Application of NK cell expressing oncolytic virus protein in tumor treatment

Abstract

The invention discloses application of an NK cell expressing oncolytic virus protein in tumor treatment, and belongs to the technical field of biology. NK cells are transfected through lentivirus, and the NK-US11 cells of the US11 protein expression cassette are integrated and recombined through puromycin pressurization screening. Through Western blot detection, the US11 protein is continuously expressed along with the proliferation of the NK cells. A common NK cell and an NK-US11 cell are respectively co-cultured with a K562 cell, the killing activity of a tumor cell is detected by a lactic dehydrogenase (LDH) release method, the killing activity of the common NK cell to the K562 cell is 64.37%, the killing activity of the NK-US11 cell to the K562 cell is 87.48%, and the killing activity is improved by 35.9%. The killing activity of the NK cells expressing the oncolytic virus HSV US11 protein on tumor cells is remarkably improved, and the NK cells can play an effective role in tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, and in particular relates to the application of NK cells expressing oncolytic virus proteins in tumor treatment. Background Technology

[0002] NK cells (natural killer cells) are key effector cells of the innate immune system. They can recognize and kill tumor cells without prior sensitization and are not restricted by MHC, thus having great potential in tumor immunotherapy.

[0003] NK cells induce tumor cell apoptosis by releasing perforin and granzyme, while simultaneously secreting IFN-γ and TNF-α to regulate the immune microenvironment and inhibit tumor angiogenesis. NK cells also enhance targeted killing by recognizing antibodies (such as rituximab and trastuzumab) that bind to tumor cells via the Fc receptor (CD16). Furthermore, NK cells can activate apoptosis signaling in tumor cells through the FasL or TRAIL pathways.

[0004] Existing research has shown potential in the treatment of hematologic malignancies (such as AmL and lymphoma) through adoptive infusion of autologous or allogeneic NK cells. NK cells can be obtained from a wide range of sources, including peripheral blood, umbilical cord blood, induced pluripotent stem cells (iPSCs), or NK cell lines (such as NK-92). Due to its safety, broad-spectrum antitumor activity, and allogeneic availability, NK cell therapy has become an important supplement to tumor immunotherapy. However, the killing effect of NK cells alone on tumor cells is limited, and it needs to be combined with other antitumor therapies to achieve the goal of clinical tumor treatment.

[0005] Oncolytic viruses (OVs) are a class of viruses that selectively infect and lyse tumor cells. Their antitumor effects depend not only on direct oncolysis but also on the interaction between viral proteins and host cells, as well as the activation of the immune system. Oncolytic viruses can induce autophagy in tumor cells through various mechanisms, with some viral proteins directly or indirectly regulating the autophagy pathway, thereby affecting tumor cell survival, viral replication, and antitumor immune responses. With the development of genetic engineering and combination strategies, modifying NK cells through genetic engineering to construct genetically engineered NK cells that express oncolytic virus proteins holds promise for playing a greater role in personalized cancer therapy. Summary of the Invention

[0006] The purpose of this invention is to provide an application of NK cells expressing oncolytic virus proteins in tumor treatment, which belongs to the field of biological technology.

[0007] To achieve the above objectives, the present invention provides the following technical solution: First, this invention provides an application of NK cells expressing oncolytic virus protein in tumor treatment, wherein the oncolytic virus protein is recombinant human herpesvirus US11 protein.

[0008] Furthermore, the amino acid sequence of the recombinant US11 protein is SEQ ID NO.2.

[0009] Furthermore, the nucleotide sequence of the gene expressing the recombinant US11 protein is SEQ ID NO.3.

[0010] Furthermore, the method for preparing NK cells expressing oncolytic virus protein includes the following steps: (1) Synthesize the recombinant US11 protein expression gene and clone it into a lentiviral packaging plasmid to construct the pCDH-EF1-US11-T2A-Puro plasmid; (2) pCDH-EF1-US11-T2A-Puro, psPAX2 and pCMV-VSVG plasmids were co-transfected into HEK293T cells to package lentivirus; (3) Lentiviral transfection of freshly isolated NK cells, followed by 48 hours of culture with 1 μg / mL puromycin under pressure for 3 days; (4) Centrifuge at 300×g for 5 minutes every 3 days, discard half of the supernatant, add fresh complete culture medium, and maintain the cell density at 0.5–2×10⁻⁶. 6 Cells / mL, continue culturing for 15 days to harvest NK-US11 cells.

