Application of gene modified NK cell in tumor treatment

Abstract

The invention discloses application of a gene modified NK cell in tumor treatment, and belongs to the technical field of biology. The recombinant NK cell for expressing the human perforin-collagenase fusion protein is constructed through a CRISPR-Cas9 technology. The killing activity of a common NK cell to a human breast cancer cell MDA-MB-231 is 59.47%, the killing activity of a recombinant NK cell is 85.71%, and the killing activity is improved by 44.12%. A treatment test on a breast cancer model mouse shows that the average volume of a common NK cell group mouse breast tumor is 117.02 mm, and the NK cell infiltration rate is 15.28%; the mouse breast tumor of the recombinant NK cell group is obviously reduced, the average volume is 86.03 mm, and the NK cell infiltration rate is increased to 22.58%. The infiltration rate and killing activity of the recombinant NK cell on solid tumors are remarkably improved, and the recombinant NK cell can play an effective role in tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biology, and particularly relates to application of genetically modified NK cells in tumor treatment. BACKGROUND

[0002] NK cells are the first line of defense in anti-tumor immune response, killing tumor cells or recruiting other immune cells through ADCC effect and secreting various cytokines. NK cells induce tumor cell apoptosis by releasing perforin and granzyme, and at the same time, secrete IFN-gamma, TNF-alpha and other immune microenvironment regulators to inhibit tumor angiogenesis. In recent years, adoptive NK cell therapy has become a new method for effective treatment of cancer and has important potential in tumor immunotherapy.

[0003] NK cells play an important role in anti-tumor immunity, but their effect on solid tumors is poor, and the main reason involves the unique microenvironment of solid tumors and their inhibition mechanism on immune cells. Solid tumors are usually wrapped by fibrotic stroma (such as collagen, fibroblasts), forming a physical barrier to hinder NK cell infiltration into the tumor core. The dense extracellular matrix of solid tumors, especially the excessive deposition of collagen, is the main barrier to hinder immune cell infiltration, drug penetration and radiotherapy effect. Collagenases, such as collagenase I / IV and MMPs, can improve the tumor microenvironment and enhance the effect of anti-tumor therapy by degrading collagen.

[0004] Perforin is a pore-forming protein mainly secreted by cytotoxic T cells (CTL) and NK cells, and is one of the core effector molecules of their killing tumor cells. By means of genetic technology to modify NK cells, combined with perforin and collagenase to attack tumor cells, it is expected to enhance the killing effect of NK cells on solid tumors and broaden the application range of NK cells in cancer treatment. SUMMARY

[0005] The purpose of the present application is to provide application of genetically modified NK cells in tumor treatment, which belongs to the technical field of biology.

[0006] To achieve the above purpose, the present application provides the following technical scheme: Firstly, the present application provides application of genetically modified NK cells in tumor treatment, wherein the genetically modified NK cells are recombinant NK cells expressing human perforin-collagenase fusion protein Further, the amino acid sequence of the human perforin-collagenase fusion protein is SEQ ID NO. 1.

[0007] Further, the genetically modified NK cells are obtained by co-electrotransferring gRNA1, gRNA2, Cas9 mRNA and Donor DNA into human NK cells, sorting and culturing by a flow cytometer.

[0008] Further, the nucleotide sequence of the gRNA1 is SEQ ID NO. 2.

[0009] Further, the nucleotide sequence of the gRNA2 is SEQ ID NO. 3.

[0010] Further, the nucleotide sequence of the Donor DNA is SEQ ID NO. 4.

[0011] Secondly, the present application provides a human perforin-collagenase fusion protein for improving the tumor cell infiltration rate and killing effect of NK cells, and the amino acid sequence of the human perforin-collagenase fusion protein is SEQ ID NO. 1.

[0012] The present application has the following beneficial effects: The present application analyzes the structure, amino acid sequence and expression gene of human perforin protein and human collagenase, designs the amino acid sequence of human perforin-collagenase fusion protein, and obtains gRNA1, gRNA2 and Cas9 mRNA for editing the genome of human NK cells by CRISPR-Cas9 through in vitro transcription, and prepares Donor DNA for homologous recombination, co-electrotransfers human NK cells, sorts and cultures by a flow cytometer to obtain recombinant NK cells capable of continuously expressing human perforin-collagenase fusion protein. The killing activity of ordinary NK cells on human breast cancer cells MDA-MB-231 is 59.47%, and the killing activity of the recombinant NK cells is 85.71%, which is increased by 44.12%. In the treatment test of breast cancer model mice, the breast tumor growth rate of the ordinary NK cell group mice is slightly reduced, the average volume is 117.02mm3, and the NK cell infiltration rate is 15.28%. The breast tumor of the recombinant NK cell group mice is significantly reduced, the average volume is 86.03mm3, and the NK cell infiltration rate is increased to 22.58%. The infiltration rate and killing activity of the recombinant NK cells on solid tumors are significantly improved, and the recombinant NK cells can play an effective role in tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a pX458-T7Cas9 plasmid map.

