A method of improving nk cell effector function

Abstract

The application belongs to the technical field of immunotherapy, and particularly relates to a method for improving the effect function of NK cells, wherein point mutation vectors PKM2-322K-R and PKM2-433K-R of a PKM2 gene are constructed, the glycolysis metabolic process is affected, the generation of lactic acid is reduced, and the lactic acidification modification level is inhibited. The application further provides a construction method of NK92MI cells of a PKM2 protein, and the steps include constructing a vector, extracting a plasmid, packaging and concentrating viruses, and transfecting target cells. The application targets lactic acidification modification, a protein modification, blocks lactic acidification modification, reverses the functional exhaustion of NK cells, makes PKM2 de-lactic acidification through point mutation, can partially reverse the function of NK cells, and restores the expression level of effect molecules of NK92MI cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immunotherapy, and particularly relates to a method for improving the effector function of NK cells. BACKGROUND

[0002] Immunotherapy is an important breakthrough in the field of tumor treatment in recent years. Existing immunotherapy techniques mainly include immune checkpoint inhibitors, chimeric antigen receptor (CAR) cell therapy, cytokine therapy, tumor vaccines, etc. However, there are still many defects such as low response rate and drug resistance, serious side effects, solid tumor barriers, and limited indications.

[0003] In the tumor microenvironment, in order to adapt to the hypoxic and nutrient-poor environment, cancer cells usually initiate metabolic reprogramming, and produce a large amount of lactic acid through aerobic glycolysis. Lactic acid affects the malignant characteristics of tumors through various ways, and the remodeling of the immune microenvironment is complex. In addition to forming an acidic environment, lactic acid can also be epigenetically regulated through post-translational modification, affecting tumor progression. Lactylation is a new type of post-translational modification discovered in recent years, and molecules and modification enzymes that undergo lactylation are continuously being identified. Targeting lactylation has become one of the directions for developing tumor treatment strategies.

[0004] Natural killer cells (NK cells) enhance anti-tumor immune responses by secreting cytokines, ADCC effect, recruitment and maturation of DC cells, and assisting CD8+ T cells. However, in the tumor microenvironment, NK cell function is severely impaired. NK cells take up a large amount of lactic acid in the tumor microenvironment, leading to lactylation of PKM2 and weakening the effector function of NK cells. How to effectively target NK cell lactylation and then reverse NK cell function exhaustion in the tumor microenvironment is a difficult problem in cancer treatment. So far, there has been no report that targeting NK cell lactylation in solid tumors can effectively improve the killing ability of NK cells on tumor cells. SUMMARY

[0005] In order to solve the problem of NK cell function exhaustion in the tumor microenvironment, the present application provides a method for improving the effector function of NK cells.

[0006] A method for improving the effector function of NK cells, the method is to construct a point mutation vector of PKM2 gene, affect the glycolytic metabolic process, reduce the production of lactic acid to inhibit the modification level of lactic acidification; the mutation vector is PKM2-322K-R (the 322th lysine is mutated to arginine), PKM2-433K-R (the 433th lysine is mutated to arginine); the coding nucleic acid of the PKM2-322K-R is SEQ ID NO: 1; the coding nucleic acid of the PKM2-433K-R is SEQ ID NO: 2.

[0007] A method for constructing NK92MI cells of PKM2 protein, comprising the following steps:

[0008] (1) Constructing a strain containing the PKM2 gene point mutation vector PKM2-322K-R or PKM2-433K-R plasmid as claimed in claim 1;

[0009] (2) Extracting plasmid: inoculate the strain containing the PKM2-322K-R or PKM2-433K-R plasmid into an ampicillin-containing solid culture medium, cultivate in a 37°C incubator for 12 h, pick up a single colony, transfer to LB liquid medium, then place on a shaker for shaking, obtain bacterial solution, then use an endotoxin-free plasmid extraction kit to extract plasmid, and determine plasmid purity and concentration;

[0010] (3) Packaging and concentrating virus: use 293T cells to package the PKM2-322K-R or PKM2-433K-R plasmid obtained in step (2), filter the culture supernatant of 293T cells, then add 4×PEG8000, invert on a shaker, centrifuge, resuspend the lower precipitate with 1×PBS, add 1000×protamine and BX795, obtain concentrated virus;

[0011] (4) Transfecting target cells: add the concentrated virus obtained in (3) to the NK92MI cell culture supernatant (virus volume is 1 / 100 of the culture supernatant); change the liquid after 8 h, obtain NK92MI cells transfected with the target plasmid.

