IL-12-CD137 chimeric protein, function-enhanced engineered NK cell as well as preparation and application of IL-12-CD137 chimeric protein and function-enhanced engineered NK cell

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an IL-12-CD137 chimeric protein, a function-enhanced engineered NK cell, and preparation and application thereof. The IL-12-CD137 chimeric protein comprises a signal peptide, an IL-12a protein molecule, a CD137 transmembrane structural domain and a CD137 intracellular structural domain, and has an amino acid sequence as shown in SEQ ID NO: 1. According to the function-enhanced engineered NK cell, the IL-12-CD137 chimeric protein sequence is introduced into an original NK cell, the function-enhanced engineered NK cell expressing Chime-IL12-CD137 is obtained, the amplification capacity and killing capacity of the NK cell can be enhanced, and the function-enhanced engineered NK cell is applied to treatment of tumor and virus-infected diseases of companion animals for the first time.

Description

Technical Field

[0001] The present invention relates to an IL-12-CD137 chimeric protein, a function-enhanced engineered NK cell, and preparation and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Tumor immunotherapy has become the fourth type of tumor treatment method after surgery, radiotherapy and chemotherapy, and is the most cutting-edge research field in applied research and clinical medical practice in recent years. NK cell therapy is a promising anti-tumor and anti-viral strategy. NK cells, or natural killer cells, are important effector lymphocytes and the "first line of defense" of the immune system. They have the most prominent antiviral activity and can directly identify and quickly kill tumor cells and virus-infected cells without antigen presensitization. They are not restricted by the major histocompatibility complex (MHC). They have a wide range of cell sources and good safety. They have received increasing attention in the field of tumor treatment and anti-infection. NK cells kill diseased cells in direct and indirect ways: when NK cells contact target cells and form immune synapses, NK cells release perforins and granzymes to directly act on target cells; at the same time, they induce cell apoptosis by binding to the target cell TRAIL receptor through the surface tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In addition, NK cells regulate innate and acquired immune cells by secreting cytokines such as interferon, thereby indirectly killing diseased cells.

[0003] Although NK cells have shown great potential for application in immune cell therapy, unlike T cells, NK cells have a shorter survival time in the host, which may affect the effectiveness of NK cells in killing tumor cells and virus-infected cells. Treatment with IL-2 (interleukin-2) can prolong the survival of NK cells in the host to a certain extent, but IL-2 can also cause a large number of Treg cells (regulatory T cells, Regulatory Tcells) that exert immunosuppressive effects.

[0004] Therefore, the problem that technicians in this field urgently need to solve is: how to develop a NK cell with longer in vivo persistence and higher efficacy, improve the proliferation and killing ability of NK cells, and enhance the application potential of NK cells. Summary of the invention

[0005] In view of the above-mentioned technical problems of the prior art, the present invention provides an IL-12-CD137 chimeric protein, a function-enhanced engineered NK cell, and its preparation and application, aiming to transform the IL12 and CD137 pathway characteristics of NK cells through membrane chimeric protein molecules, thereby improving the proliferation and killing ability of NK cells and enhancing the application potential of NK cells.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] One of the purposes of the present invention is to provide an IL-12-CD137 chimeric protein, comprising a signal peptide, an IL-12 protein molecule, a CD137 transmembrane domain, and a CD137 intracellular domain, having an amino acid sequence as shown in SEQ ID NO: 1, and having a molecular structure as shown in Figure 1 shown.

[0008] On the basis of the above technical solution, the present invention can also make the following improvements:

[0009] Furthermore, the IL-12 protein molecule is a cat IL-12a protein, and the amino acid sequence of the cat IL-12a protein molecule is shown in SEQ ID NO:2.

[0010] Furthermore, the amino acid sequence of the signal peptide is shown in SEQ ID NO:3.

[0011] Furthermore, the CD137 transmembrane domain is derived from cat CD137 protein, and the amino acid sequence of the CD137 transmembrane domain is shown in SEQ ID NO:4.

[0012] Furthermore, the CD137 intracellular domain is derived from cat CD137 protein, and the amino acid sequence of the CD137 intracellular domain is shown in SEQ ID NO:5.

