Method for enhancing target cell removal capability of immune cells and application

Abstract

The invention relates to a method for enhancing target cell removal capacity of immune cells and application. The method comprises the following steps: recombining CD58 and TP53 genes with a granzyme B gene so as to obtain recombinant genes NFAT-GZMB-CD58 and NFAT-GZMB-TP53, and meanwhile, adding a transcription factor NFAT binding site into a regulation region of the obtained recombinant genes. Then, the two recombinant genes are directionally inserted into specific gene loci of immune cells through a gene editing technology. Expression of the two recombinant genes is regulated and controlled by an immune cell activation signal pathway, and translation products of the two recombinant genes can be delivered to target cells of immune cells through a granzyme-perforin pathway, so that the scavenging capacity of the immune cells to the target cells is enhanced, and apoptosis and escape of the target cells are inhibited. The method can be applied to the field of cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and application for enhancing the ability of immune cells to clear target cells. Background Art

[0002] Cancer has always been one of the major health problems in the world. Immunotherapy has become an effective means of treating many types of tumors, among which CAR-T technology has attracted much attention. CAR-T has achieved remarkable success in tumor treatment, especially in the treatment of hematological tumors, and can cure some patients. CAR-T uses artificially synthesized receptors or receptor recombination technology to give ordinary T cells the ability to recognize and eliminate tumors, thereby enhancing the patient's immune system's attack on tumors.

[0003] However, tumor cells also have anti-immune mechanisms to resist the attack of modified immune cells, which may lead to the failure or slow progress of immunotherapy. Some common immune escape mechanisms include: reduced immunogenicity, loss of antigen processing and presentation pathways, production of immunosuppressive molecules, cell apoptosis escape, and obstacles to immune cell infiltration. Therefore, solving the tumor cell escape mechanism is one of the main challenges facing current immunotherapy.

[0004] In order to overcome the immune escape effect caused by tumor cells expressing immunosuppressive molecules, the current CAR-T cell preparation technology uses gene editing technology to knock out the PD-1 receptor gene of CAR-T cells or make CAR-T cells secrete antibody molecules of immunosuppressive molecules to inhibit related signaling pathways. However, both technologies have corresponding safety risks. PD-1 is an inhibitory co-stimulatory receptor that binds to its ligand PD-L1 to inhibit the immune response of T cells. Knockout or blockade of PD-1 of T cells may trigger excessive activation of the immune response, resulting in autoimmune diseases or cytokine release syndrome (CRS). Therefore, this strategy still needs more research and data to verify its safety and effectiveness.

[0005] The CD28 molecule is an important co-stimulatory molecule expressed on the surface of T lymphocytes and plays a key role in the activation of T cells. It binds to the ligand molecules CD80 and CD86, provides activation signals, mediates the proliferation of T cells and produces cytokines. T cell infiltration can be detected in many solid tumors, however, T cells in tumor tissues usually show downregulation or loss of CD28 expression, which may be due to the effects of immunosuppressive molecules and cytokines in the tumor microenvironment. For example, tumor cells can overexpress PD-L1, which binds to PD-1 on the surface of T cells and inhibits its activity. The CD2 molecule is another important co-stimulatory molecule for T cells. The CD58 molecule is a transmembrane protein, also known as lymphocyte function-associated antigen 3 (LFA-3). The CD58 molecule can serve as a ligand molecule for the CD2 molecule. When the CD28 molecule is missing, the combination of the CD2 molecule and the CD58 molecule can replace the role of the CD28 molecule and provide activation signals for T cells. The combination of CD2 and CD58 can initiate multiple signal transduction pathways, including Lck, Fyn, ZAP-70 and Vav, thereby promoting the activation and proliferation of T cells. However, studies have shown that the expression of CD58 molecules is significantly reduced in various malignant tumors, such as non-small cell lung cancer, renal cancer and gastric cancer, and this downregulation is usually associated with tumor invasion, metastasis and poor prognosis. This suggests that tumor cells inhibit the immune attack of T cells by regulating the expression of CD58, thereby escaping immune surveillance.

[0006] Therefore, delivering CD58 molecules into tumor cells will help T cells in tumor tissues fight against tumor cells. The specific mechanism is as follows:

[0007] First, the immune synapse is an immune complex formed when immune cells meet target cells (such as cancer cells). This is a key step for immune cells to recognize and kill target cells. In the process of immune synapse formation, the interaction between various molecules plays an important role, among which CD2 molecules are enriched at the outer edge of the immune synapse and dynamically interact with CD58 molecules on the surface of tumor cells. Therefore, the combination of CD2 molecules and CD58 molecules is considered to be one of the key factors in stabilizing the immune synapse. The delivery of CD58 molecules into tumor cells will increase the expression level of CD58 on the surface of tumor cells, which will help form a more stable immune synapse between immune cells and tumor cells.