[0012] Preferably, the shaking temperature in step (2) is 37°C, the rotation speed is 2000 rpm, and the shaking time is 12 h.

[0013] Preferably, the plasmid purity and concentration requirement in step (2) is that the absorbance 260nm: 280nm is within the interval of 1.8-2.0, and the absorbance 260nm: 230nm is between 2.0-2.2; the plasmid concentration is greater than 2 μg / μl.

[0014] Preferably, the filtration in step (3) is performed using a filter with a pore size of 0.45 μm.

[0015] Preferably, the shaking in step (3) is performed at a temperature of 4°C for 12 h.

[0016] Preferably, the centrifugation in step (3) is performed at a centrifugal force of 1600 g for 1 h.

[0017] The present application has the following advantages:

[0018] The present application targets the lactylation modification of the protein, blocks the lactylation modification to reverse the functional exhaustion of the NK cells, and partially reverses the NK cell function by de-lactylation of PKM2 through point mutation to restore the expression level of the effector molecules of the NK92MI cells. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The structure of the target fragment of the PKM2-322K-R plasmid is shown in the following figure.

[0020] Figure 2 The GFP fluorescence map after transfection of the virus liquid into 293T cells is shown in the following figure.

[0021] Figure 3 A is the expression level of the NK cell effector molecule TNF-α of GFP+. Figure 3 B is the expression level of the NK cell effector molecule granzyme B of GFP+. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be further described in detail below with reference to the accompanying drawings of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0023] Embodiment 1

[0024] A method for constructing an NK92MI cell of a PKM2 protein comprises the following steps:

[0025] (1) Construct a strain of a point mutation vector PKM2-322K-R plasmid of a PKM2 gene, and the structure of the target fragment of the vector is shown in the following figure. Figure 1

[0026] ​(2) Quality improvement: the bacteria containing PKM2-322K-R plasmid were inoculated in the solid culture medium containing ampicillin, and cultured in a 37°C incubator for 12 h. A single colony was picked and transferred to LB liquid medium, and then placed on a shaker for shaking. The bacterial solution was obtained, and the plasmid was extracted using a TIANGEN endotoxin-free plasmid extraction kit. The plasmid purity and concentration were determined. The shaking temperature was 37°C, the rotation speed was 2000 rpm, and the shaking time was 12 h. The plasmid purity and concentration requirements were as follows: the absorbance at 260 nm: 280 nm was within the range of 1.8-2.0, the absorbance at 260 nm: 230 nm was between 1.8-2.0, and the plasmid concentration was greater than 2 μg / μl;

[0027] (3) Virus packaging and concentration: the PKM2-322K-R plasmid obtained in step (2) was packaged using 293T cells, filtered using a filter with a pore size of 0.45 μm, and then 1 / 3 of the volume of PEG8000 was added. The supernatant was shaken at 4°C for 12 h, centrifuged at 1600 g for 1 h, resuspended with 150 μL 1×PBS, and added with 0.15 μL protamine and BX795 to obtain concentrated virus;

[0028] (4) Transfection of target cells: 50 μL of the concentrated virus obtained in step (3) was added to the NK92MI cell culture supernatant (1 million / 1 ml), and the virus volume was 1 / 100 of the culture supernatant. After 8 h, the liquid was changed to obtain NK92MI cells transfected with the target plasmid.

[0029] Preferably, the plasmid purity and concentration requirements in step (2) are as follows: the absorbance at 260 nm: 280 nm is within the range of 1.8-2.0, the absorbance at 260 nm: 230 nm is between 1.8-2.0, and the plasmid concentration is greater than 2 μg / μl.