[0011] Secondly, the present invention provides a lentivirus for expressing recombinant human herpesvirus US11 protein, wherein the lentivirus packages an expression gene for the recombinant US11 protein, and the nucleotide sequence of the expression gene for the recombinant US11 protein is SEQ ID NO.3.

[0012] The present invention also provides a recombinant human herpesvirus US11 protein with autophagy-dependent tumor cell killing activity, wherein the amino acid sequence of the recombinant US11 protein is SEQ ID NO.2.

[0013] Furthermore, the nucleotide sequence of the gene expressing the recombinant US11 protein is SEQ ID NO.3.

[0014] The beneficial effects of this invention are as follows: This invention analyzes the structure of the human herpesvirus US11 protein, eliminates the innate immune-suppressing activity of the US11 protein while retaining its autophagy-dependent tumor-killing activity, designs recombinant US11 protein, and constructs a lentivirus expressing the recombinant US11 protein. Lentiviral cells are transfected with the lentivirus, and NK-US11 cells integrating the recombinant US11 protein expression cassette are selected using puromycin-induced compression. Western blot analysis shows that the US11 protein is continuously expressed as NK cells proliferate. Conventional NK cells and NK-US11 cells are co-cultured with K562 cells, and the tumor cell killing activity is detected by the lactate dehydrogenase (LDH) release assay. The killing activity of ordinary NK cells against K562 cells is 64.37%, while that of NK-US11 cells is 87.48%, representing a 35.9% increase in killing activity. NK cells expressing the oncolytic virus HSV US11 protein show significantly enhanced tumor cell killing activity and may play an effective role in tumor treatment. Attached Figure Description

[0015] Figure 1 Results of SDS-PAGE electrophoresis for detecting US11 protein expression in Escherichia coli.

[0016] Figure 1 Indication numbers: M: Protein ladder, 1: Protein detection before IPTG induction, 2: Protein detection after IPTG induction.

[0017] Figure 2 Results of Western Blot analysis of US11 protein expression in NK cells.

[0018] Figure 3 The results show the cytotoxic activity of NK cells against K562 cells. Detailed Implementation

[0019] The following is a more detailed description of the present invention, illustrated by examples. It should be understood that these examples are merely illustrative of the invention and are intended to explain the principles and functions of the invention, and are not intended to limit the scope of protection of the invention.

[0020] Example 1: Isolation and Culture of NK Cells (1) Dilute human peripheral blood (heparin sodium or EDTA anticoagulated) with PBS at a ratio of 1:1.

[0021] (2) Add Ficoll solution (about 3:4 volume ratio of diluted blood) to the centrifuge tube, slowly spread the diluted blood on the surface of the Ficoll solution, avoiding mixing, and centrifuge at 400 ×g for 30 minutes.

[0022] (3) Use a pipette to aspirate the white membrane layer at the interface between plasma and Ficoll, resuspend it in 10 times the volume of PBS, centrifuge at 300 ×g for 10 minutes, collect the cell pellet, resuspend it in 10 times the volume of PBS, count the cells, centrifuge at 300 ×g for 10 minutes, and collect the cell pellet.

[0023] (4) Resuspend the cell pellet in buffer (90 μL buffer / 1×10⁻⁶). 7 Cells). Add NK Cell Biotin-Antibody Cocktail (10 μL / 1×10⁻⁶ cells). 7 (Cells), mix well. Incubate at 4°C for 5 minutes (avoid light).

[0024] (5) Add Anti-Biotin MicroBeads (20 μL / 1×10 7 (Cells), mix well. Incubate at 4°C for 10 minutes.

[0025] (6) Add Buffer (1–2 mL / 1×10 7 (Cells), centrifuge at 300×g for 10 minutes, and discard the supernatant.

[0026] (7) Resuspend the cells in 500 μL buffer. Place the LS sorting column in the magnetic rack and rinse with 500 μL buffer.

[0027] (8) Load the cell suspension onto the column and collect the flow-through fluid containing unlabeled NK cells. Wash the column three times with 500 μL buffer and combine the flow-through fluids.