[0013] The beneficial effect of the IL-12-CD137 chimeric protein of the present invention is that the chimeric protein molecular sequences of the present invention are all selected from protein sequences in cat species, wherein the IL-12 protein molecular sequence is the full length of the cat IL-12a factor, and the CD137 part only contains the transmembrane region and the intracellular region of the cat CD137 molecule. This chimeric protein molecular sequence is introduced into NK cells through a lentiviral system for expression to form membrane-localized IL12-CD137 molecules. At this time, the NK cells are IL12-CD137-NK cells (engineered NK cells). The IL-12 protein molecules outside the NK cell membrane will bind to the IL-12R (IL12 receptor) molecules in the adjacent NK cells, resulting in the activation of adjacent NK cells; at the same time, since this molecule also includes the CD137 intracellular domain, the extracellular segment (IL-12a) in the chimeric molecule binds to the I L-12R (IL12 receptor) molecules in the adjacent NK cells. When the I L12 receptor binds, the neighboring NK cells will activate the CD137 intracellular region in the chimeric molecule to form a CD137-mediated cell activation pathway, so the I L12-CD137-NK cells themselves will also be activated, and the activated CD137 signaling pathway is quite different from the I L12-I L12R pathway signal. The activation of CD137 is very necessary in the activation of immune cells such as T cells. The CD137 (also known as 4-1BB) activation pathway is conducive to T cells maintaining a "stem" state and better persistence in the body. The CD137 signal of the I L12-CD137-NK cells themselves is not only conducive to the activation of I L12-CD137-NK cells, but also inhibits the exhaustion phenotype of I L12-CD137-NK cells, thereby avoiding the rapid exhaustion of conventional NK cells in the body and enhancing the tumor killing ability of I L12-CD137-NK cells.

[0014] The second object of the present invention is to provide a functionally enhanced engineered NK cell, comprising the IL-12-CD137 chimeric protein.

[0015] Specifically, by introducing the IL-12-CD137 chimeric protein sequence into the original NK cells using a lentiviral system for expression, functionally enhanced engineered NK cells expressing Chime-IL12-CD137 are obtained. The functionally enhanced engineered NK cells can enhance the proliferation and killing capabilities of NK cells, and are used for the first time to treat companion animal tumors and viral infectious diseases.

[0016] The beneficial effect of the functionally enhanced engineered NK cells of the present invention is as follows: the present invention innovatively combines the transmembrane region and intracellular region of the cat IL12a protein and the cat CD137 protein to form functionally enhanced engineered NK cells, which can activate the IL12R pathway of neighboring NK cells, and their own CD137 signals can also be activated by neighboring NK cells, thereby improving the expansion of NK cells and the ability of NK cells to kill tumors, solving the problem of the difficulty of NK cell expansion in vitro, especially the current situation that the activation of cat NK cells still requires human IL12 activation, thereby improving the in vivo killing ability of NK cells against tumors.

[0017] The third object of the present invention is to provide a preparation of the above-mentioned functionally enhanced engineered NK cells, after synthesizing the DNA fragment encoding the above-mentioned IL-12-CD137 chimeric protein, after enzyme digestion, constructing into a lentiviral plasmid, obtaining a lentiviral plasmid expressing the target protein, and co-transfecting the target plasmid with the lentiviral packaging plasmid in 293T cells for 48 hours to 72 hours to obtain a culture medium containing a lentivirus expressing the target protein molecule. After the culture medium containing the lentivirus is added to the NK cell culture system, primary NK cells are infected, and the NK cells infected with the lentivirus express the IL-12-CD138 protein to obtain the functionally enhanced engineered NK cells.

[0018] Furthermore, the lentiviral plasmid is Plv-EF1alpha.

[0019] Furthermore, the primary NK cells are peripheral blood-derived NK cells, umbilical cord blood-derived NK cells or stem cell-derived NK cells; the primary NK cells are non-genetically modified NK cells or genetically modified NK cells; the primary NK cells include autologous NK cells or allogeneic NK cells.

[0020] Furthermore, the enzyme digestion is performed by double digestion with BamH I and SalI.

[0021] Furthermore, the lentiviral packaging plasmids are pCMV-VSV-G, pMD2.G and pRSV-Rev4.

[0022] The fourth object of the present invention is to provide the application of the above-mentioned function-enhanced engineered NK cells, which are used to prepare drugs for treating feline viral infectious diseases or tumors; the feline viral infectious diseases are feline infectious peritonitis, viral respiratory infectious diseases, feline panleukopenia or feline rhinotracheitis; the tumor is feline lymphoma, breast tumor, ovarian tumor, breast tumor, digestive tract tumor, kidney cancer, prostate cancer or fibrosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the IL-12-CD137 chimeric protein structure;

[0024] Figure 2 It is the change of the number of live cells during the proliferation of NK cells;

[0025] Figure 3 It is the change of total cell number during NK cell proliferation;

[0026] Figure 4 Changes in SAA values ​​before and after treatment of cats with FIP;

[0027] Figure 5 This is an in vivo imaging image of mice in an anti-tumor experiment. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0029] Example 1: Construction and expression of IL-12-CD137 chimeric protein (Chime-IL12-CD137)

[0030] The IL-12-CD137 chimeric protein is named as Chime-IL12-CD137 (wherein Chime is the abbreviation of chimera), and its structure is designed as follows:

[0031] Signal peptide + cat IL-12a protein molecule (signal peptide domain removed) + cat CD137 transmembrane domain + cat CD137 intracellular protein sequence.

