in the logarithmic growth phase were used for experiments.

[0035] ② Cell preparation: 293T cells were resuspended in high-glucose DMEM medium containing 10% FBS in 10cm culture dishes and placed in a 37°C incubator containing 5% CO2. After the cells adhered to the wall and the density reached about 80%-90%, they were ready for transfection. The medium was changed 2 hours before transfection (6-8ml of fresh complete medium replaced the old medium).

[0036] ③ Plasmid transfection: Mix the core plasmid and packaging plasmid. Take a sterile 1.5ml EP tube, add 500ul Opti-MEM, and add 20ug of mixed plasmid (Target plasmid: psPAX2: pMD2.G = 4:3:1). Take another sterile 1.5ml EP tube, add 500ul Opti-MEM, and add 40ul Lipofectamine 2000 (Lipofectamine 2000: plasmid = 2:1). Mix thoroughly and incubate at room temperature for 5 minutes. Slowly add Lipofectamine 2000 to the plasmid tube, mix thoroughly, and incubate at room temperature for 15-20 minutes. Add 1ml of the mixture dropwise to the culture medium of 293T cells, gently shake the petri dish to mix, and incubate at 37 degrees Celsius with 5% CO2.

[0037] ④ Virus collection: After 4-6 hours, remove the old culture medium, add 12-15 ml of fresh, preheated 37°C complete culture medium, and continue to incubate the cells in an incubator. After about 72 hours, collect the supernatant into a 15 ml centrifuge tube, filter the supernatant through a 0.45 μm filter, add 4× lentivirus concentrate, incubate overnight on a shaker at 4°C, centrifuge at 3500 g for 30 minutes, resuspend the supernatant with 1 / 100 volume of PBS, aliquot and store at -80°C for later use.

[0038] ⑤ Viral titer determination (dilution counting method): Titer unit: TU / ml, which refers to the number of biologically active viral particles contained in each milliliter. "TU" is an abbreviation for "transducing units", which represents the number of viral genomes that can infect and enter target cells.

[0039] Day 1 Cell Preparation: Digest and count healthy 293T cells, then dilute to 1×10⁴ / ml and add to 96-well plates, 100 μL / well, preparing 10 wells for each virus. Incubate at 37°C in a 5% CO₂ incubator.

[0040] On the second day, add the virus: Perform 8-fold serial dilutions in EP tubes, creating 8 consecutive dilutions. The dilution method is as follows: Prepare 8 1.5ml EP tubes for each virus. Add 100ul of culture medium to each tube. Add 50ul of the original virus solution to the first tube, mix well, and then add 50ul to the second tube and mix well. Continue in this manner to create 8 dilutions. Add 50ul of the diluted virus solution to each well of a 96-well container, and bring the culture medium to a final volume of 200ul. Label the container accordingly.

[0041] On the fifth day, observe the results and calculate the titer: Flow cytometry was used to detect the infection efficiency, and the number of fluorescent cell clones with an infection efficiency of 10%-20% was selected. The titer (TU / ml) was calculated as 1000 × infection efficiency × dilution factor × 20.

[0042] 5. Extraction of peripheral blood mononuclear cells (PBMCs) from healthy adults and construction of CD19 CAR-T cells.

[0043] ①PBMC Extraction: Mix 3ml of fresh anticoagulated blood from a healthy adult with 3ml of sample diluent. In a separate 15ml centrifuge tube, add 6ml of lymphocyte separation medium. Carefully spread the diluted blood sample evenly on the separation medium. Centrifuge at 2000 rpm for 20 minutes (1 step up, 1 step down). After centrifugation, the centrifuge tube will separate into four layers from top to bottom. Collect the white ring layer, which contains mononuclear cells. Add 3 times the volume of washing buffer and mix well. Centrifuge at 2000 rpm for 5 minutes. Add 1ml of erythrocyte lysis buffer, mix well by pipetting, and incubate at room temperature for 10 minutes. Add 3 times the volume of washing buffer, mix well, and centrifuge at 2000 rpm for 5 minutes. PBMC cells can then be obtained. The cells are used for magnetic bead sorting to obtain CD3+. + T cells.