[0008] Second, T cells are an important type of immune cell in the body that can protect the body from disease by identifying and attacking abnormal cells. However, T cell activation requires multiple signals, the most important of which is provided by the interaction between the T cell receptor (TCR) and co-stimulatory molecules (such as CD28) and ligands. The first signal of T cell activation comes from TCR recognition of the antigen-MHC complex. This process initiates transmembrane signals and triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domain (CD3CD) of CD3, thereby activating a series of reactions downstream of T cells and mediating specific immune responses. This signal is necessary for T cell activation, but it cannot cause T cell proliferation and cytokine release. On this basis, a second signal is also required. The CD28 molecule can bind to the co-stimulatory molecules CD80 and CD86 and provide a second signal to activate and enhance the activity of T cells. When CD28 binds to CD80 / CD86, it can activate multiple signaling pathways in the cell, including NF-κB, MAPK, and PI3K / Akt, thereby enhancing the activity of T cells. However, the expression of CD28 molecules on T cells in the tumor immune microenvironment is missing, so the combination of CD2 molecules and CD58 molecules is needed to replace the binding of CD28 molecules and CD80 / CD86 to provide a second activation signal for T cells. However, tumors downregulate the expression of CD58, making it difficult for T cells to receive this important second signal. Therefore, delivering CD58 molecules into tumor cells will help restore this signal, thereby enhancing the activity and attack power of T cells.

[0009] Third, PD-L1 is a co-stimulatory molecule that can inhibit the immune response of T cells by binding to the receptor PD-1 molecule on T cells. In tumor cells, the overexpression of PD-L1 molecules is a strategy for tumor cells to evade immune surveillance. CMTM6 (CKLF-like MARVEL transmembrane domain-containing 6) is a membrane protein that is widely expressed in tumor cells and is an important factor in tumor immune escape. CMTM6 is a key protein that regulates the expression of PD-L1. It co-localizes with PD-L1 molecules on the cell membrane and recycling endosomes. By blocking the interaction between STUB1 and PD-L1 molecules, CMTM6 can inhibit the ubiquitination and degradation of PD-L1, thereby directly inhibiting the endosomal degradation of PD-L1. CD58 molecules and PD-L1 have similar domains. By delivering CD58 molecules into tumor cells, the binding of CD58 molecules to CMTM6 can compete for the binding of CMTM6 to PD-L1, thereby reducing the expression level of PD-L1 on the surface of tumor cell membranes.

[0010] During the process of immune cells attacking tumor cells through the immune synapse, a dense molecular structure is formed in the contact area between cells. Immune cells release a series of molecules such as perforin, regulatory protein ase-1 and granzyme through the immune synapse, and the interaction of these molecules promotes the destruction of tumor cell membranes and cell death.

[0011] Granzymes can induce apoptosis through direct and indirect pathways. In the direct pathway, granzymes directly enter target cells and activate Caspase. Once inside the cell, granzymes can directly act on key cysteine ​​proteases, such as Caspase-3 and Caspase-7, thereby activating the apoptosis signaling pathway. In the indirect pathway, granzymes activate intracellular calcium ions (Ca 2+ ) signaling pathway to promote apoptosis. Granzymes can act on calcium ion channels on the cell membrane, leading to the influx of calcium ions. These calcium ions can further activate protein kinases such as protein kinase C (PKC) and protein kinase A (PKA), thereby activating Caspase and promoting apoptosis.

[0012] However, tumor cells also have some mechanisms to resist granzymes injected by immune cells. One of these mechanisms involves intracellular protease inhibitors, such as IAP family proteins. IAP family proteins can directly inhibit the activity of caspase enzymes, thereby preventing the activation of apoptosis signaling pathways. This may make it impossible to kill tumor cells even if immune cells inject granzymes into tumor cells. After immunotherapy, tumor cells may upregulate the expression of IAP family proteins. This is because some tumor cells are able to increase the expression of IAP genes through genetic or epigenetic changes, thereby inhibiting cell apoptosis and increasing the survival of tumor cells. In addition, some anti-tumor treatments, such as radiotherapy and chemotherapy, may also lead to the upregulation of IAP proteins, which may increase the drug resistance and treatment failure rate of tumor cells. Therefore, inhibitors targeting IAP family proteins have become an important tumor treatment strategy.

