An IL-12-CD137 chimeric protein, a function-enhanced engineered NK cell, and its preparation and application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to an IL-12-CD137 chimeric protein, a functionally enhanced engineered NK cell, and its preparation and application. The IL-12-CD137 chimeric protein includes a signal peptide, an IL-12a protein molecule, a CD137 transmembrane domain, and a CD137 intracellular domain, and has an amino acid sequence as shown in SEQ ID NO: 1. The functionally enhanced engineered NK cell, by introducing the above-mentioned IL-12-CD137 chimeric protein sequence into the original NK cell, obtains a functionally enhanced engineered NK cell expressing Chime-IL12-CD137, which can enhance the amplification ability and killing ability of NK cells, and is used for the first time to treat companion animal tumors and viral infectious diseases.

Description

Technical Field

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

[0002] Tumor immunotherapy has become the fourth type of cancer treatment, following surgery, radiotherapy, and chemotherapy, and has been at the forefront of applied research and clinical practice in recent years. NK cell therapy is a promising anti-tumor and antiviral strategy. NK cells, or natural killer cells, are important effector lymphocytes and the immune system's "first line of defense." They possess prominent antiviral activity and can directly recognize and rapidly kill tumor cells and virus-infected cells without antigen presensitization, unrestricted by the major histocompatibility complex (MHC) molecule. Their broad availability and safety have garnered increasing attention in the fields of cancer therapy and anti-infection. NK cells kill diseased cells through both direct and indirect pathways. When NK cells contact target cells and form an immune synapse, they release perforins and granzymes, which act directly on the target cells. Simultaneously, they induce apoptosis by binding to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor on the target cells through their surface expression. Furthermore, NK cells indirectly kill diseased cells by secreting cytokines such as interferon, regulating innate and adaptive immune cells.

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

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

[0005] In response to 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. The invention aims to transform the IL-12 and CD137 pathway characteristics of NK cells through membrane chimeric protein molecules, thereby improving the expansion 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 objects 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 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 feline IL-12a protein, and the amino acid sequence of the feline 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 cat IL-12a factor, and the CD137 portion only contains the transmembrane region and 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 adjacent NK cells, leading to the activation of adjacent NK cells; at the same time, because this molecule also includes the CD137 intracellular domain, the extracellular segment (IL-12a) in the chimeric molecule binds to the I L12R (IL12 receptor) molecules in adjacent NK cells, leading to the activation of adjacent NK cells. When the IL12 receptor binds, neighboring NK cells activate the CD137 intracellular region of the chimeric molecule, forming a CD137-mediated cell activation pathway. Consequently, IL12-CD137-NK cells themselves are activated, and the activated CD137 signaling pathway differs significantly from the IL12-IL12R pathway. CD137 activation is essential for the activation of immune cells such as T cells. The CD137 (also known as 4-1BB) activation pathway helps T cells maintain their "stemness" state and better persist in vivo. The CD137 signaling on IL12-CD137-NK cells not only facilitates their activation but also inhibits their exhaustion phenotype, thereby preventing the rapid exhaustion that commonly occurs with conventional NK cells and enhancing their tumor-killing ability.

[0014] A second object of the present invention is to provide a function-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 effects of the functionally enhanced engineered NK cells of the present invention are 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. The functionally enhanced engineered NK cells 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] A third object of the present invention is to provide a preparation for the above-mentioned functionally enhanced engineered NK cells. After synthesizing a DNA fragment encoding the above-mentioned IL-12-CD137 chimeric protein, the DNA fragment is digested and then constructed into a lentiviral plasmid to obtain a lentiviral plasmid expressing the target protein. After this target plasmid and the lentiviral packaging plasmid are co-transfected into 293T cells for 48 to 72 hours, a culture medium containing the lentivirus expressing the target protein molecule is obtained. After the culture medium containing the lentivirus is added to the NK cell culture system, primary NK cells are infected. The lentiviral-infected NK cells 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 using BamH I and SalI for double digestion.