[0044] ②CD3 + T cell sorting: used to construct CD19 CAR-T cells.

[0045] The acquired PBMC count, every 10 7 Resuspend cells in 40 μL MACS Buffer, then add 10 μL Pan TCell Biotin-Antibody Cocktail, mix well, and incubate at 4°C in the dark for 5 minutes. Repeat for every 10 cells. 7Add 30 μL MACS Buffer and 20 μL Pan T Cell MicroBead Cocktail to the cells, mix well, incubate at 4°C in the dark for 10 minutes, add 3 ml MACS Buffer, centrifuge at 300 g for 10 minutes, and resuspend the cells in 3 ml MACS Buffer.

[0046] Prepare an LS sorting column in advance. Rinse the column with 3 ml of MACS Buffer, add cell suspension, and wash the column twice, adding 3 ml of MACS Buffer each time. Collect the liquid flowing out of the sorting column and centrifuge. Resuspend the cells in 1 ml of 1640 medium containing 10% FBS. After resuspending, count the cells and adjust the density to 1×10^6 / ml. Culture the cells into wells, adding the corresponding amount of CD3 / CD28 activator per ml of cells.

[0047] ③Construction of CD19 CAR-T cells.

[0048] T cells were infected 24 hours after CD3 / CD28 activation with the corresponding viral concentrate at an MOI of 5. For T cells requiring SQLE and LSS overexpression or knockout, both SQLE and LSS overexpression or knockout viruses were added simultaneously. After viral addition, the culture medium was replaced with fresh complete medium every two days. T cell infection efficiency was assessed by flow cytometry after 7-9 days, and luciferase killing assays were performed.

[0049] 6. Firefly luciferase killing assay to detect CAR-T cell killing efficiency.

[0050] The firefly luciferase killing assay is a mature scientific method for indicating the degree of cell killing. Luciferase is pre-transferred into the Nalm6B cell line, and after the killing experiment, firefly luciferase substrate is added. Chemiluminescence values ​​are then measured. The higher the number of surviving target cells and the higher the chemiluminescence value, the more CAR-T cells have been killed. Conversely, the lower the number of surviving target cells and the lower the chemiluminescence value, the more successfully the CAR-T cells have killed the target cells.

[0051] ①Nalm6 cell culture: Nalm6 cells were purchased from Stemer Biotechnology Co., Ltd., catalog number STM-CL-5240. They were placed in 1640 medium containing 10% inactivated fetal bovine serum and incubated at 37°C in a 5% CO2 incubator according to standard methods. Cells in the logarithmic growth phase were used for experiments.

[0052] ②Nalm6 cell plating: Adjust the density of the Nalm6 B cell line to 1×10⁻⁶ cells / cells. 4 / ml, cells were seeded in a white opaque microplate for chemiluminescence detection. 100ul of cells were added to each well. A portion of the cells without T cells served as a negative control, and another portion of the cells were centrifuged and resuspended in sterile water as a positive control.

[0053] ③ Add CD19 CAR-T cells, CD19 CAR-T+SQLE overexpress cells, CD19 CAR-T+LSS overexpress cells, CD19 CAR-T+sgSQLE cells, and CD19 CAR-T+sgLSS cells respectively according to an E:T ratio of 5:1, and make up the culture volume of each well to 200ul. Place the co-cultured cells in a 37-degree, 5% CO2 incubator.

[0054] ④ The killing efficiency was determined by detecting the chemiluminescence values ​​using a multi-functional microplate reader after 24 hours of co-culture. Killing efficiency = (Negative well value - Target well value) / (Negative well value - Positive well value).