[0013] TP53 protein is a natural inhibitor of IAP family proteins. TP53 protein can effectively inhibit the activity of IAP family proteins through direct and indirect effects. In direct action, TP53 protein can bind to IAP family proteins, thereby interfering with the binding of IAP family proteins to caspase enzymes and inhibiting the anti-apoptotic activity of IAP family proteins. In indirect action, TP53 protein can induce the expression of certain genes, which can inhibit the expression of IAP family proteins. For example, TP53 protein can induce the expression of genes such as Bax and PUMA, and the proteins encoded by these genes can bind to IAP family proteins, thereby reducing the availability of IAP family proteins and further promoting the occurrence of apoptosis. The dysfunction or loss of TP53 gene is closely related to the occurrence and development of many tumors. Many cancer patients have mutations in the TP53 gene, so delivering TP53 protein to tumor cells will enhance the ability of immune cells to clear tumors.

[0014] Promoting apoptosis is an important way for immune cells to kill tumor cells, but due to the widespread mutations in tumor cells, some tumor cells acquire the ability to escape apoptosis through mutations. Summary of the invention

[0015] Based on this, the object of the present invention is to develop a method for enhancing the ability of immune cells to clear target cells, especially tumor cells. To achieve this goal, the present invention uses gene recombination technology to recombinate the CD58 and TP53 genes with the granzyme B gene respectively, and add the binding site of the transcription factor NFAT to the regulatory region of the gene, thereby obtaining the recombinant genes NFAT-GZMB-CD58 and NFAT-GZMB-TP53. Then, through gene editing technology, these two recombinant genes are directed to be inserted into specific gene sites of immune cells to enhance the ability of immune cells to clear target cells, especially tumor cells.

[0016] According to an embodiment of the present application, a method for enhancing the ability of immune cells to eliminate target cells comprises the following steps:

[0017] S1: Using a lentiviral packaging system to package a CRISPR / Cas9 gene editing lentiviral vector that can cut at a specific gene site, and purifying to obtain a lentivirus that can edit a specific gene site;

[0018] S2: using a recombinant adeno-associated virus packaging system to package and purify the NFAT-GZMB-CD58 and NFAT-GZMB-TP53 recombinant gene vectors, and obtaining recombinant adeno-associated viruses carrying the NFAT-GZMB-CD58 and NFAT-GZMB-TP53 recombinant genes;

[0019] S3: Use the lentivirus obtained in step S1 and the recombinant adeno-associated virus obtained in step S2 to infect immune cells to obtain immune cells in which the NFAT-GZMB-CD58 and NFAT-GZMB-TP53 recombinant genes are inserted into specific gene sites.

[0020] According to an embodiment of the present application, the lentiviral vector used in the lentiviral packaging system in step S1 includes lentiCRISPR v2, pX260 and pX330, and the sgRNA sequence of the lentiviral vector is SEQ ID NO: 1 in the sequence table.

[0021] According to an embodiment of the present application, the specific gene is the SafeHarbor gene of the immune cell, and the SafeHarbor gene includes: AAVS1, located in the q13.3 region of human chromosome 19; hROSA26, located in the q27 region of human chromosome 6; H11, located in the q11.2 region of human chromosome 22; HPRT1, located in the q26.1 region of human chromosome X.

[0022] According to an embodiment of the present application, the nucleic acid sequence of the NFAT-GZMB-CD58 recombinant gene carried by the recombinant adeno-associated virus in step S2 is SEQ ID NO: 2 in the sequence list, and the nucleic acid sequence of the NFAT-GZMB-TP53 recombinant gene carried by the recombinant adeno-associated virus is SEQ ID NO: 3 in the sequence list.

[0023] According to an embodiment of the present application, the immune cells include T cells, NK cells, macrophages, dendritic cells, B cells, neutrophils, CAR-T, CAR-NK and TCR-T.

[0024] According to an embodiment of the present application, the lentiviral packaging system in step S1 includes a shuttle plasmid pX333-AAVS1-CCR5-KO, a viral packaging helper plasmid pMD2.G, psPAX2 and a transfection reagent.

[0025] According to an embodiment of the present application, the recombinant adeno-associated virus packaging system in step S2 includes a shuttle plasmid pAAV6-AAVS1-NFAT-GZMB-CD58 or pAAV6-CCR5-NFAT-GZMB-TP53, a recombinant capsid packaging plasmid pAAV-RC6, an auxiliary plasmid pAdDeltaF6 and a transfection reagent.

[0026] On the other hand, the present application discloses the application of the above-mentioned method for enhancing the ability of immune cells to eliminate target cells in CAR-T therapy.