[0030] Example 2

[0031] The point mutation vector of the PKM2 gene is PKM2-433K-R, and the other steps are the same as in Example 1.

[0032] Comparative Example 1

[0033] The vector of the PKM2 gene is PKM2-433K-WT, and the coding nucleic acid of PKM2-433K-WT is SEQ ID NO: 3. The other steps are the same as in Example 1.

[0034] Test Example 1

[0035] The concentrated virus liquid obtained in Example 1-2 and Comparative Example 1 was added to 293T cell culture supernatant (2 million / 1 ml) 50 μL; 8 h later, the liquid was changed, and 293T cells transfected with the plasmid of interest were obtained, and GFP fluorescence was observed using immunofluorescence microscopy. After the virus liquid transfected 293T cells, green fluorescence was observed under fluorescence microscopy, as shown in Figure 2 , indicating that plasmid transfection was successful.

[0036] Test Example 2

[0037] NK92MI cells transfected with the plasmid of interest obtained in Example 1-2 and Comparative Example 1 were treated with sodium lactate 15 mM for 48 h, and the proportion of NK cells positive for TNF-α (A) and granzyme B (B) and the mean fluorescence intensity were analyzed by flow cytometry.

[0038] As shown in Figure 3 , it was found by flow cytometry that the de-lactation mutations of 322 / 433 restored the expression levels of TNF-α and granzyme B to varying degrees under the high lactate background.

Claims

1. A method for constructing NK92MI cells containing PKM2 protein, characterized in that, The construction method includes the following steps: (1) Construct a bacterial strain containing a point mutation vector PKM2-322K-R or PKM2-433K-R plasmid containing the PKM2 gene; the nucleic acid encoded by PKM2-322K-R is SEQ ID NO:1; the nucleic acid encoded by PKM2-433K-R is SEQ ID NO:2; (2) Plasmid extraction: The bacterial strain containing PKM2-322K-R or PKM2-433K-R plasmid was inoculated into a solid medium containing ampicillin resistance and cultured in a 37°C incubator for 12 h. A single colony was picked and transferred to LB liquid medium and shaken on a shaker to obtain bacterial solution. Then, the plasmid was extracted using an endotoxin-free plasmid extraction kit, and the purity and concentration of the plasmid were determined. (3) Packaging and concentrating the virus: The PKM2-322K-R or PKM2-433K-R plasmids obtained in step (2) were packaged using 293T cells. The culture supernatant of the 293T cells was collected and filtered. Then 4×PEG8000 was added, the mixture was turned on a shaker, centrifuged, and the lower precipitate was resuspended with 1×PBS. 1000×protamine and BX795 were added to obtain the concentrated virus. (4) Transfection of target cells: Add the concentrated virus obtained in step (3) to the culture supernatant of NK92MI cells (the volume of the virus is 1 / 100 of the culture supernatant); change the medium after 8 h to obtain NK92MI cells transfected with the target plasmid.

2. The construction method according to claim 1, characterized in that, The oscillation temperature in step (2) is 37°C, the rotation speed is 2000 rpm, and the oscillation time is 12 h.

3. The construction method according to claim 1, characterized in that, The plasmid purity requirements in step (2) are as follows: absorbance 260nm: 280nm in the range of 1.8-2.0; absorbance 260nm: 230nm in the range of 2.0-2.

2.

4. The construction method according to claim 1, characterized in that, The plasmid concentration in step (2) is greater than 2 μg / μl.

5. The construction method according to claim 1, characterized in that, In step (3), a filter with a pore size of 0.45 μm is used for filtration.

6. The construction method according to claim 1, characterized in that, In step (3), the shaking temperature of the shaker is 4°C and the shaking and turning time is 12 h.

7. The construction method according to claim 1, characterized in that, In step (3), the centrifugal force is 1600g and the centrifugation time is 1 h.

8. NK92MI cells containing PKM2 protein obtained by the construction method according to any one of claims 1-7.

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