[0032] Schematic diagram of the structure of IL-12-CD137 chimeric protein is shown in Figure 1 shown.

[0033] in,

[0034] The IL-12-CD137 chimeric protein has the amino acid sequence shown in SEQ ID NO: 1, and the DNA encoding the IL-12-CD137 chimeric protein has the nucleotide sequence shown in SEQ ID NO: 6.

[0035] The amino acid sequence of the feline IL-12a protein is shown in SEQ ID NO:2, and the DNA encoding the feline IL-12a protein has the nucleotide sequence shown in SEQ ID NO:7.

[0036] The amino acid sequence of the signal peptide is shown as SEQ ID NO:3, and the DNA encoding the signal peptide has the nucleotide sequence shown as SEQ ID NO:8.

[0037] The amino acid sequence of the cat CD137 transmembrane domain is shown in SEQ ID NO:4, and the DNA encoding the CD137 transmembrane domain has a nucleotide sequence shown in SEQ ID NO:9.

[0038] The amino acid sequence of the cat CD137 intracellular domain is shown in SEQ ID NO:5, and the DNA encoding the CD137 intracellular domain has the nucleotide sequence shown in SEQ ID NO:10.

[0039] The DNA sequence corresponding to the designed IL-12-CD137 chimeric protein was synthesized, and BamH I and SalI restriction sites were added to the 5' and 3' ends, respectively. After double digestion with BamH I and SalI, the synthesized DNA sequence was linked to the Plv-EF1alpha plasmid. This plasmid can be used for lentiviral packaging, and the packaged lentivirus can be used to infect NK cells. After infection, NK cells express IL-12 chimeric protein under the regulation of EF1a promoter.

[0040] Example 2: Preparation and proliferation comparison of functionally enhanced engineered NK cells

[0041] 1. Preparation of IL12-CD137 chimeric protein overexpression lentivirus and blank lentivirus:

[0042] (1) After synthesizing the DNA fragment encoding the IL-12-CD137 chimeric protein, it was double-digested with BamHI and SalI and constructed into the Plv-EF1alpha lentiviral plasmid to obtain the lentiviral plasmid Plv-EF1alpha-IL12-CD137 expressing the target molecule (IL12-CD137 chimeric protein). The plasmid without the DNA sequence encoding the IL12-CD137 chimeric protein was the blank lentiviral plasmid Plv-EF1alpha, which was used for the preparation of blank gene lentivirus.

[0043] (2) 293T cells were transfected with lentiviral packaging plasmids (pCMV-VSV-G, pMD2.G and pRSV-Rev) and target plasmid (Plv-EF1alpha-IL12-CD137) for lentiviral packaging. The transfection kit used was Lipofectamine 3000 transfection kit (Invitrogen, catalog number L3000-015). The specific process is as follows:

[0044] First, prepare 293T cells cultured in a 10 cm dish. The cells were used for transfection when the confluence was 90%. At the same time, 15 mL of DMEM culture medium containing 3% (volume fraction) fetal bovine serum (FBS) was prepared. Subsequently, in the clean bench, 1.5 mL of Opti-MEM culture medium and 56 μL of Lipo3000 liposome solution in the transfection kit were added to a 15 mL centrifuge tube marked as "tube A". 1.5 mL of Opti-MEM culture medium, 48 μL of P3000 solution in the transfection kit, 5.7 μg of Plv-EF1alpha-IL12-CD137 plasmid, 5.7 μg of pCMV-VSV-G plasmid, 5.7 μg of pMD2.G plasmid and 5.7 μg of pRSV-Rev plasmid were added to a 15 mL centrifuge tube marked as "tube B". After mixing, the solution in tube A was slowly added to tube B with a 1 mL pipette tip, then gently mixed and allowed to stand for 15 minutes. After completion, 3 mL of 3% (volume fraction) fetal bovine serum (FBS) DMEM medium was added to the mixing tube. The culture medium in the dish was removed, and a total of 6.5 mL of plasmid-liposome-culture medium in the tube was transferred to the dish. After the cells were cultured in an incubator at 37 ° C and 5% CO2 concentration for 5 hours, all the culture medium was aspirated, and 8 mL of fresh culture medium was added. After culturing for 48 hours, the cell culture medium supernatant was collected. At this time, there was a packaged lentivirus expressing IL12-CD137 chimeric protein in the supernatant. After the culture medium supernatant was collected, it was centrifuged at 3000 rpm for 5 minutes. After removing the cells in the culture medium, a syringe was used to filter the centrifuged supernatant with a 0.45 μm filter to obtain a lentiviral suspension that can directly infect NK cells.