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

[0022] A fourth object of the present invention is to provide an application of the above-mentioned function-enhanced engineered NK cells, which is used to prepare a drug 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, mammary tumors, digestive tract tumors, 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 living cells during NK cell proliferation;

[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 for 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 with reference to 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 Chime-IL12-CD137 (where 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 in SEQ ID NO: 3, and the DNA encoding the signal peptide has the nucleotide sequence shown in 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 the nucleotide sequence shown in SEQ ID NO: 9.

[0038] The amino acid sequence of the feline 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 BamHI and SalI restriction sites were added at the 5' and 3' ends, respectively. After double digestion with BamHI and SalI, the synthesized DNA sequence was ligated into the Plv-EF1alpha plasmid. This plasmid can be used for lentiviral packaging, which can then be used to infect NK cells, where the IL-12 chimeric protein is expressed under the control of the EF1alpha 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 a DNA fragment encoding an IL-12-CD137 chimeric protein, the fragment was double-digested with BamHI and SalI and then constructed into a Plv-EF1alpha lentiviral plasmid to obtain a 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 called a blank lentiviral plasmid Plv-EF1alpha and was used for blank gene lentiviral preparation.

[0043] (2) 293T cells were transfected with lentiviral packaging plasmids (pCMV-VSV-G, pMD2.G, and pRSV-Rev) and the target plasmid (Plv-EF1alpha-IL12-CD137) for lentiviral packaging. The transfection kit used was the Lipofectamine 3000 transfection kit (Invitrogen, Cat. No. L3000-015). The specific procedure was as follows:

[0044] First, prepare 293T cells cultured in a 10 cm dish. When the cell confluence is 90%, use for transfection. At the same time, prepare 15 mL of DMEM medium containing 3% (volume fraction) fetal bovine serum (FBS); then, in the clean bench, add 1.5 mL of Opti-MEM medium and 56 μL of Lipo3000 liposome solution in the transfection kit to a 15 mL centrifuge tube marked "tube A"; add 1.5 mL of Opti-MEM 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 to a 15 mL centrifuge tube marked "tube B". After mixing, the solution in tube A was slowly added dropwise to tube B using 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 medium in the dish was removed, and a total of 6.5 mL of plasmid-liposome-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 medium was aspirated and 8 mL of fresh medium was added. After culturing for 48 hours, the cell culture supernatant was collected. At this time, the supernatant contained packaged lentivirus expressing the IL12-CD137 chimeric protein. After the culture supernatant was collected, it was centrifuged at 3000 rpm for 5 minutes. After removing the cells in the culture medium, the supernatant was filtered through a 0.45 μm filter using a syringe to obtain a lentiviral suspension that can directly infect NK cells.

[0045] Similarly, replace the target plasmid Plv-EF1alpha-IL12-CD137 in the step with the blank plasmid Plv-EF1alpha. The obtained lentivirus is used as the control lentivirus. Since the plasmid does not have an exogenous sequence inserted, the control lentivirus is a blank virus and will 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 no 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 through a 0.45μm filter and store sterile until 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 to filter the solution through a 0.45 μm filter in a clean bench and store it aseptically until use.

[0050] 2.1 Feline PBMC Isolation

[0051] (1) Add an equal volume of PBS + 2% BSA solution to fresh blood, dilute and mix thoroughly. Generally, 50cc-100cc of fresh blood (collected in an anticoagulant blood collection tube) is used 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 layer;

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

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

[0055] (5) Discard 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, for 15 min.

[0056] (6) Discard 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 magnetic bead sorting.

[0058] 2.2 Magnetic bead sorting

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

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

[0061] Table 1 Magnetic bead separation 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 160 μL washing buffer + 40 μL antibody (CD5 Monoclonal Antibody (YKIX322.3), PE, eBioscience M) + 20 μL FCR blocker for every 1x10^7 cells. Incubate at 4°C in the dark for 10-20 minutes, pipetting every 5 minutes.

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

[0067] (2) Incubate cells with magnetic beads and separate 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 buffer, add 40μL magnetic beads, mix well, and place in the dark at 4℃ for 15 minutes, pipetting every 5 minutes;

[0070] ② After incubation, add 2 mL of washing solution per 1 x 10^7 cells, centrifuge at 4°C, 300 g for 10 min, repeat the wash twice, and resuspend the cells in 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 and pass through the column. After the liquid level shows that it has entered the separation area of ​​the LD column and no droplets continue to drip, add 1 mL of washing solution.