[0028] (9) Centrifuge the flow-through solution at 300×g for 10 minutes, resuspend the NK cells in complete culture medium, and adjust the density to 1×10⁻⁶. 6 cells / mL. The complete culture medium was RPMI-1640 medium containing 10% FBS (heat inactivated), 2 mM L-glutamine, 1000 IU / mL IL-2, 50 ng / mL IL-15, and 10 ng / mL IL-12.

[0029] (10) Centrifuge at 300×g for 5 minutes every 2–3 days, discard half of the supernatant, add fresh complete culture medium, and maintain cell density at 0.5–2×10⁻⁶. 6 Cells / mL, avoid overcrowding. Incubate at 37°C and 5% CO2 for 14–21 days to achieve a fold increase of 10–100 times.

[0030] Example 2: Construction of NK cells expressing HSV US11 protein (1) The amino acid sequence of the human herpesvirus US11 protein is SEQ ID NO.1 in GenBank: UKD60368.1. To eliminate the innate immune-suppressing activity of the US11 protein while retaining its autophagy-dependent tumor-killing activity, 21 amino acids qtqppapvgpgdpdvylkgvp were deleted from its amino terminus, resulting in the recombinant US11 protein amino acid sequence SEQ ID NO.2. The recombinant US11 protein expression gene was synthesized according to SEQ ID NO.3 and cloned into the pCDH-EF1-MCS-T2A-Puro vector between the EcoRI / NotI sites to construct pCDH-EF1-US11-T2A-Puro.

[0031] (2) The pCDH-EF1-US11-T2A-Puro, psPAX2 and pCMV-VSVG plasmids were amplified in DH5α Escherichia coli, and the three plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0032] (3) 48 hours before transfection, HEK293T cells in logarithmic growth phase were digested with trypsin and adjusted to approximately 5 × 10⁶ cells / day with medium containing 10% serum. 6 The cells were re-seeded in T25 cell culture flasks and cultured at 37 ℃ in a 5% CO2 incubator for 48 h. When the cell density reached 70%–80%, the cells were ready for transfection.

[0033] (4) Add 517 μL of ultrapure water, 10 μL each of pCDH-CMV-US11, psPAX2 and pCMV-VSVG plasmids to a sterile centrifuge tube, add 78 μL of CaCl2 and vortex thoroughly, then add 625 μL of HEPES and vortex thoroughly. Let stand at room temperature for 30 minutes.

[0034] (5) Add the above reagents dropwise to a T25 cell culture flask and incubate at 37 ℃ in a 5% CO2 incubator. Change the medium 12 hours after transfection and collect the virus after another 48 hours. When collecting the virus, use a syringe to aspirate the supernatant from the cell flask, filter it through a 0.22nm filter, and collect the filtrate as the packaged lentivirus solution.

[0035] (6) Take freshly isolated NK cells and adjust the cell number to 2 × 10⁻⁶ using NK cell complete culture medium. 6 Inoculate 1 mL / well of each cell culture plate with 1 mL of lentivirus solution, then add 1 mL of lentivirus solution, followed by polybrene at a final concentration of 8 μg / mL. Incubate at 37 °C in a 5% CO2 incubator for 48 hours.

[0036] (7) Centrifuge at 300×g for 5 minutes, discard half of the supernatant, add fresh complete culture medium, and maintain the cell density at 2×10⁻⁶. 6cells / mL. Add puromycin to a final concentration of 1 μg / mL and incubate at 37°C with 5% CO2 for 3 days.

[0037] (8) Centrifuge at 300×g for 5 minutes every 3 days, discard half of the supernatant, add fresh complete culture medium, and maintain the cell density at 0.5–2×10⁻⁶. 6 Cells / mL should be kept low to avoid overcrowding. Cultured at 37°C and 5% CO2 for 15 days, and named NK-US11 cells.

[0038] Example 3 Preparation of HSV US11 protein antibody (1) Expression plasmid construction: pCDH-EF1-US11-T2A-Puro and pET28a plasmids were double-digested with restriction endonucleases EcoRI / NotI. The EF1 fragment was recovered by gel and ligated with linearized pET28a to construct pET28a-US11 plasmid.

[0039] (2) The recombinant expression strain was used to construct the pET28a-US11 plasmid and transform it into E. coli BL21 competent cells. Single colonies were picked and positive transformants were identified by PCR and named E. coli BL21 / pET28a-US11.