[0014] Figure 2 It is the result of Western Blot detection of human perforin-collagenase fusion protein.

[0015] Figure 3Results of detection of killing activity of NK cells on MDA-MB-231 cells.

[0016] Figure 4 Results of detection of tumor volume of breast cancer model mice affected by NK cells.

[0017] Figure 5 Results of detection of NK cell infiltration rate in breast cancer model mice tumors. DETAILED DESCRIPTION

[0018] The following is a more detailed description of the application, which is illustrated by examples. It is to be understood that these examples are merely set forth in order to illustrate the principles and application of the present application and are not intended to limit the scope of the application.

[0019] Example 1 Design of human perforin-collagenase fusion protein The human collagenase amino acid sequence AAB36941.1 was retrieved in GenBank, and its structure was analyzed. The 27-261 amino acid fragment of the collagen binding region and the enzyme active center region structure in human collagenase was selected. The human perforin protein amino acid sequence AAA60167.1 was retrieved, and its structure was analyzed. A GGSSGG flexible peptide sequence was added at the carboxy terminus, followed by fusion of the 27-261 amino acid fragment of human collagenase, to design a human perforin-collagenase fusion protein, with an amino acid sequence of SEQ ID NO. 1.

[0020] Example 2 Design of gRNA and gene repair Donor fragment for gene editing The human perforin protein DNA sequence M31951.1 was retrieved in GenBank, and a gRNA was designed for guiding Cas9 protein to cut the genomic DNA upstream and downstream of the mRNA stop codon. The gRNA coding fragment structure was designed as follows: T7 promoter followed by Hammerhead ribozyme, gRNA, gRNA scaffold, HDV ribozyme and T7 terminator. The gRNA1 coding fragment SEQ ID NO. 2 and the gRNA2 coding fragment SEQ ID NO. 3 were synthesized by a gene synthesis company and cloned into pUC57 plasmid to obtain pUC57-gRNA1 and pUC57-gRNA2, respectively.

[0021] Approximately 30 bp homologous arms were designed upstream and downstream of the two cleavage sites for homologous recombination of the Donor fragment. The middle of the Donor fragment was the DNA encoding the 27-261 amino acid fragment of human collagenase, and the nucleotide sequence of the Donor was SEQ ID NO. 4. SEQ ID NO. 4 was synthesized by a gene synthesis company and cloned into pUC57 plasmid to obtain pUC57-Donor.

[0022] Example 3 Preparation of gRNA and Cas9 mRNA pUC57-gRNA1, pUC57-gRNA2 and pX458-T7Cas9 plasmid (see plasmid map in Figure 1 ) were respectively used for in vitro transcription by Invitrogen MAXIscript™ T7 Transcription Kit, and the transcription products were purified by RNA purification kit. Both Cas9 mRNA and gRNA were dissolved in 20 μL TE buffer and stored at -80℃.

[0023] Example 4 Preparation of insertion fragment Donor DNA Donor DNA fragment was amplified by PCR using NEBNext Q5 Hot Start HiFi PCR Master Mix (New England Biolabs) high-fidelity enzyme system with pUC57-Donor as template. The PCR reaction was 50 μL system, and the primer concentration was 0.5 μM, 1 μL pUC57-Donor as template. The PCR program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 20 s, and 72℃ extension for 20 s; finally 72℃ extension for 5 min. The PCR product was electrophoresed on 1.5% agarose gel, and the target band was recovered and purified by agarose gel recovery kit as the gene repair insertion fragment Donor DNA.

[0024] Example 5 Isolation and culture of NK cells (1) Dilute the anticoagulant whole blood (sodium heparin or EDTA) with PBS at a ratio of 1:1.

[0025] (2) Add Ficoll solution (about 3:4 volume ratio with diluted blood) to the centrifuge tube, slowly spread the diluted blood on the surface of the Ficoll solution, avoid mixing, and centrifuge at 400 ×g for 30 minutes.