[0045] Similarly, the target plasmid Plv-EF1alpha-IL12-CD137 in the step was replaced with a blank plasmid Plv-EF1alpha, and the obtained lentivirus was used as a control lentivirus. Since no exogenous sequence was inserted into the plasmid, the control lentivirus was a blank virus and would not express the IL12-CD137 chimeric protein.

[0046] 2. Isolation, culture, expansion and identification of NK cells

[0047] NK cells are isolated from cat peripheral blood mononuclear cells (PBMCs). After culture and expansion, a cell population with a purity of not less than 90% can be obtained.

[0048] Preparation of washing solution: Add bovine serum albumin (BSA) and EDTA stock solution (0.05M concentration) to PBS buffer to prepare PBS buffer containing 0.5% BSA (mass / volume) and 2mM EDTA. Use a syringe in a clean bench to filter the solution with a 0.45μm filter and store it aseptically for later use.

[0049] Preparation of 2% BSA-PBS solution: Add 2 g of bovine serum albumin (BSA) to every 100 mL of PBS buffer. After dissolving, use a syringe in a clean bench to filter the solution with a 0.45 μm filter and store it aseptically for later use.

[0050] 2.1 Feline PBMC Isolation

[0051] (1) Add an equal volume of PBS + 2% BSA solution to fresh blood, dilute and mix, and generally select 50cc-100cc of fresh blood (collected in an anticoagulant blood collection tube) for cell separation;

[0052] (2) Add 15 mL of Ficoll lymphocyte separation solution (abbreviated as Ficoll) to a 50 mL test tube, and then slowly add 35 mL of diluted blood along the tube wall, taking care not to disrupt the Ficoll liquid surface layer;

[0053] (3) Centrifugation: 750 g for 20 min;

[0054] (4) Discard the first layer of supernatant, carefully transfer the buffy coat solution into a new 50 mL test tube, add PBS + 2% BSA solution to 50 mL, and centrifuge at 350 g, 4 °C, 10 min;

[0055] (5) Aspirate the supernatant, add 1 mL of PBS + 2% BSA solution to resuspend the cells, add PBS / BSA to 50 mL, and centrifuge at 160 g, 4 °C, and 15 min.

[0056] (6) Aspirate the supernatant, resuspend the cells in 50 mL of PBS + 2% BSA solution, and centrifuge at 300 g, 4°C, for 10 min.

[0057] (7) After centrifugation, resuspend with an appropriate amount of washing buffer and continue with magnetic bead sorting.

[0058] 2.2 Magnetic bead sorting

[0059] (1) Antibody labeling of feline NK cells

[0060] The relevant reagents used are shown in Table 1:

[0061] Table 1 Magnetic bead sorting reagents

[0062]

[0063] The antibody used in this step is the anti-CD5 Monoclonal Antibody (clone number YKIX322.3) of the PE fluorescent group, brand eBioscience TM .

[0064] The specific steps include:

[0065] ① Count the cells, add 160μL washing solution + 40μL antibody (CD5 Monoclonal Antibody (YKIX322.3), PE, eBioscienceM) + 20μL FCR blocker for every 1x10^7 cells, place at 4℃ away from light for 10-20min, and pipette every 5min;

[0066] ②After incubation, add 2 mL of washing solution for every 1x10^7 cells, centrifuge at 4°C and 300g for 10 min, repeat the washing twice, resuspend with washing solution and count;

[0067] (2) Incubation of magnetic beads with cells and separation of cells using LD columns

[0068] The magnetic beads used were anti-PE UltraPure MACS microbeads, brand number Miltenyi130-105-639;

[0069] ① According to the cell number, resuspend every 1x10^7 cells with 160μL washing solution, add 40μL magnetic beads, mix well, place at 4℃ away from light for 15min, and pipette every 5min;

[0070] ②After incubation, add 2 mL of washing solution for every 1x10^7 cells, centrifuge at 4°C and 300g for 10 min, repeat the wash twice, and resuspend the cells with 500 μL of washing solution;

[0071] ③ Install the cell sorting device, add 2 mL of washing solution to the LD columns (LD columns separation columns), and add 500 μL of resuspended cells to the LD columns;

[0072] ④Observe the liquid level. After the cells have completely entered the LD column, add 1 mL of washing solution to pass through the column. After the liquid level shows that it has entered the separation part of the LD column and ensures that there are no droplets dripping, add 1 mL of washing solution.