[0027] The beneficial effects of the technical solution of the present invention compared with the prior art are:

[0028] 1. The present invention provides a new immunotherapy. This therapy inserts two recombinant genes into the Safe Harbor gene site of immune cells (such as T cells and NK cells) through gene editing technology. The two recombinant genes encode CD58 and TP53 proteins fused with granzyme B, respectively. The granzyme B protein sequence carried by the gene expression product can guide itself to enter the target cell through the granzyme-perforin pathway.

[0029] These two recombinant genes are regulated by the transcription factor NFAT. These two recombinant protein genes are usually silent or low-expression in host immune cells. Only when the host immune cells are activated will these two genes be highly activated.

[0030] CD58 and TP53 proteins have powerful anti-tumor effects. Delivering these two proteins into tumor cells can enhance the ability of immune cells to eliminate tumor cells.

[0031] 2. The present invention uses a lentiviral vector for transfection, which has the advantages of high transfection efficiency, large capacity to accommodate exogenous target gene fragments, low immune response, and can achieve stable long-term expression of specific genes.

[0032] 3. The present invention provides a new method for enhancing the anti-tumor ability of immune cells. It improves the anti-tumor ability of immune cells by modifying the genes of immune cells and can be used in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the CAR positive rate prepared by CD58-TP53-CD19-CAR-T in the example of the present invention;

[0034] Figure 2 Schematic diagram of the expression ability of CD19 and CD58 proteins in Raji cell line and Human CD58 knockout Raji cell line respectively in the examples of the present invention;

[0035] Figure 3 It is a schematic diagram of the killing rate of tumor cells by CD19-CAR-T cells and CD58-TP53-CD19-CAR-T cells in the common Raj i cell line and the Human CD58 knockout Raj i cell line respectively in the examples of the present invention;

[0036] Figure 4 Schematic diagram of the residual tumor cells in the co-incubation system of CD19-CAR-T cells, CD58-TP53-CD19-CAR-T cells and HumanCD58 knockout Raj i cell line cells in the examples of the present invention;

[0037] Figure 5 It is a schematic diagram comparing tumor cells and all living cells in the co-incubation system of CD19-CAR-T cells, CD58-TP53-CD19-CAR-T cells and HumanCD58 knockout Raj i cell line cells in the example of the present invention;

[0038] Figure 6 A diagram showing the working principle of a method for enhancing the ability of immune cells to eliminate tumor cells in an example of the present invention. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0040] The present invention inserts two recombinant genes into the SafeHarbor gene site of the human genome, and the translation products of the two genes are directedly delivered into tumor cells through the granzyme-perforin pathway, and the two genes are regulated by the transcription factor NFAT.

[0041] SafeHarbor gene loci refer to relatively stable regions in the human genome, usually located on chromosomes, where gene expression is rarely affected by position effects. These gene loci are considered suitable sites for the integration of foreign genes without adversely affecting the growth and development of host cells. SafeHarbor gene loci include but are not limited to:

[0042] AAVS1, located in the q13.3 region of human chromosome 19;

[0043] hROSA26, located in the q27 region of human chromosome 6;

[0044] H11, located in the q11.2 region of human chromosome 22;

[0045] HPRT1 is located on the q26.1 region of human chromosome X.

[0046] These SafeHarbor gene sites have been widely used for the integration and expression of foreign genes.

[0047] GZMB (granzyme B) can act as a molecular chaperone to guide the localization of proteins, and proteins fused to GZMB can be delivered to target cells via the granzyme-perforin delivery pathway.

[0048] like Figure 6 As shown, the present invention provides a method for enhancing the ability of immune cells to clear tumor cells, wherein CD58 and TP53 genes are recombined with granzyme B gene respectively, and transcription factor NFAT binding site is added to the regulatory region of the gene to obtain recombinant genes NFAT-GZMB-CD58 and NFAT-GZMB-TP53, and two recombinant genes are inserted into specific gene sites of immune cells through gene editing technology. The expression of the two recombinant genes is regulated by the immune cell activation signal pathway; the translation products of the two recombinant genes will enter the target cells of immune cells through the granzyme-perforin pathway, enhance the ability of immune cells to clear target cells, and inhibit the apoptosis escape of target cells.