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

[0074] NK cell culture

[0075] NK cell culture and expansion were performed using NK cell culture medium kit ( NK cell expansion kit, Yikesai), 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), draw 45 μL cytokine I into PBS (if cytokine I is used up at one time, it is recommended to draw 1 mL PBS from the 50 mL centrifuge tube to rinse the cytokine I tube once and add it back to the centrifuge tube), turn it upside down to mix, and add it to a 75 cm bottom area. 2 Place the solution in a T75 cell culture flask, shake it back and forth to disperse the liquid at the bottom of the flask, and activate it at 4°C overnight. If not used immediately, store it in a refrigerator at 4°C. 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, and resuspend the PBMC in 15 mL of NK culture medium (supplemented with 10% heat-inactivated autologous plasma) and inoculate it into a T75 flask at a seeding density of 2×10^6 cells / mL. Add 150 μL of cytokine II, shake it back and forth, and place it in a 37°C, 5% CO2 incubator for culture.

[0078] (3) On the third day, slowly add 15 mL of fresh NK culture medium along the side of the culture flask. Be careful not to touch the bottom of the culture flask and 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 flask 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 cells into a cell culture bag according to the volume of the cell suspension, and reduce the content of heat-inactivated autologous plasma in the fresh NK culture medium to 1% starting from the 7th day.

[0081] (6) Harvest cells on day 18 of culture and perform viral transduction.

[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 seed 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°C, 5% CO2 incubator and culture them. 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 rehydration is performed. Cell proliferation accelerates after day 7, and cells are centrifuged and the fluid is replaced every 3 days.

[0087] ④ After 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 NK cell proliferation, the function-enhanced engineered NK cells of the present invention (labeled as "NK-IL12-CD137 cells", which are NK cells expressing IL12-CD137 chimeric protein prepared in this example), control group 1 (labeled as "NK cells + IL12 factor" group, which are NK cells infected with control blank lentivirus, and human IL12 factor (200 U / mL concentration) was added during the amplification process) and control group 2 (labeled as "NK cells", which are NK cells infected with 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 statistics were performed. 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". During the culture process, IL12 factor was not added; the control group 1 ("NK cells + IL12 factor" group) was NK cells infected with the empty lentivirus prepared in step 2.4 (for the preparation of blank lentivirus, see "Preparation of IL12-CD137 chimeric protein overexpressing lentivirus and blank lentivirus" in Example 1). During the culture process, human recombinant IL12 factor (200 U / mL concentration) was always used; the control group 2 (labeled as "NK cells") was NK cells infected with the control blank lentivirus, and human recombinant IL12 factor was not 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), except for control group 1, IL12 factor was not added during the culture process of other groups.

[0091] (2) During the 9 days after inoculation, the culture medium was half-changed every two days. The total cell number and the number of viable cells were counted daily using AOP I 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 on a Counstar Altair instrument. Cell proliferation was recorded, and bright field photography was taken to record the morphology of NK cells.

[0092] Results see 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 the feline infectious peritonitis virus (FIPV) that primarily 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 forms based on its pathological characteristics, with clinical manifestations including fatigue, loss of appetite, and fever. Currently, the main treatments for FIP are immunosuppressive 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 the disease. FIP is also considered 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 isolation 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 Groups and Treatment Plan:

[0101] NK cell treatment group (intravenous infusion of IL2-CD137-NK cells): 15 cats received intravenous injection of a mixture of 1 mL of IL2-CD137-NK cell suspension and 9 mL of normal saline. The treatment was once every two weeks for a total of three 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 the NK cells are tested to ensure purity is above 85% and cell viability is above 90%. During infusion, 1 mL of cell suspension is injected into a bag containing 9 mL of saline solution, mixed, and then infused. The cell infusion rate is adjusted to 10 mL / h. In other words, the cell suspension mixed with saline solution 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, the medication was stopped.