[0040] (3) Expression and identification of the target protein: A single colony of E. coli BL21 / pET28a-US11 was picked and inoculated into 10 ml of LB medium containing 50 μg / ml kanamycin. The culture was incubated at 37°C and 250 r / min for 16 hours with shaking. 1% of the colony was transferred to LB medium containing 50 μg / ml and incubated at 37°C and 250 r / min until the OD600nm reached 0.6-0.8. 0.5 mol / L α-lactose was added to a final concentration of 0.03 mol / L, and the culture was incubated at 37°C and 250 r / min for 6 hours with shaking. The cells were then collected by centrifugation at 12000 r / min for 10 minutes. The cells were resuspended in PBS at 10 times their weight and sonicated. The supernatant was collected by centrifugation at 4°C and 12000 r / min for 10 minutes. SDS-PAGE electrophoresis was used to detect the expression of US11 protein. The electrophoresis results are shown in [Figure number missing]. Figure 1 .

[0041] (4) Purification of recombinant protein: Transfer the supernatant to a new tube, filter it through a 0.22 μm filter, and then add it to a Ni-NTA chromatography column treated with binding buffer. Shake at low speed for 30 min at room temperature, and then let it stand at room temperature until the resin has completely settled, allowing the liquid to flow out naturally. Then wash the chromatography column thoroughly with 10 column volumes of binding buffer and 6 column volumes of washing buffer (8 M urea, 0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9). Finally, elute the target protein with 6 column volumes of elution buffer (6 M urea, 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Collect the eluent, which is the US11 protein, and store it at 4°C for later use.

[0042] (5) Antibody preparation: US11 protein solution and 201 adjuvant were mixed at a mass ratio of 1:1 and emulsified at 3000 r / min for 15 min to prepare an oil emulsion vaccine. Five female BALB / c mice aged 6-8 weeks were subcutaneously injected with 0.1 mL of US11 protein vaccine per mouse. 21 days later, the mice were immunized again with 0.1 mL per mouse. 14 days after the second immunization, blood was collected from the orbital vein and serum was separated to obtain mouse anti-US11 protein antibodies.

[0043] Example 4: Detection of US11 protein expression in NK-US11 cells (1) Take 100 μL of NK-US11 cells expressing HSV US11 protein that were cultured for 6, 9, 12 and 15 days in Example 2, centrifuge at 3000 r / min for 5 minutes, resuspend the cell pellet in 10 μL PBS, add 10 μL of 2×SDS loading buffer, treat in a boiling water bath for 10 min, and take 10 μL for SDS-PAGE gel electrophoresis.

[0044] (2) After SDS-PAGE electrophoresis is completed, carefully remove the gel, cut off the stacking gel, immerse the separating gel in the electrophoresis solution, and install the electrophoresis device at the same time.

[0045] (3) Take a PVDF membrane about the size of a gel and 6 sheets of filter paper. Soak the PVDF membrane in methanol for 1-2 minutes, and then soak the PVDF membrane and 6 sheets of filter paper in the electroporation solution.

[0046] (4) Assemble the electro-splitter in sequence: single-layer sponge (on the black side of the sandwich panel), three-layer filter paper, SDS-PAGE gel, PVDF membrane, three-layer filter paper, and single-layer sponge.

[0047] (5) Add a small amount of transfer solution to drive out air bubbles. After assembling the transfer jacket, place it in the transfer cell (with the membrane at the positive electrode and the gel surface at the negative electrode). Add 1,000 mL of pre-cooled transfer solution to the transfer cell. Transfer the membrane for 50 min at a constant voltage of 100 V.

[0048] (6) After the transfer is completed, the PVDF membrane is removed, washed three times with 0.01 M PBST, and then immersed in blocking solution containing 1% skim milk powder for 2 h at 37°C.

[0049] (7) Discard a portion of the blocking solution, leaving enough to cover the membrane. Add 100 μL of mouse anti-US11 protein antibody (1:500 dilution), mix well, and incubate overnight at 4°C.

[0050] (8) Discard the primary antibody, wash the membrane 3 times with PBST, 5-10 min each time. Add HRP-labeled rabbit anti-mouse IgG diluted 1:1000 and bind at 37℃ for 30 min.