[0026] (3) Use a pipette to suck the white membrane layer at the interface between plasma and Ficoll, resuspend with 10 times the volume of PBS, centrifuge at 300 ×g for 10 minutes, resuspend the cell precipitate with 10 times the volume of PBS, count the cells, centrifuge at 300 ×g for 10 minutes, and collect the cell precipitate.

[0027] (4) Resuspend the cell precipitate in Buffer (90 μL Buffer / 1×10 7 cells). Add NK Cell Biotin-Antibody Cocktail (10 μL / 1×10 7Cells), mix well. Incubate at 4°C for 5 min (avoid light).

[0028] (5) Add Anti-Biotin MicroBeads (20 μL / 1 x 10 7 cells), mix well. Incubate at 4°C for 10 min.

[0029] (6) Add Buffer (1-2 mL / 1 x 10 7 cells), centrifuge at 300 x g for 10 min, discard the supernatant.

[0030] (7) Resuspend the cells in 500 μL Buffer. Place the LS column in a magnetic stand and rinse with 500 μL Buffer.

[0031] (8) Apply the cell suspension to the column and collect the flow-through containing unlabeled NK cells. Rinse the column 3 times with 500 μL Buffer and pool the flow-through.

[0032] (9) Centrifuge the flow-through at 300 x g for 10 min and resuspend the NK cells in complete medium at a density of 1 x 10 6 cells / mL. Complete medium is RPMI-1640 medium containing 10% FBS (heat-inactivated), 2 mM L-glutamine, 1000 IU / mL IL-2, 50 ng / mL IL-15, 10 ng / mL IL-12.

[0033] (10) Centrifuge at 300 x g for 5 min every 2-3 days, discard half of the supernatant, and supplement with fresh complete medium to maintain a cell density of 0.5-2 x 10 6 cells / mL, avoiding over-crowding. Culture at 37°C, 5% CO2 for 14-21 days.

[0034] Example 6 Construction of NK cells expressing human perforin-collagenase fusion protein (1) After counting the cultured NK cells, centrifuge at 1000 rpm for 10 min, resuspend the cells in NK cell serum-free medium, and adjust the cell density to 1 x 10 6 cells / mL.

[0035] (2) Take 100 μL cells into a 1.5 mL EP tube, add Donor DNA 2.5 μg, gRNA1, gRNA2, Cas9 mRNA each 1 μg, mix with a pipette, then take 100 μL mixture into a 4 mm electrotransformation cup. Place the electrotransformation cup in the ECM 830 electrotransformation instrument, set the electrotransformation parameters to 250 V, 6 msec, 625 V / cm, and electroshock 1 time. After electroshocking, immediately transfer the cells to a 6-well cell culture plate and place in a 5% CO2, 37°C saturated humidity CO2 incubator for 24 hours. Sort the cells with green fluorescence into a 6-well plate using a flow cytometer. Replace half of the medium every 7 days, count the cells and expand the culture, and sample the cells after 21 days, extract total RNA, and perform RT-PCR amplification detection using the primer pair perforinf / collagenase r.

[0036] As a result, the NK cells cultured after transfection can be amplified to a 2439 bp band, proving that the Donor DNA is correctly inserted into the NK cell genome and can transcribe full-length human perforin-collagenase fusion protein mRNA, and the recombinant NK cell (rNK cell) expressing human perforin-collagenase fusion protein is successfully constructed.

[0037] Example 7 Detection of recombinant NK cell human perforin-collagenase fusion protein expression (1) Take 100 uL of recombinant NK cells cultured for 7, 14, and 21 days in Example 6, centrifuge at 3000 r / min for 5 minutes, resuspend the cell pellet with 10 ul PBS, add 2x SDS loading buffer 10 μL, and treat in a boiling water bath for 10 min. After treatment, take 10 μL for SDS-PAGE gel electrophoresis.

[0038] (2) After SDS-PAGE electrophoresis, carefully remove the gel, cut off the concentrated gel, and immerse the separated gel part in the electrotransfer solution. At the same time, install the electrotransfer device.

[0039] (3) Take one PVDF membrane of the size of the gel and six filter papers. Soak the PVDF membrane in methanol for 1-2 min, then soak the PVDF membrane and six filter papers in the electrotransfer solution.

[0040] (4) Assemble the electrotransfer sandwich in order: single layer of sponge (on the black side of the clamp), three layers of filter paper, SDS-PAGE gel, PVDF membrane, three layers of filter paper, single layer of sponge.