[0055] Figure 1 This document illustrates the detection results of the killing efficiency of CD19 CAR-T cells co-incubated with Nalm6 under different treatment conditions in Example 1 of this application. Specifically, CD19 CAR-T+SQLE overexpression refers to CD19 CAR-T cells overexpressing SQLE; CD19 CAR-T+SQLE knockout refers to CD19 CAR-T cells with SQLE knockout; CD19 CAR-T+LSS overexpression refers to CD19 CAR-T cells overexpressing LSS; and CD19 CAR-T+sgLSS refers to CD19 CAR-T cells with LSS knockout. Figure 1 As shown, CD19 CAR-T cells overexpressing SQLE and CD19 CAR-T cells with LSS knockout exhibited weaker cytotoxic effects than the CD19 CAR-T cell group. However, knocking out SQLE or overexpressing LSS in CD19 CAR-T cells increased the cytotoxic effect of CD19 CAR-T cells. This indicates that both SQLE and LSS, two key enzymes in the cholesterol synthesis pathway, can serve as key targets for enhancing the cytotoxic effect of CAR-T cells.

[0056] To further investigate whether SQLE knockout or LSS overexpression requires CAR-T loading to enhance cytotoxicity, we proceeded to the next step of cytotoxicity testing, adjusting the Nalm6 B cell line density to 1×10⁻⁶ cells / year. 4Cells were seeded in opaque white microplates for chemiluminescence detection at / ml. 100µl of cells were added to each well. A portion of the cells without T cells served as a negative control, while another portion was centrifuged and resuspended in sterile water as a positive control. CD19 CAR-T cells, CD19 CAR-T+LSS overexpressed cells, CD19 CAR-T+sgSQLE cells, NTD-T cells, NTD-T cells+LSS overexpressed cells, and NTD-T cells+sgSQLE cells were added at different effector-to-target ratios (1:1, 2:1, 5:1). Culture medium was added to bring the volume to 200µl, and the co-cultured cells were placed in a 37°C, 5% CO2 incubator. When CD19 CAR-T cells were co-incubated with Nalm6 cells, the CD19 CAR-T cells recognized CD19 in the Nalm6 cell line and thus exerted a killing function. NTD-T cells did not carry a sequence recognizing the CD19 antigen and therefore could not exert a killing function. This patent investigates the effects of LSS overexpression and SQLE knockout in CD19 CAR-T cells and NTD-T cells, respectively, to determine whether LSS overexpression and SQLE knockout enhance T cell cytotoxicity only when T cells are capable of cytotoxic activity. Furthermore, if LSS overexpression and SQLE knockout require T cells to possess cytotoxic function to exert their enhancing effect, then LSS overexpression and SQLE knockout in CD19 CAR-T cells enhance CD19 CAR-T cell cytotoxicity, while LSS overexpression and SQLE knockout in NTD-T cells do not affect NTD-T cell cytotoxicity. Conversely, if LSS overexpression and SQLE knockout do not require T cells to possess cytotoxic function, then both LSS overexpression and SQLE knockout will enhance T cell cytotoxicity to the same extent in both NTD-T cells and CD19 CAR-T cells.