[0073] ⑤ The collected cells are centrifuged, resuspended in culture medium and then counted. The cells obtained at this time are cat NK cells, which are used for subsequent NK cell culture.

[0074] 2.3 NK cell culture

[0075] NK cell culture and expansion were performed using the NK cell culture medium kit ( NK cell expansion kit, Icosai), the specific method is as follows:

[0076] (1) Culture flask pretreatment: melt cytokine I at room temperature, take a 50 mL centrifuge tube, add 15 mL PBS (phosphate buffer solution), pipette 45 μL cytokine I into PBS (if cytokine I is used up at one time, it is recommended to pipette 1 mL PBS in the 50 mL centrifuge tube to rinse the cytokine I tube once and add it back into the centrifuge tube), turn it upside down to mix, and add it to a 75 cm bottom area. 2 Place the solution in a cell culture flask (T75) and shake it back and forth to disperse the liquid at the bottom of the flask. Activate it at 4°C overnight. If not used immediately, store it in a 4°C refrigerator. It is recommended to use it within 3 days.

[0077] (2) On day 0, remove the culture flask that was activated overnight at 4°C, discard the coating solution, use 15 mL of NK culture medium (added with 10% heat-inactivated autologous plasma) to resuspend the PBMCs and inoculate them into a T75 flask at a seeding density of 2×10^6 cells / mL. Add 150 μL of cytokine II, shake the cells back and forth and left and right, and culture them in a 37°C, 5% CO2 incubator.

[0078] (3) On the third day, slowly add 15 mL of fresh NK medium along the side of the culture flask. Be careful not to touch the bottom of the culture flask. Do not blow the cells. Minimize counting and observation operations to avoid affecting the initial growth of cells.

[0079] (4) On the 5th day, take samples and count them, add fresh NK culture medium, adjust the cell density to 1.0×10^6-1.5×10^6 cells / mL, and expand the bottle or transfer the cell suspension into a cell culture bag for culture according to the volume of the cell suspension.

[0080] (5) After the 7th day, take samples and count the cells every one or two days for rehydration, adjust the cell density to 0.5×10^6-1.0×10^6 cells / mL, expand the flask or transfer the cell suspension into a cell culture bag according to the volume of the cell suspension. Starting from the 7th day, reduce the content of heat-inactivated autologous plasma in the fresh NK culture medium to 1%.

[0081] (6) The cells were harvested on day 18 and viral transduction was performed.

[0082] 2.4 Preparation of Chime-I L12-CD137 Functionally Enhanced Engineered NK Cells

[0083] (1) Lentiviral transduction of NK cells

[0084] ① Take the NK cells cultured in 2.3, centrifuge the cells, change the medium, take 3x10^5 cells, resuspend them in 200μL NK medium, and plant them in a 24-well plate. Add 100μL of lentivirus and Polybrene (polybrene, final concentration 8ug / mL), adjust the total system to 1mL, and centrifuge at 1000g for 40min. After centrifugation, place the cells in a 37℃, 5% C02 incubator for culture. After 6-8h, centrifuge the system and change the medium; 24h after the medium change, add lentivirus and Polybrene to the system again for secondary infection. The system and operation are the same as above;

[0085] ② After the secondary infection, centrifuge the system, change the medium, and calculate the total number of cells in the system;

[0086] ③Cultivate cells in a 37°C, 5% CO2 incubator. Cell proliferation is slow in the early stage, so only fluid replenishment is performed. Cell proliferation accelerates after day 7, and cells are centrifuged and fluid is replaced every 3 days.

[0087] ④After 2 weeks of culture, the cells were harvested for purity testing and functional evaluation.