[0049] Experimental material description:

[0050] Example 1 Packaging of CRISPR / Cas9 gene editing lentivirus capable of cutting AAVS1 gene locus and CCR5 gene locus

[0051] 1. Preparation of lentiviral packaging system: The full sequence of pX333-AAVS1-CCR5-KO was commissioned to Shanghai Sangon Biotechnology Co., Ltd. for synthesis, and the host bacteria DH5α was transformed. The monoclonal bacteria with correct sequencing were picked and amplified in large quantities. According to the instructions of the endotoxin-free plasmid extraction kit (Shanghai Sangon Biotechnology Co., Ltd.), the shuttle plasmid pX333-AAVS1-CCR5-KO, the viral packaging plasmid pMD2.G and psPAX2, and the empty control group plasmid were extracted in large quantities;

[0052] 2. Cell preparation: Take out the frozen HEK-293T cells from liquid nitrogen and thaw them. Prepare a 15mL centrifuge tube, add 5mL DMEM complete medium (Beijing Xigong Biological Co., Ltd.) in advance, aspirate the thawed cell freezing solution into the medium and mix thoroughly; wash the cells twice at room temperature and 650rpm for 5 minutes, and discard the supernatant; then add 10mL DMEM complete medium to the centrifuge tube, swirl and blow repeatedly to form a cell suspension to avoid bubbles. Culture in a 37℃, 5% CO2 incubator. Count the HEK-293T cells in good condition in advance to about 2×10 6 Spread onto a 100 mm cell culture dish so that the cell density reaches 80% to 90% after culture;

[0053] 3. Packaging of AAVS1-CCR5-KO lentivirus: According to the requirements of jetPRIME Polyplus transfection reagent (Polyplus Transfection Company), shuttle plasmid pX333-AAVS1-CCR5-KO, packaging auxiliary plasmid pMD2.G and psPAX2 were co-transfected into HEK293T cells to obtain lentivirus carrying the target gene; at the same time, empty vector group lentivirus was packaged as a control. The packaging system is shown in Table 1 below.

[0054] Table 1: AAVS1-CCR5-KO lentiviral packaging system

[0055]

[0056]

[0057] The transfected HEK-293T cells were placed in an incubator at 37°C and 5% CO2 for 48-72 hours, and the culture supernatant was collected as the virus stock solution.

[0058] 4. Concentration of lentivirus: The collected virus stock solution was filtered into a 50 mL centrifuge tube with a 0.45 μm filter membrane, and centrifuged at 4°C and 12000×g for 15 minutes to remove cell debris to obtain a crude virus extract; a pre-cooled sterile disposable Amicon Ultra-15100K centrifugal filter (Bio-Rad, USA) was prepared, and the obtained crude virus extract was added, and centrifuged at 4°C and 3000×g for 30 minutes, so that the filter membrane panel of the centrifugal filter was facing upward to obtain a virus concentrate, named pX333-AAVS1-CCR5-KO lentivirus concentrate. The sgRNA sequence of the pX333-AAVS1-CCR5-KO vector sequence is shown in the sequence table SEQ ID NO: 1.

[0059] Example 2 Packaging and concentration of recombinant adeno-associated virus rAAV-AAVS1-NFAT-GZMB-CD58

[0060] 1. Plasmid extraction: The full sequences of pAAV6-AAVS1-NFAT-GZMB-CD58 and pAAV6-CCR5-NFAT-GZMB-TP53 were commissioned to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis, and the host bacteria DH5α were transformed. The monoclonal bacteria with correct sequencing were picked and amplified and cultured in large quantities. The E. coli containing the constructed AAV vector shuttle plasmids (pAAV6-AAVS1-NFAT-GZMB-CD58 and pAAV6-CCR5-NFAT-GZMB-TP53), recombinant capsid packaging plasmid (pAAV-RC6) and auxiliary plasmid (pAdDeltaF6) were extracted using the Tiangen endotoxin-free plasmid extraction kit (for specific operations, refer to the instructions of the Tiangen endotoxin-free plasmid extraction kit). The plasmid concentration is required to be greater than 1μg / μL, A260 / 280 is greater than 1.8, and A260 / 230 is greater than 2.7 before the virus can be packaged;

[0061] 2. Preparation of 293 cells, an AAV packaging cell line: amplify 293 cells in large quantities, aspirate the culture medium in the 100mm culture dish where 293 cells are cultured, wash the cells twice with DPBS, remove the DPBS and add 2mL of 0.25% trypsin solution to evenly cover the bottom of the bottle, place it in a 37°C incubator for 2 minutes, take it out, shake it and you will find that the cells are detached from the bottom, add 3mL of DMEM culture medium (containing 10% FBS) preheated in a 37°C water bath, use a 10mL pipette to blow all the cells off, and place them in a 15mL centrifuge tube. Then centrifuge at 1000rpm for 5 minutes, divide the cells into 100mm culture dishes, and spread about 2×106 cells into 100mm cell culture dishes; observe the cell density on the day of transfection, and transfection can be performed when the density reaches more than 80%;