[0051] (9) Discard the secondary antibody, wash the membrane three times with PBS, 5-10 min each time. Add chemiluminescence reagent to the membrane, and detect the target protein band using a chemiluminescence imaging system. The results are shown in [Figure number missing]. Figure 2 It can be seen that the US11 protein is continuously expressed as NK cells proliferate.

[0052] Example 5: Detection of the cytotoxic activity of NK-US11 cells against K562 cells (1) Target cell preparation: K562 cells, a human chronic myeloid leukemia cell line in logarithmic growth phase, were prepared and their concentration was adjusted to 1×10⁻⁶ cells using RPMI-1640 + 10% FBS medium. 5 Add 100 μL of K562 cell suspension (i.e., 1×10⁶ cells / mL) to each well. 4 (cells / well) to 96-well plate. Set 3 duplicate wells / group.

[0053] (2) NK cell preparation: NK cells and NK-US11 cells expressing HSV US11 protein were serially diluted separately, with NK:K562=10:1, that is, 100 μL of NK cell suspension (1×10⁻⁶) was added to each well. 5 (cells / mL).

[0054] Blank control: K562 cells only + culture medium (no NK cells).

[0055] (3) Control group setup Spontaneous release control: K562 cells + culture medium (no NK cells).

[0056] Maximum release control: K562 cells + lysis buffer (final concentration 1% Triton X-100).

[0057] Background control: Culture medium only (cell-free).

[0058] (4) Incubate at 37°C and 5% CO2 for 4 hours.

[0059] (5) For LDH detection, centrifuge at 1000 rpm for 5 minutes, carefully aspirate 50 μL of the supernatant and transfer it to a new 96-well plate (avoiding cell aspiration). Add 50 μL of LDH substrate mixture to each well and incubate at room temperature in the dark for 15 minutes. Add 50 μL of stop solution to each well to terminate the reaction. Measure the absorbance (OD value) at 490 nm (main wavelength) and 630 nm (reference wavelength) using a microplate reader.

[0060] (6) Data calculation Correct OD value: Corrected OD = OD490nm – OD630nm Calculate cytotoxicity (%): Killing activity = (Experimental group OD – Spontaneous release OD) / (Maximum release OD – Spontaneous release OD) × 100% Spontaneous OD release: LDH release from target cells cultured alone.

[0061] Maximum OD release: LDH release upon complete lysis of target cells.

[0062] Depend on Figure 3 The results show that the killing activity of ordinary NK cells against K562 cells was 64.37%, while that of NK-US11 cells against K562 cells was 87.48%, representing an increase of 35.9%.

Claims

1. The application of NK cells expressing oncolytic virus protein in tumor treatment, characterized in that, The oncolytic virus protein is recombinant human herpesvirus US11 protein, and the amino acid sequence of the recombinant US11 protein is SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene expressing the recombinant US11 protein is SEQ ID NO.

3.

3. The application according to claim 1, characterized in that, The method for preparing NK cells expressing oncolytic virus protein includes the following steps: (1) Synthesize the recombinant US11 protein expression gene and clone it into a lentiviral packaging plasmid to construct the pCDH-EF1-US11-T2A-Puro plasmid; (2) pCDH-EF1-US11-T2A-Puro, psPAX2 and pCMV-VSVG plasmids were co-transfected into HEK293T cells to package lentivirus; (3) Lentiviral transfection of freshly isolated NK cells, followed by 48 hours of culture with 1 μg / mL puromycin under pressure for 3 days; (4) Centrifuge at 300×g for 5 minutes every 3 days, discard half of the supernatant, add fresh complete culture medium, and maintain the cell density at 0.5–2×10⁻⁶. 6 Cells / mL, continue culturing for 15 days to harvest NK-US11 cells.

4. A lentivirus for expressing recombinant human herpesvirus US11 protein, characterized in that, The lentivirus packages an expression gene for the recombinant US11 protein, the nucleotide sequence of which is SEQ ID NO.

3.

5. A recombinant human herpesvirus US11 protein with autophagy-dependent tumor cell killing activity, characterized in that, The amino acid sequence of the recombinant US11 protein is SEQ ID NO.

2.

6. The recombinant human herpesvirus US11 protein according to claim 5, characterized in that, The nucleotide sequence of the gene expressing the recombinant US11 protein is SEQ ID NO.3.

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