[0088] Effect of 2.5I L12-CD137 chimeric molecule on NK cell proliferation

[0089] In order to verify the effect of the improved IL-12-CD137 chimeric protein molecule of the present invention on the proliferation of NK cells, the functionally enhanced engineered NK cells of the present invention (labeled as "NK-IL12-CD137 cells", which are NK cells expressing IL12-CD137 chimeric proteins prepared in this example), the control group 1 (labeled as "NK cells + IL12 factor" group, which are NK cells infected with the control blank lentivirus, and human IL12 factor (200U / mL concentration) was added during the amplification process) and the control group 2 (labeled as "NK cells", which are NK cells infected with the control blank lentivirus, and human IL12 factor was not added during the amplification process. The cells in each group were counted at different culture days, and the proliferation was statistically analyzed. The specific process is as follows:

[0090] (1) The NK cells infected with the IL12-CD138 chimeric protein expression lentivirus prepared in step 2.4 were taken as the experimental group, labeled as "NK-IL12-CD137 cells", and no IL12 factor was added during the culture process; the control group 1 ("NK cells + IL12 factor" group) was NK cells infected with the empty lentivirus during the preparation process in 2.4 (the preparation of blank lentivirus is shown in "Preparation of IL12-CD137 chimeric protein overexpression lentivirus and blank lentivirus" in Example 1), and human recombinant IL12 factor (200 U / mL concentration) was used during the culture process; the control group 2 (labeled as "NK cells") was NK cells infected with the control blank lentivirus, and no human recombinant IL12 factor was added during the amplification process. 0.5x10^5 viable cells were taken from each group of cells for subsequent culture, and the culture medium was the same as the amplification kit used in "NK cell culture" in 2.1 ( NK cell expansion kit (Ecocell, Inc.), except for control group 1, no IL12 factor was added during the culture process of other groups.

[0091] (2) During the 9 days after inoculation, the medium was half-changed every two days, and the total cell number and the number of live cells were counted every day using AO / PI staining: 20 μL of cells were placed in a 1.5 mL centrifuge tube, AO / PI (Counstar brand, catalog number C0212) dye was added, and the cells were counted in a Counstar Altair instrument. Cell proliferation was recorded, and bright field photography was performed to record the morphology of NK cells.

[0092] Results Figure 2 , Figure 3 It can be seen that in this embodiment, the functionally enhanced engineered NK cells of the genetically modified IL-12 of the present invention can significantly promote the division and proliferation of NK cells.

[0093] Example 3: Functional study of enhanced engineered NK cells in the treatment of feline infectious peritonitis

[0094] Feline infectious peritonitis (FIP) is a highly lethal disease caused by feline infectious peritonitis virus (FIPV), which mainly affects cats and is referred to as FIP. FIPV is a single-stranded RNA virus belonging to the Coronaviridae family. The disease can be divided into dry and wet types according to its pathological characteristics, and its clinical manifestations include fatigue, loss of appetite, fever, etc. At present, the main treatment drugs for FIP are immunosuppressive drugs and anti-inflammatory drugs, such as corticosteroids and interferon drugs with immunomodulatory effects. Although these drugs can prolong the life of sick cats to a certain extent, they cannot cure it. FIP is also considered to be one of the most challenging feline infectious diseases known to veterinarians. Therefore, the development of safe and effective drugs for the treatment of FIP has important social significance and economic value.

[0095] 1. Isolate NK cells from the peripheral blood of healthy adult British shorthair cats and prepare engineered NK cells expressing I L2-CD137 chimeric molecules;

[0096] Specifically, about 30 mL of peripheral blood was collected from a healthy British shorthair cat weighing 5 kg. The NK cell separation and virus infection processes were the same as those in Example 1 and Example 2 and are not described in detail.

[0097] 2. Infuse the expanded and activated NK cells back into the cat with FIP;

[0098] 2.1 Experimental animals

[0099] The 30 cases used in this example were all cats naturally infected with FIP.

[0100] 2.2 Trial Grouping and Treatment Plan:

[0101] NK cell treatment group (intravenous reinfusion of IL2-CD137-NK cells): 15 cats were intravenously injected with a mixture of 1 mL of IL2-CD137-NK cell suspension and 9 mL of normal saline. The treatment time was once every 2 weeks, for a total of 3 times.

[0102] Non-NK cell treatment group: intravenous injection of 9 mL of normal saline, once every 2 weeks, for a total of 3 times;

[0103] 2.3 Engineered NK cell injection method:

[0104] Before NK cell transfusion, the purity and activity of NK cells are tested to ensure that the purity is above 85% and the cell viability is above 90%. During transfusion, 1 mL of cell suspension is injected into a bag containing 9 mL of saline and mixed before infusion. The cell infusion rate is adjusted to 10 mL / h. That is, the cell suspension mixed with saline is infused within 1 hour.