[0062] 3. Packaging of recombinant adeno-associated virus: Polyethyleneimine (PEI) (1 mg / mL) was used to transfect 293 cells with plasmids and package the recombinant adeno-associated virus rAAV-AAVS1-NFAT-GZMB-CD58. The packaging system is shown in Tables 2 and 3 below:

[0063] Table 2: Recombinant adeno-associated virus rAAV-AAVS1-NFAT-GZMB-CD58 packaging system

[0064] Transfection system 100mm culture dish Dosage pAAV-AAVS1-NFAT-GZMB-CD58 6μg pAAV-RC6 10 μg pAdDeltaF6 12 μg PEI transfection reagent 45μL

[0065] Table 3: Recombinant adeno-associated virus rAAV-CCR5-NFAT-GZMB-CD58 packaging system

[0066] Transfection system 100mm culture dish Dosage pAAV-CCR5-NFAT-GZMB-CD58 6μg pAAV-RC6 10 μg pAdDeltaF6 12 μg PEI transfection reagent 45μL

[0067] After 72 hours of culture, start collecting virus particles. Virus particles exist in both packaging cells and culture supernatant. Both cells and culture supernatant can be collected to obtain the best yield. Prepare a dry ice ethanol bath and a 37°C water bath to collect the virus-producing cells together with the culture medium into a 15mL centrifuge tube. Transfer the cell suspension repeatedly between a dry ice ethanol bath and a 37°C water bath, freeze and thaw three times, and shake slightly after each thawing (Note: Each coagulation and thawing takes about ten minutes); centrifuge the treated cell fluid at 10,000g to remove cell debris, filter the supernatant with a 0.22μm filter membrane, and then transfer the supernatant to a new centrifuge tube to collect the virus particles of the recombinant adeno-associated virus;

[0068] 4. Purification of recombinant adeno-associated virus: Add 15%, 25%, 40%, and 60% iodixanol solutions to ultracentrifuge tubes in sequence, of which 25% and 60% iodixanol solutions contain phenol red solution. After the gradient solution is prepared, slowly add the supernatant of the step lysis to the density gradient centrifugation medium. After balancing, use an ultracentrifuge at 360000g, 16°C, and centrifuge for 2h. Use a needle to pierce the intersection of the 40%-60% layer and recover the 40% layer solution. Then use a Millpore100kd ultrafiltration tube for concentration, centrifuge at 3500rpm for 30min, and use 300-500μL PBS solution to repeatedly blow the retained virus particles and then suck them into a centrifuge tube for packaging, so as to obtain the purified recombinant adeno-associated virus, which is stored in a -80°C refrigerator.

[0069] The nucleic acid sequence of the NFAT-GZMB-CD58 recombinant gene carried by the recombinant adeno-associated virus rAAV-AAVS1-NFAT-GZMB-CD58 is SEQ ID NO: 2 in the sequence list, and the nucleic acid sequence of the NFAT-GZMB-TP53 recombinant gene carried by the recombinant adeno-associated virus rAAV-CCR5-NFAT-GZMB-CD58 is SEQ ID NO: 3 in the sequence list.

[0070] Example 3 Preparation of CD19-CAR-T cells

[0071] 1.CD19-CAR-T lentivirus packaging: 293T cells were cultured with DMEM medium containing 10% fetal bovine serum; HEK293T cells were transferred to a culture dish with a diameter of 15 cm at a density of 3×105 / cm2 and cultured for 20 hours to ensure that the cell confluence was 80%-90% during transfection; serum-free DMEM medium was used for replacement and standby; according to the requirements of jetPRIME Polyplus transfection reagent (Polyplus Transfection Company), the shuttle plasmid pSLCAR-CD19-28z, the packaging auxiliary plasmid pMD2.G and psPAX2 were co-transfected into HEK 293T cells to obtain CD19-CAR-T lentivirus, and the packaging system is as shown in Table 4:

[0072] Table 4: CD19-CAR lentiviral packaging system

[0073]

[0074]

[0075] After continuing to culture for 48h-72h, the cell supernatant was collected to obtain a crude CD19-CAR lentiviral extract.

[0076] 2. CD19-CAR-T lentivirus purification: The CD19CAR virus crude extract obtained in step 1 above was filtered with a 0.45 μm filter membrane, and the filtrate was collected in a 50 ml ultrafiltration tube and centrifuged at 3000 g / min for 45 min; the remaining upper virus concentrate was transferred to an EP tube and stored at -80°C for later use.