[0057] Figure 2 This paper illustrates the killing results of co-incubation of CD19 CAR-T and Nalm6 under different treatment conditions in Example 1 of this application, wherein... Figure 2 A represents the test result of lethality; Figure 2 B represents the enhanced cytotoxicity of different T cell types through overexpression of LSS or knockout of SQLE. For example... Figure 2As shown in Figure A, in the NTD-T cell setting, T cells cannot kill target cells, while CD19 CAR-T cells can successfully kill target cells. Similarly, knocking out SQLE or overexpressing LSS in NTD-T cells does not enhance T cell killing function, while knocking out SQLE or overexpressing LSS in CD19 CAR-T cells can improve CD19 CAR-T cell killing ability. Furthermore, knocking out SQLE or overexpressing LSS only enhances the killing effect when CAR-T cells are loaded. Therefore, the killing effect of CD19 CAR-T SQLE knockout = (CD19 CAR-T) + (SQLE knockout) Figure 2 The ED in the middle) and the killing effect of NTD CAR-T SQLE knockout = (NTD-T) + (SQLE knockout) Figure 2 If BA is involved, then (CD19 CAR-T + SQLE knockout killing effect) - (CD19 CAR-T killing effect) = (NTD - T SQLE knockout killing effect) - (NTD - T), i.e., ED = BA. The same logic applies to overexpression LSS. Figure 2 B shows that the enhanced T-cell killing effect from SQLE knockout or LSS overexpression was achieved only when CAR-T cells were loaded. In other words, since NTD-T cells themselves do not have killing function while CD19 CAR-T cells do, knocking out SQLE or overexpressing LSS alone in NTD-T cells does not induce NTD-T cell killing. However, in CD19 CAR-T cells loaded with CD19 CARs, both SQLE knockout and LSS overexpression alone significantly enhanced the killing ability of CD19 CAR-T cells.

[0058] Example 2: Knockout of SQLE and overexpression of LSS can significantly enhance the killing ability of AQP4 CAAR-T cells.

[0059] 1. Construct a CAAR-T lentiviral plasmid vector overexpressing AQP4 antigen.

[0060] The full-length sequences of human AQP4 M1 (protein ID P55087-1, amino acid sequence M1-V323) and M23 (protein ID P55087-2, amino acid sequence M1-V301) were selected from the Uniprot website as the antigen recognition regions of CAAR-T cells. The CD8α transmembrane region, co-stimulatory domain 4-1BB, and activation domain CD3ξ, which are the same as those of CD19 CAR-T, were selected. The CD8α transmembrane region base sequence is shown in SEQ ID NO: 5, the full-length AQP4 M1 base sequence is shown in SEQ ID NO: 6, the full-length AQP4 M23 base sequence is shown in SEQ ID NO: 7, the 4-1BB base sequence is shown in SEQ ID NO: 8, and the CD3ξ base sequence is shown in SEQ ID NO: 9. After sequence design, the pCDH GFP plasmid vector, which was synthesized by Genewiz Biotechnology Co., Ltd., was ligated with the pCDH GFP plasmid vector digested with XbaI and BamHI to obtain the AQP4 CAAR-T lentiviral plasmid vector, which is the plasmid vector used in this embodiment. M1 CAAR-T and M23 CAAR-T successfully bound to the Nalm6 B cell line expressing rAb53 and successfully killed the target cells.

[0061] 2. Construct a lentiviral plasmid vector overexpressing rAb53.

[0062] The sequence of the rAb53 antibody was obtained from a patent (patent number: WO 2016 / 033509 A1). The heavy chain variable region VH of the rAb53 antibody sequence was linked to the heavy chain constant region IgG1, and the light chain variable region VL was linked to the κ chain of the human light chain constant region. The secreted peptide base sequence is shown in SEQ ID NO:10, the rAb53 heavy chain variable region base sequence is shown in SEQ ID NO:11, the heavy chain constant region IgG1 base sequence is shown in SEQ ID NO:12, the rAb53 light chain variable region base sequence is shown in SEQ ID NO:13, and the light chain constant region base sequence is shown in SEQ ID NO:14. After sequence design, the rAb53 antibody was synthesized by Genewiz Biotechnology Co., Ltd. and ligated into a pCDH plasmid vector digested with XbaI and BamHI to obtain a lentiviral plasmid vector overexpressing the AQP4 antibody rAb53, which is the plasmid vector used in this embodiment.

[0063] 3. Construct SQLE and LSS gene overexpression and knockout lentiviral vectors.

[0064] The construction method is the same as steps 2 and 3 in Example 1.

[0065] 4. Virus packaging and obtaining stable transfected cell lines.