[0041] (5) Add a little electrotransfer solution to drive out the bubbles, assemble the electrotransfer sandwich, and place it in the electrotransfer tank (membrane at the positive electrode and gel side at the negative electrode). Add 1 000 mL of pre-cooled electrotransfer solution to the electrotransfer tank. Electrotransfer at 100 V constant voltage for 50 min.

[0042] (6) After the end of the transfer film, take out the PVDF membrane, wash it with 0.01 M PBST for 3 times, and then immerse it in a blocking solution containing 1% skimmed milk powder, and block it at 37°C for 2 h.

[0043] (7) Discard part of the blocking solution, leave enough to cover the membrane, add 100 μL of mouse anti-human collagen protein antibody (1:500 dilution), mix well, and then incubate at 4°C overnight.

[0044] (8) Discard the primary antibody, wash the membrane with PBST for 3 times, each time for 5-10 min. Add HRP-labeled rabbit anti-mouse IgG diluted 1:1000, and combine at 37°C for 30 min.

[0045] (9) Discard the secondary antibody, wash the membrane with PBS for 3 times, each time for 5-10 min. Add chemiluminescence developing reagent on the membrane, and detect the protein band of interest with a chemiluminescence imager. The results are shown in Figure 2 It can be seen that the human perforin-collagenase fusion protein is continuously expressed with the proliferation of the recombinant NK cells.

[0046] Example 8: Detection of the killing activity of recombinant NK cells on MDA-MB-231 cells (1) Target cell preparation: logarithmically growing human breast cancer cells MDA-MB-231 were adjusted to a concentration of 1×10 5 cells / mL with DMEM + 10% FBS medium. Add 100 μL of MDA-MB-231 cell suspension (1×10 4 cells / well) to a 96-well plate. Set 3 replicate wells / group.

[0047] (2) NK cell preparation: Gradient dilute NK cells and recombinant NK cells expressing human perforin-collagenase fusion protein, NK:MDA-MB-231=10:1, i.e. add 100 μL of NK cell suspension (1×10 5 cells / mL) per well.

[0048] Blank control: only MDA-MB-231 cells + medium (without NK cells).

[0049] (3) Control group setting Spontaneous release control: MDA-MB-231 cells + medium (without NK cells).

[0050] Maximum release control: MDA-MB-231 cells + lysis solution (final concentration 1% Triton X-100).

[0051] Background control: only medium (without cells).

[0052] (4) Co-incubate in 37°C, 5% CO2 incubator for 4 hours.

[0053] (5) LDH detection Centrifuge at 1000 rpm for 5 minutes, carefully pipette 50 μL supernatant into a new 96-well plate (avoid sucking into cells). Add 50 μL LDH substrate mixture to each well, avoid light, react at room temperature for 15 minutes. Add 50 μL stop solution to each well to stop the reaction. Detect absorbance (OD value) at 490 nm (main wavelength) and 630 nm (reference wavelength) by microplate reader.

[0054] (6) Data calculation Corrected OD value: Corrected OD = OD490nm-OD630nm Calculate cytotoxicity (%): Killing activity = (experimental group OD-spontaneous release OD) / (maximum release OD-spontaneous release OD) x 100% Spontaneous release OD: LDH release of target cells cultured alone.

[0055] Maximum release OD: LDH release of completely lysed target cells.

[0056] From Figure 3 It can be seen that the killing activity of ordinary NK cells on MDA-MB-231 cells is 59.47%, and the killing activity of recombinant NK cells expressing human perforin-collagenase fusion protein on MDA-MB-231 cells is 85.71%, which is increased by 44.12%.

[0057] Example 9 Preparation of breast cancer mouse model (1) Select 60 NSG immunodeficient female mice with a body weight of about 20 g, and the feeding environment is: temperature 25~30℃, relative humidity 45~55%, light and dark cycle 12 h and 12 h, free feeding and drinking water.

[0058] (2) Immune system humanization Irradiate (1-2 Gy) the mice to eliminate endogenous immune cells, and inject human CD34+HSCs (1×10^5 cells / mouse) into the tail vein. After 8-12 weeks, detect the proportion of human CD45+cells by flow cytometry (>20% is successful).

[0059] (3) Breast cancer cell inoculation MDA-MB-231 cells cultured to the logarithmic growth phase were resuspended in PBS:Matrigel (1:1, operated on ice). The mice were anesthetized by intraperitoneal injection of sodium pentobarbital, the fourth mammary fat pad was disinfected, the skin was incised, and 1×10^6 cells / 50 μL were microinjected, the wound was sutured, and recovery was monitored.