[0077] 3. Preparation of CD19-CAR-T cells: Cultivate frozen and revived human T cells in RPMI1640 complete medium with 10% fetal bovine serum, and the cultured cell concentration is 1×107 T cells per milliliter. On the first day of culture, add 1μg / mL of CD3 and CD28 monoclonal antibodies and 500 IU / mL recombinant human IL-2 to 1×107 T cells to activate T cells; the day after T cell activation, prepare a 15μg / ml Retronectin solution in PBS and coat a non-tissuetreated 24-well culture plate; after 24 hours, remove the coated 24-well plate and discard the coating solution. Add HBSS containing 2% BSA and block for 30 minutes at room temperature. The volume of blocking solution is 500 μL / well. Aspirate and discard the blocking solution. Wash the plate twice with HBSS containing 2.5% HEPES. Add 100 μL of CD19-CAR lentivirus concentrate to each well and centrifuge at 32°C, 2000g, 2h. Discard the supernatant and add 5×10 activated human T cells to each well. 5The culture plates were placed in a 37°C, 5% CO2 incubator for three days.

[0078] Example 4 Preparation of CD19-CAR-T cells capable of delivering CD58 and TP58 proteins to target cells via the granzyme-perforin pathway

[0079] On the fourth day of culture of T cells transfected with CD19-CAR lentivirus in Example 3, a 15 μg / mL Retronectin solution was prepared with PBS and a non-tissuetreated 24-well culture plate was re-coated; 24 hours later, the coated 24-well plate was taken out and the coating solution was discarded; HBSS containing 2% BSA was added for blocking at room temperature for 30 minutes, and the volume of the blocking solution was 500 μL / well; the blocking solution was discarded, and the plate was washed twice with HBSS containing 2.5% HEPES; 20 μL of the pX333-AAVS1-CCR5-KO lentivirus obtained in Example 1 was added to each well in equal amounts; 50 μL of the rAAV-NFAT-GZMB-TP53 recombinant adeno-associated virus concentrate and 50 μL of the rAAV-AAVS1-NFAT-GZMB-CD58 recombinant adeno-associated virus concentrate obtained in Example 2 were obtained. Centrifuge at 32°C, 2000g for 2h; then discard the supernatant, add 5×105 T cells transfected with CD19-CAR lentivirus to each well, volume 1mL; centrifuge at 32°C, 1000g for 20min; then culture plate is placed in a 37°C, 5% CO2 incubator for culture; after 16h, the culture medium is replaced with RPMI1640 complete medium containing 50IU / mL recombinant human IL-2. Among them, pX333-AAVS1-CCR5-KO lentivirus plays a role in genome site-directed cutting, while rAAV-NFAT-GZMB-TP53 and rAAV-AAVS1-NFAT-GZMB-CD58 recombinant AAV provide gene editing homologous recombination repair templates.

[0080] According to the medium replacement operation every two days, the cell concentration was maintained at 1×106 / mL according to the cell count. The culture medium was RPMI1640 complete medium containing 50IU / mL recombinant human IL-2. After continuous culture for 8-10 days, CD19-CAR-T cells that can deliver CD58 and TP53 proteins to target cells through the granzyme-perforin pathway were obtained, which are hereinafter referred to as CD58-TP53-CD19-CAR-T. The positive rate of CAR prepared by CD58-TP53-CD19-CAR-T is as follows Figure 1 shown.

[0081] Example 5 Comparison of killing ability between common CD19-CAR-T cells and CD58-TP53-CD19-CAR-T

[0082] Human CD58 knockout Raj i cell line (Abcam Plc) was selected, such as Figure 2 As shown, flow cytometry detection showed that both the common Raj i cell line and the Human CD58 knockout Raj i cell line highly expressed CD19 (>90%), while there were obvious differences in CD58 protein.

[0083] CD19-CAR-T cells and CD58-TP53-CD19-CAR-T cells were co-incubated in the common Raj i cell line and the HumanCD58 knockout Raj i cell line at the effector-target ratio of 1:1, 5:1, and 10:1 for 4 hours, and the apoptosis and death of tumor cells were detected by an apoptosis kit (PI / Annexin-V). The results are shown in Figure 3 It shows that CD58-TP53-CD19-CAR-T cells have stronger killing ability than ordinary CD19-CAR-T cells.

[0084] For further verification, CD19-CAR-T cells and CD58-TP53-CD19-CAR-T cells were co-incubated with Human CD58 knockout Raj i cell line cells at an effector-target ratio of 1:4 for 7 days. Figure 4 , Figure 5 As shown, flow cytometry detected that CD58-TP53-CD19-CAR-T cells could almost completely kill tumor cells, while tumor cells still remained in the co-culture system of ordinary CD19-CAR-T cells and Human CD58knockout Raj i cells.