[0066] Lentiviral cells expressing the target gene were packaged using the CaCl2 method. The viral suspension was collected and used to infect Nalm6 cells in the presence of 6 μg / ml polybrene. After one week, positive and stably transfected cells were sorted. Further passage culture yielded a 100% transfected Nalm6 cell line stably expressing the fusion protein.

[0067] ① Virus packaging: The method is the same as steps 4①-⑤ in Example 1.

[0068] ② Viral infection and construction of the rAb53 Nalm6 cell line: Cell lines in logarithmic growth phase were infected with a concentrated virus solution (containing 6 μg / ml Polybrene) with an MOI of 5. The multiplicity of infection (MOI) is the ratio of the number of viruses capable of infecting cells to the total number of cells in a system. After 48 hours, 2 μg / ml puromycin was added. The selection medium was changed to fresh medium approximately every 2 days. After culturing for about 2 weeks, the relative expression level of the target gene was detected using quantitative PCR.

[0069] 5. Extraction of PBMCs from NMOSD patients, and construction of M1 CAAR-T cells and M23 CAAR-T cells.

[0070] The steps for extracting PBMCs and constructing CAAR-T cells are the same as step 5 in Example 1.

[0071] 6. Firefly luciferase killing assay to detect CAR-T cell killing efficiency.

[0072] ① After plating Nalm6 cells according to step 6 in Example 1, M1 / M23 CAAR-T cells, M1 / M23 CAAR-T+LSS overexpress cells, M1 / M23 CAAR-T+sgSQLE cells, and NMOSD CD3 cells were added according to different E:T ratios (1:1, 2:1, 5:1). + T cells, NMOSD CD3 + T+LSS overexpressed cells, NMOSD CD3 + T+sgSQLE cells were co-cultured, and the culture volume of each well was increased to 200 μL. The cells were then placed in a 37°C, 5% CO2 incubator.

[0073] ②The killing efficiency was detected by measuring the chemiluminescence value using a multi-functional microplate reader after 24 hours of co-culture.

[0074] It should be noted that NMOSD CD3 + T represents CD3 extracted from peripheral blood of NMOSD patients. +T cells; NMOSD CD3 + T+SQLE knockout refers to peripheral blood CD3 in NMOSD patients who have had SQLE knocked out. + T cells; NMOSD CD3 + T+LSS overexpression refers to peripheral blood CD3 in NMOSD patients who overexpress LSS. + T cells; M1 CAAR-T is AQP4 M1 CAAR-T cells constructed in Example 2; M1CAAR-T+SQLE knockout is AQP4 M1 CAAR-T cells with SQLE knocked out; M1 CAAR-T+LSS overexpression is AQP4 M1 CAAR-T cells with LSS overexpression.

[0075] When M1 CAAR-T cells were co-incubated with Nalm6 rAb53, the M1 and M23 subtypes were observed to be two subtypes of the AQP4 protein, the main pathogenic antigen in NMOSD patients. Considering the presence of CD3 in NMOSD patients... + The T cells themselves are in an activated state. To investigate whether overexpression of LSS or SQLE knockout enhances cytotoxicity only in cells loaded with CAR-T or CAAR-T cells, or in activated CD3 cells from NMOSD patients... + CD3 can also be enhanced in T cells. + Therefore, we selected CD3 from the peripheral blood of NMOSD patients to assess T cell killing function. + T cells were used as a negative control, while M1 CAAR-T or M23 CAAR-T cells were used as positive controls. LSS was overexpressed or SQLE was knocked out in these two cell types, respectively, to observe whether the overexpression of LSS or the knockout of SQLE enhanced the killing function only in CAAR-T cells.

[0076] Figure 3 This paper illustrates the killing results of co-incubation of M1 CAAR-T and Nalm6 rAb53 under different treatment conditions in Example 2 of this application. Figure 3 A represents the test result of lethality; Figure 3 B represents the presence of LSS overexpression or SQLE knockout in M1 CAAR-T cells and NMOSD CD3. + Enhanced killing function in T cells. Figure 4 This paper illustrates the killing results of co-incubation of M23CAAR-T and Nalm6 rAb53 under different treatment conditions in Example 2 of this application. Figure 4 A represents the test result of lethality; Figure 4 B represents the expression of LSS or knockout of SQLE in M23 CAAR-T cells and NMOSD CD3. + Enhanced killing function in T cells.