[0060] (4) Tumor monitoring: Tumor size was measured every 3 days with a caliper, and volume was calculated (V = length x width2x 0.5). Mice with tumor volume reaching 100 mm3were selected as successfully modeled mice.

[0061] Example 10 Therapeutic effect of recombinant NK cells on breast cancer model mice (1) 30 breast cancer model mice with tumor volume of about 100 mm3were randomly divided into 3 groups. The first group was tail vein transfused with IL-15 activated human primary NK cells 5x10^6 / mouse, the second group was tail vein transfused with IL-15 activated recombinant NK cells 5x10^6 / mouse, and the third group was tail vein transfused with the same volume of normal saline. Tumor volume was monitored twice a week.

[0062] (2) After the mice were sacrificed on the 14th day, the tumor tissue was aseptically removed, weighed and recorded.

[0063] (3) The tissue was placed in pre-cooled PBS and cut into 1-2 mm3pieces. Collagenase IV (1 mg / mL) + DNase I (20 μg / mL) PBS solution was added, and the digestion was carried out at 37°C for 30-40 minutes, with vortexing every 10 minutes. The enzyme activity was neutralized by adding 10% FBS in DMEM to stop the digestion.

[0064] (4) Filtered with a 70 μm cell strainer, and the filtrate was collected. Centrifuged at 300g for 5 min, the supernatant was discarded, and the red blood cells were removed by treating with red blood cell lysis solution for 5 minutes. Washed twice with PBS and resuspended in flow buffer (PBS + 1% FBS).

[0065] (5) Add 1x10^6 cells per tube, and label the following combinations respectively: Panel 1 (phenotype analysis): CD45+CD3-CD56+CD16± Panel 2 (functional analysis): CD45+CD56+CD107a+IFN-γ+Granzyme B+ (6) Incubate at 4°C for 30 minutes in the dark.

[0066] (7) Add 2 mL PBS, centrifuge at 300g for 5 min, discard the supernatant, and repeat twice.

[0067] (8) Fixation and membrane rupture: Use Foxp3 / Transcription Factor Buffer Set (BD Biosciences) and follow the instructions.

[0068] (9) Add anti-IFN-γ, Granzyme B antibodies, and incubate in the dark for 30 minutes (room temperature).

[0069] (10) Flow cytometry detection Adjust fluorescence compensation with CompBeads, and set gate strategy: Step 1: FSC-A vs SSC-A (exclude debris, circle lymphocyte population).

[0070] Step 2: FSC-H vs FSC-A (exclude doublet).

[0071] Step 3: CD45+ (screen human white blood cells).

[0072] Step 4: CD3-CD56+ (circle NK cells).

[0073] Step 5: Analyze CD16, CD107a, IFN-γ, etc. subgroups.

[0074] From Figure 4 , Figure 5 it can be seen that the control group of mice transfused with normal saline had continuously growing breast tumors, with an average volume of 132.47 mm3 and an NK cell infiltration rate of 0.58%; the ordinary NK cell group of mice had slightly reduced breast tumor growth, with an average volume of 117.02 mm3 and an NK cell infiltration rate of 15.28%; the recombinant NK cell group of mice had significantly reduced breast tumors, with an average volume of 86.03 mm3 and an NK cell infiltration rate of 22.58%.

Claims

1. The application of a genetically modified NK cell in tumor treatment, characterized in that, The genetically modified NK cells are recombinant NK cells expressing human perforin-collagenase fusion protein, the amino acid sequence of which is SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The genetically modified NK cells were obtained by electroporation of gRNA1, gRNA2, Cas9 mRNA and Donor DNA into NK cells, followed by sorting and culture using flow cytometry.

3. The gRNA1 according to claim 2, characterized in that, The nucleotide sequence of the gRNA1 is SEQ ID NO.

2.

4. The gRNA2 according to claim 2, characterized in that, The nucleotide sequence of the gRNA2 is SEQ ID NO.

3.

5. The Donor DNA according to claim 2, characterized in that, The nucleotide sequence of the Donor DNA is SEQ ID NO.

4.

6. A human perforin-collagenase fusion protein for improving the infiltration rate and killing effect of NK cells against tumor cells, characterized in that, The amino acid sequence of the human perforin-collagenase fusion protein is SEQ ID NO.1.

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