[0085] In summary, the technical solution of the present application has the following beneficial effects:

[0086] 1. The present invention provides an innovative immunotherapy by inserting two recombinant genes into the Safe Harbor gene site of immune cells (such as T cells and NK cells). The two recombinant genes respectively encode CD58 and TP53 proteins fused with molecular chaperone proteins (GZMB). Molecular chaperone proteins can guide the localization of proteins. CD58 and TP53 proteins carrying molecular chaperone proteins have acquired the function of being delivered to tumor cells through the granzyme-perforin pathway, and the two fusion genes are regulated by the transcription factor NFAT. Therefore, the two genes are only in an activated state when immune cells are activated. CD58 and TP53 proteins have a strong effect of clearing tumor cells. Delivering these two proteins to tumor cells will improve the ability of immune cells to clear tumor cells.

[0087] 2. The present invention uses a lentiviral vector for transfection, which has the advantages of high transfection efficiency, large capacity to accommodate exogenous specific gene fragments, low immune response, and can achieve stable long-term expression of specific genes.

[0088] 3. The present invention provides a new method for enhancing the ability of immune cells to eliminate tumor cells. It improves the ability of immune cells to eliminate tumor cells by modifying the genes of immune cells and can be used in the field of tumor cell treatment.

[0089] The above are only preferred embodiments of the present invention, and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for enhancing the ability of immune cells to eliminate target cells, characterized in that: First, through gene recombination technology, the CD58 and TP53 genes are recombined with the granzyme B gene, and the binding sites of the immune cell activation transcription factors are added to the regulatory regions of the obtained recombinant genes; then, through gene editing technology, the two recombinant genes are directed into specific gene sites of immune cells.

2. The method according to claim 1, characterized in that The method comprises the following steps: S1: Using a lentiviral packaging system to package a CRISPR / Cas9 gene editing lentiviral vector that can cut at a specific gene site, and purifying to obtain a lentivirus that can edit a specific gene site; S2: using a recombinant adeno-associated virus packaging system to package and purify the NFAT-GZMB-CD58 and NFAT-GZMB-TP53 recombinant gene vectors, and obtaining recombinant adeno-associated viruses carrying the NFAT-GZMB-CD58 and NFAT-GZMB-TP53 recombinant genes; S3: Use the lentivirus obtained in step S1 and the recombinant adeno-associated virus obtained in step S2 to infect immune cells to obtain immune cells in which the NFAT-GZMB-CD58 and NFAT-GZMB-TP53 recombinant genes are inserted into specific gene sites.

3. The method according to claim 2, characterized in that The lentiviral vector used in the lentiviral packaging system in step S1 includes lentiCRISPR V2, pX260 and pX330, and the sgRNA sequence of the lentiviral vector is SEQ ID NO: 1 in the sequence table.

4. The method according to claim 2, characterized in that: The specific gene locus is the SafeHarbor gene of immune cells, and the SafeHarbor gene includes: AAVS1, located in the q13.3 region of human chromosome 19; hROSA26, located in the q27 region of human chromosome 6; H11, located in the q11.2 region of human chromosome 22; HPRT1, located in the q26.1 region of human chromosome X.

5. The method according to claim 2, characterized in that: The nucleic acid sequence of the NFAT-GZMB-CD58 recombinant gene carried by the recombinant adeno-associated virus in step S2 is SEQ ID NO: 2 in the sequence list, and the nucleic acid sequence of the NFAT-GZMB-TP53 recombinant gene carried by the recombinant adeno-associated virus is SEQ ID NO: 3 in the sequence list.

6. The method according to claim 1, characterized in that The immune cells include T cells, NK cells, macrophages, dendritic cells, B cells, neutrophils, CAR-T, CAR-NK and TCR-T.

7. The method according to claim 2, characterized in that The lentiviral packaging system in step S1 shown includes the shuttle plasmid pX333-AAVS1-CCR5-KO, the virus packaging helper plasmid pMD2.G, psPAX2 and a transfection reagent.

8. The method according to claim 2, characterized in that: The recombinant adeno-associated virus packaging system in step S2 includes shuttle plasmids pAAV6-AAVS1-NFAT-GZMB-CD58 and pAAV6-CCR5-NFAT-GZMB-TP53, recombinant capsid packaging plasmid pAAV-RC6, auxiliary plasmid pAdDeltaF6 and transfection reagent.

9. The method according to claim 1, characterized in that: Target cells include tumor cells.

10. Use of the method according to any one of claims 1 to 9 in CAR-T therapy.

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