[0077] like Figure 3-4 As shown, CD3 can be extracted simply from the peripheral blood of NMOSD patients. + T cells exhibit mild target cell killing activity (approximately 5%-10%), and knocking out SQLE or overexpressing LSS does not enhance this killing effect. Furthermore, the killing efficiency of M1 / M23 CAAR-T cells is comparable to... Figure 2 The positive control CD19 CAR-T cells were similar, and knocking out SQLE or overexpressing LSS enhanced their CAAR-T cell killing effect. Figure 3 B and Figure 4 Both B results indicate that the enhanced killing effect of SQLE knockout or LSS overexpression occurred under CAAR-T cell loading conditions, while in NMOSD CD3 + In the presence of T cells, their killing effect cannot be enhanced. That is to say, in NMOSD patients, CD3... + Knocking out SQLE alone or overexpressing LSS alone in T cells does not enhance their killing ability against target cells (Nalm6 cells overexpressing AQP4 antibody rAb53), while knocking out SQLE alone or overexpressing LSS alone in CAAR-T cells loaded with AQP4-M1 (M23) antigen can significantly enhance the killing ability of M1 CAAR-T cells or M23 CAAR-T cells.

[0078] Example 3: Knockout of SQLE and overexpression of LSS can significantly enhance the killing ability of MOG CAAR-T cells.

[0079] 1. Construct a CAAR-T lentiviral plasmid vector overexpressing the extracellular domain of the MOG antigen.

[0080] The extracellular sequence of the human MOG antigen (protein number G16653-1, amino acid sequence G30-G154) was selected from the Uniprot website. The secretory peptide, CD8α transmembrane region, co-stimulatory domain 4-1BB, and activation domain CD3ξ (sequences same as in Example 2) were also selected, and the MOG extracellular sequence is shown in SEQ ID NO: 15. After sequence design, the sequence was synthesized by Genewiz Biotechnology Co., Ltd., and ligated into a pCDH GFP plasmid vector digested with XbaI and BamHI to obtain the lentiviral plasmid vector of MOG CAAR-T, which is the plasmid vector used in this example. This plasmid vector enables the MOG antigen expressed on the surface of T cells to bind to Nalm6 target cells expressing its specific antibody 8-18C5, successfully killing the target cells.

[0081] 2. Construct a lentiviral plasmid vector overexpressing 8-18C5.

[0082] The 8-18C5 antibody sequence was obtained from NCBI. The heavy chain variable region VH of the 8-18C5 antibody sequence was linked to the human heavy chain constant region IgG1, and the light chain variable region VL was linked to the κ chain of the human light chain constant region. The secreted peptide base sequence is shown in SEQ ID NO:16, the 8-18C5 heavy chain variable region base sequence is shown in SEQ ID NO:17, the heavy chain constant region IgG1 base sequence is shown in SEQ ID NO:18, the 8-18C5 light chain variable region base sequence is shown in SEQ ID NO:19, and the light chain constant region base sequence is shown in SEQ ID NO:20. After sequence design, the lentiviral plasmid vector was synthesized by Genewiz Biotechnology Co., Ltd. and ligated into a pLV plasmid vector digested with XbaI and BamHI to obtain a lentiviral plasmid vector overexpressing the MOG antibody 8-18C5, which is the plasmid vector used in this embodiment.

[0083] 3. Construct SQLE and LSS gene overexpression and knockout lentiviral vectors.

[0084] The construction method is the same as steps 2 and 3 in Example 1.

[0085] 4. Virus packaging and obtaining stable transfected cell lines.