[0105] 2.4 Treatment cycle

[0106] Each group received medication for 50 days, and if the cats achieved clinical cure within 50 days, medication was stopped.

[0107] Principles of symptomatic treatment: antibacterial and anti-inflammatory, blood-tonifying and liver-protecting, regulation of water, electrolyte, and acid-base balance, and nutritional support.

[0108] 3. Therapeutic effect of engineered NK cells

[0109] 3.1 Changes in blood biochemical indicators

[0110] Serum amyloid A (SAA) is an acute phase protein that is usually produced by the liver in response to inflammation or infection. In the diagnosis of feline infectious peritonitis, serum amyloid A is used as a biomarker to help assess the inflammatory status of cats. Since FIP is an inflammatory disease, SAA levels will be elevated in cats with FIP.

[0111] The SAA values ​​of 30 cats were monitored, and the changes in indicators before and after treatment in each group were statistically analyzed. The results are shown in Figure 4 Compared with before treatment, the SAA value of the NK cell treatment group decreased, and the difference was extremely significant. The SAA value of the non-NK cell treatment group decreased, but the difference was not significant. It can be seen that the use of the engineered NK cells of the present invention can significantly reduce the inflammatory response in the affected cat.

[0112] Example 4: Anti-tumor evaluation of functionally enhanced engineered IL12-CD137-NK cells

[0113] The therapeutic effect of the functionally enhanced engineered N cells of the present invention in the leukemia K562-Luciferase tail vein tumor-bearing mouse model was explored.

[0114] 1. The experimental animals and feeding conditions are shown in Table 2 below.

[0115] Table 2 Experimental animals and feeding conditions

[0116]

[0117] 2. Tumor cell inoculation (disease model mouse construction)

[0118] Experimental cells: K562-Luc cells were revived, and the revived generation was P14. K562-Luc cells in the logarithmic growth phase were collected on the same day, the culture medium was removed and washed twice with PBS before inoculation (the survival rates of K562-Luc cells before and after tumor loading were 97.8% and 95.9%, respectively), inoculation volume: 1×10^6 / 200μL / mouse, inoculation site: mouse tail vein.

[0119] 3. Group Dosing

[0120] On day 5 after tumor cell inoculation, in vivo fluorescence imaging was used to detect tumor load after inoculation and data analysis was performed. The mean photon value (mean Rad iance) was 5.79×10^4 (p / sec / cm 2 / sr), 8 mice were randomly divided into 2 groups, 4 mice in each group, and NK cell treatment was started on the same day (D5). The mouse grouping information is as follows:

[0121] Engineered NK cell group (NK-IL12-CD137 cell administration group): Injection of engineered cat NK cells (NK cells expressing IL2-CD137 chimeric protein), injection dose of 2x10^7 / mouse, tail vein injection, injection volume of 200μL. The cell preparation process is the same as that in Example 1 and Example 2.

[0122] Control group (NK control cell group): Tumor model mice were injected with conventional NK cells that had not been engineered. The injection dose was 2x10^7 / mouse, injected through the tail vein, and the injection volume was 200μL.

[0123] 4. In vivo imaging to detect the tumor burden of each group of mice:

[0124] The process of photon numerical detection of tumor load of each group of animals at D5, D8, D11, D14, D17, D20, D23, D26, D32, and D34 after tumor inoculation is as follows:

[0125] 4.1 Prepare the stock solution of luciferin: the concentration is 15 mg / mL. Dissolve 1.5 g of luciferin powder in 100 mL of PBS phosphate solution. After it is fully dissolved, filter it in a clean bench using a 0.2 μm filter to remove impurities.

[0126] 4.2 The injection dose is 10 μL of Luciferin solution per gram of body weight. For a 20 g mouse, 200 μL of Luciferin solution (containing 3 mg Luciferin) needs to be injected.

[0127] 4.3 Use small animal in vivo imaging equipment (SI Ami X three-in-one multi-mode long-line system, SI imaging manufacturer, USA) to detect and monitor tumor growth and metastasis.

[0128] 5. Experimental Results

[0129] Results Figure 5, from the results of the mouse live imaging diagram, it is shown that compared with conventional cat NK control cells, the functionally enhanced engineered NK cells (NK-I L12-CD137) of the present invention have better anti-tumor effects. When NK cells cannot suppress tumors, the mouse tumor load will be higher and higher, eventually leading to the death of mice. From the point of view of death, the control group was at the D26 time point, all of which had died, so there was no live image, and all mice in the experimental group (injected with NK-I L12-CD137 mice) were in a state of survival, and the engineered NK cell treatment group died at the D32 time point, and only one engineered NK treated mouse was left alive at D34, and the engineered NK treated mice died at D35. From the tumor load of live imaging in the figure, the engineered NK treatment group mice maintained a low tumor load at D8-D23 (the red in the imaging figure indicates that the tumor load is large). From the point of view of therapeutic effect, engineered NK cells can significantly prolong the life of mice, proving that the killing ability of engineered NK in mice to tumor cells is much stronger than that of the control group NK.