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

[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 typically produced by the liver in response to inflammation or infection. In the diagnosis of feline infectious peritonitis, serum amyloid A serves as a biomarker to help assess the inflammatory status of cats. Because FIP is an inflammatory disease, SAA levels are elevated in cats with FIP.

[0111] The SAA values ​​of 30 cats were monitored, and the changes of 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. This shows that the use of the engineered NK cells of the present invention can significantly reduce the inflammatory response in the 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 their 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 at passage P14. K562-Luc cells were harvested on the same day of logarithmic growth, the culture medium was removed, and the cells were washed twice with PBS before inoculation (K562-Luc cell viability before and after tumor inoculation was 97.8% and 95.9%, respectively). The inoculation volume was 1×10^6 / 200 μL per mouse, and the inoculation site was the tail vein of the mouse.

[0119] 3. Grouped Dosing

[0120] On day 5 after tumor cell inoculation, in vivo fluorescence imaging was used to detect the tumor burden and data were analyzed. The mean photon radiation value was 5.79×10^4 (p / sec / cm 2 8 mice were randomly divided into 2 groups, 4 mice in each group, and NK cell therapy 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): Injected with engineered feline NK cells (NK cells expressing IL2-CD137 chimeric protein) at a dose of 2 x 10^7 / animal via tail vein injection in a volume of 200 μL. The cell preparation process was the same as in Examples 1 and 2.

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

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

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

[0125] 4.1 Prepare a 15 mg / mL stock solution of luciferin by dissolving 1.5 g of luciferin powder in 100 mL of PBS. Once fully dissolved, filter the solution using a 0.2 μm filter in a clean bench 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 of Luciferin) needs to be injected.

[0127] 4.3 Use a small animal in vivo imaging instrument (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] See the results Figure 5, from the results of mouse live imaging, it is shown that compared with conventional cat NK control cells, the functionally enhanced engineered NK cells (NK-IL12-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 the mice. From the perspective of mortality, the control group was at the D26 time point, all of which had died, so there was no live image. All mice in the experimental group (injected with NK-IL12-CD137 mice) were in a living state. Death occurred in the engineered NK cell treatment group at the D32 time point. Only one engineered NK treated mouse remained alive at D34, and all 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 (red indicates a large tumor load in the imaging figure). From the perspective of therapeutic effect, engineered NK cells can significantly prolong the lifespan 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 promotes NK cell proliferation, enhances cytotoxicity, and produces IFN-γ. In the in vitro expansion of human and mouse NK cells, the combination of IL-12 with 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 conventionally 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 of membrane-bound rhIL21 K562 feeder cells, supplemented with 100IU / mL human recombinant-IL2 factor, can also effectively expand cat NK cells, suggesting that 4-1BB (CD137) signaling may contribute to the proliferation of NK cells. This information suggests that transforming NK cells from the IL2 / IL12 / IL15 / IL21 and CD137 signaling pathways may be a better strategy to improve the in vitro expansion ability and in vivo killing ability of NK cells, solving 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, thereby enhancing the expansion and killing ability of NK cells and enhancing 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 principles of the present invention should be included in the scope of protection 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; its amino acid sequence is shown in SEQ ID NO:

1.

2. The IL-12-CD137 chimeric protein according to claim 1, wherein The IL-12 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, wherein 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, wherein 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, wherein 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 also comprises the IL-12-CD137 chimeric protein according to any one of claims 1 to 5.

7. A preparation of functionally enhanced engineered NK cells according to claim 6, characterized in that: After synthesizing a DNA fragment encoding the IL-12-CD137 chimeric protein according to 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 into 293T cells for 48 to 72 hours to obtain a culture medium containing a lentivirus expressing the target protein molecule. 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-EF1alpha.

9. The preparation of functionally enhanced engineered NK cells according to claim 7, characterized in that: The primary NK cells are derived from peripheral blood, umbilical cord blood 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.

10. The use of the functionally enhanced engineered NK cells according to claim 6, wherein: Used for preparing medicine for treating feline viral infectious diseases or tumors; the feline viral infectious disease is feline infectious peritonitis; the tumor is feline lymphoma.

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