[0086] ① Virus packaging: The method is the same as steps 4①-⑤ in Example 1.

[0087] ② Viral infection and construction of 8-18C5 Nalm6 cell line: The method is the same as step 4② in Example 2.

[0088] 5. Extraction of PBMCs from MOGAD patients and construction of MOG CAAR-T cells.

[0089] The steps for extracting PBMCs and constructing CAAR-T cells are the same as in Example 1, step 5.

[0090] 6. Firefly luciferase killing assay to detect CAR-T cell killing efficiency.

[0091] ① After seeding Nalm6 cells according to step 6 in Example 1, MOG CAAR-T cells, MOG CAAR-T+LSS overexpress cells, MOG CAAR-T+sgSQLE cells, and MOGAD CD3 cells were added according to different E:T ratios (1:1, 2:1, 5:1). + T cells, MOGAD CD3 + T+LSS overexpressed cells, MOGAD CD3 + T+sgSQLE cells were co-cultured, and the culture volume of each well was increased to 200 μL. The cells were then placed in a 37°C, 5% CO2 incubator.

[0092] ②The killing efficiency was detected by measuring the chemiluminescence value using a multi-functional microplate reader after 24 hours of co-culture.

[0093] It should be noted that MOGAD CD3 + T represents CD3 extracted from peripheral blood of MOGAD patients. + T cells; MOGAD CD3 + T+SQLE knockout cells are peripheral blood CD3 cells from MOGAD patients who have had SQLE knocked out. + T cells; MOGAD CD3 + T+LSS overexpression in MOGAD patients with overexpressing LSS in peripheral blood CD3 + T cells; MOG CAAR-T is CAAR-T cell overexpressing the extracellular domain of MOG antigen; MOG CAAR-T+SQLE knockout is MOG CAAR-T cell with SQLE knocked out; MOG CAAR-T+LSS overexpression is MOG CAAR-T cell overexpressing LSS.

[0094] Similarly, when co-incubating MOG CAAR-T cells with Nalm6 8-18C5, the CD3 levels in MOGAD patients should be taken into account. + The T cells themselves are in an activated state. The aim is to investigate whether overexpression of LSS or knockout of SQLE enhances cytotoxicity only in cells loaded with CAR-T or CAAR-T cells, or in activated CD3 cells from MOGAD patients. + CD3 can also be enhanced in T cells. + Therefore, we selected CD3 from the peripheral blood of MOGAD patients to measure T cell killing function. + T cells were used as a negative control, while MOG CAAR-T cells were used as a positive control. LSS was overexpressed or SQLE was knocked out in these two cell types, respectively, to observe whether the overexpression of LSS or the knockout of SQLE enhanced the killing function only in CAAR-T cells.

[0095] Figure 5 This paper illustrates the killing results of co-incubation of MOG CAAR-T and Nalm6 8-18C5 under different treatment conditions in Example 3 of this application. Figure 5 A represents the test result of lethality. Figure 5 B represents the presence of LSS overexpression or SQLE knockout in MOG CAAR-T cells and MOGAD CD3. + Enhanced killing function in T cells. For example... Figure 5 As shown, CD3 isolated solely from peripheral blood of MOGAD patients +T cells possess only a weak target cell killing ability (approximately 5%-10%). Therefore, neither SQLE knockout nor LSS overexpression enhances their killing effect. In contrast, our constructed MOG CAAR-T cells exhibit significantly enhanced killing activity, with a killing efficiency comparable to... Figure 2 Similar to the positive control CD19 CAR-T cells, knocking out SQLE or overexpressing LSS significantly enhanced the killing ability of MOG CAAR-T cells. These results indicate that in unmodified MOGAD patients, CD3... + In T cells, knocking out SQLE alone or overexpressing LSS alone did not enhance their killing ability against target cells (Nalm6 cells overexpressing MOG antibody 8-18C5); however, in CAAR-T cells loaded with MOG extracellular antigen, knocking out SQLE alone or overexpressing LSS alone significantly enhanced the killing ability of MOG CAAR-T cells.