[0130] IL-12 is a cytokine that can promote NK cell proliferation, enhance cytotoxicity and produce IFN-γ. In the in vitro expansion of human and mouse NK cells, the combination of IL-12 and other cytokines (such as IL-2 and IL-15) can more effectively expand NK cells and enhance their function.

[0131] In cat NK cell culture, recombinant human IL-2, canine IL-21, recombinant human IL-15 and recombinant human IL12 are routinely used in the prior art. Such cytokines provide proliferation activation signals to NK. At the same time, studies have also shown that only 4-1BBL (CD137L) protein coating and expression membrane-bound rhIL21 K562 feeder cells, supplemented with 100IU / mL human recombinant-IL2 factor, can also effectively amplify cat NK cells, suggesting that 4-1BB (CD137) signals may contribute to the proliferation of NK cells. This information suggests that it may be a good strategy to transform NK cells from the IL2 / IL12 / IL15 / IL21 and CD137 signaling pathways to improve the in vitro expansion ability and in vivo killing ability of NK cells, and solve the problems that those skilled in the art urgently need to solve. The present invention aims to transform the IL12 and CD137 pathway characteristics of cat NK cells through membrane chimeric protein molecules, improve the expansion and killing ability of NK cells, and enhance the application potential of NK cells. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An IL-12-CD137 chimeric protein, characterized in that It includes a signal peptide, an IL-12 protein molecule, a CD137 transmembrane domain, and a CD137 intracellular domain, and has an amino acid sequence as shown in SEQ ID NO:

1.

2. The IL-12-CD137 chimeric protein according to claim 1, characterized in that The IL-12a protein molecule is a feline IL-12a protein, and the amino acid sequence of the feline IL-12a protein molecule is shown in SEQ ID NO:

2.

3. The IL-12-CD137 chimeric protein according to claim 1, characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID NO:

3.

4. The IL-12-CD137 chimeric protein according to claim 1, characterized in that The CD137 transmembrane domain is derived from cat CD137 protein, and the amino acid sequence of the CD137 transmembrane domain is shown in SEQ ID NO:

4.

5. The IL-12-CD137 chimeric protein according to claim 1, characterized in that The CD137 intracellular domain is derived from cat CD137 protein, and the amino acid sequence of the CD137 intracellular domain is shown in SEQ ID NO:

5.

6. A functionally enhanced engineered NK cell, characterized in that: The invention comprises an IL-12-CD137 chimeric protein as described in any one of claims 1 to 5.

7. A preparation of functionally enhanced engineered NK cells as claimed in claim 6, characterized in that: After synthesizing a DNA fragment encoding the IL-12-CD137 chimeric protein as described in any one of claims 1 to 5, after enzyme digestion, it is constructed into a lentiviral plasmid to obtain a lentiviral plasmid expressing the target protein, and the target plasmid and the lentiviral packaging plasmid are co-transfected in 293T cells for 48 hours to 72 hours to obtain a culture medium containing a lentivirus expressing the target protein molecule, and the culture medium containing the virus is added to the NK cell culture system to infect primary NK cells to obtain the functionally enhanced engineered NK cells.

8. The preparation of functionally enhanced engineered NK cells according to claim 7, characterized in that: The lentiviral plasmid is Plv-EF1 alpha.

9. The functionally enhanced engineered NK cell according to claim 7, characterized in that: The primary NK cells are derived from peripheral blood, umbilical cord blood or stem cell; the primary NK cells are non-genetically modified NK cells or genetically modified NK cells; the primary NK cells include autologous NK cells or allogeneic NK cells.

10. The use of the functionally enhanced engineered NK cells according to claim 6, characterized in that: Used for preparing medicines for treating feline viral infectious diseases or tumors; the feline viral infectious diseases are feline infectious peritonitis, viral respiratory infectious diseases, feline panleukopenia or feline rhinotracheitis; the tumors are feline lymphoma, breast tumors, ovarian tumors, breast tumors, digestive tract tumors, kidney cancer, prostate cancer or fibrosarcoma.

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