[0096] It should be noted that, compared with untreated CD19 CAR-T cells, overexpression of LSS or knockout of SQLE significantly enhanced the cytotoxic effect of CD19 CAR-T cells. Under the same cytotoxic efficiency conditions, the required effector-to-target ratio was significantly reduced. For example, the cytotoxic efficiency that originally required a 10:1 effector-to-target ratio could be achieved with only an 8:1 or 7:1 ratio after LSS overexpression or SQLE knockout; the cytotoxic efficiency that originally required a 5:1 effector-to-target ratio could be reduced to approximately 4:1 or 3:1. These results indicate that after LSS overexpression or SQLE knockout, the number of effector cells required to achieve the same cytotoxic effect is reduced, thus achieving equivalent cytotoxicity under lower effector-to-target ratio conditions. Figure 2-5 As shown in Figure A, a straight line parallel to the X-axis is drawn on a certain kill efficiency coordinate axis, which can intuitively show that at the same kill efficiency level, the number of CAR-T cells or CAAR-T cells required for the SQLE knockout or LSS overexpression group is less than that for the untreated group.

[0097] Using a lower target-to-efficiency ratio has the following advantages: (1) The demand for effector cells is reduced, thereby reducing the initial cell preparation scale and helping to reduce costs. (2) The total amount of effector cells infused is reduced, which can reduce the risk of related toxic side effects.

[0098] In this application, the CAR-T cells include, but are not limited to, B lymphocyte antigen CD19 CAR-T (CD19 CAR-T), B cell maturation antigen CAR-T (BCMA CAR-T), Fc receptor-like 5 CAR-T (FcRL5 CAR-T), B cell activating factor receptor CAR-T (BAFF-R CAR-T), G protein-coupled receptor family C group 5 member D CAR-T (GPRC5D CAR-T), human epidermal growth factor 2 CAR-T (HER2 CAR-T), epidermal growth factor receptor CAR-T (EGFR CAR-T), differentiation cluster 38 CAR-T (CD38 CAR-T), sialic acid-binding Ig-like lectin CAR-T (CD22 CAR-T), and differentiation cluster 20 CAR-T (CD20 CAR-T cells).

[0099] The CAAR-T cells include, but are not limited to, aquaporin 4 CAAR-T (AQP4 CAAR-T), myelin oligodendrocyte glycoprotein CAAR-T (MOG CAAR-T), N-methyl-D-aspartate receptor CAAR-T (NMDAR CAAR-T), acetylcholine receptor CAAR-T (AchR CAAR-T), muscle-specific receptor tyrosine kinase CAAR-T (MuSK CAAR-T), desmosome core glycoprotein 3 CAAR-T (Dsg3 CAAR-T), leucine-rich G protein-coupled receptor family member 1 CAAR-T (LGI1 CAAR-T), glial fibrillary acidic protein CAAR-T (GFAP CAAR-T), glutamate decarboxylase 65 CAAR-T (GAD65 CAAR-T), and myelin basic protein CAAR-T (MBP CAAR-T cells).

[0100] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. The application of knocking out the SQLE gene alone or overexpressing the LSS gene alone in the preparation of products for enhancing the cytotoxic function of CAR-T cells and their derivatives, characterized in that, The method for knocking out the SQLE gene is achieved using an sgRNA that targets SQLE, the sequence of which is shown in SEQ ID NO:3; the method for overexpressing the LSS gene is achieved using an LSS base sequence, the sequence of which is shown in SEQ ID NO:

2. The CAR-T cells are CD19 CAR-T cells, the derivative is CAAR-T cells, and the CAAR-T cells are AQP4CAAR-T or MOG CAAR-T cells.

2. The application according to claim 1, characterized in that, The AQP4 CAAR-T cells are either AQP4 M1 CAAR-T cells or AQP4 M23 CAAR-T cells.

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