Method for improving NK cell effector function

Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to a method for improving the effector function of NK (Natural Killer) cells, which is characterized in that a glycolysis metabolism process is influenced and the generation of lactic acid is reduced by constructing point mutation vectors PKM2-322K-R and PKM2-433K-R of a PKM2 gene so as to inhibit the level of lactic acid modification. The invention also provides a construction method of the NK92MI cell of the PKM2 protein. The construction method comprises the following steps: constructing a vector, extracting plasmids, packaging concentrated viruses and transfecting target cells. According to the present invention, the protein targeting lactylation modification is adopted to modify and block the lactylation modification so as to reverse the function depletion of the NK cell, the PKM2 delactylation is performed through the point mutation so as to partially reverse the NK cell function, and the expression level of the effector molecule of the NK92MI cell is recovered;

Description

TECHNICAL FIELD

[0001] The 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 technologies mainly include immune checkpoint inhibitors, chimeric antigen receptor (CAR) cell therapy, cytokine therapy, tumor vaccine, etc. However, there are still many defects such as low response rate and drug resistance, serious side effects, solid tumor barrier and limited indications. In the tumor microenvironment, in order to adapt to the hypoxic and nutrient-poor environment, cancer cells usually start 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 identified. Targeting lactylation has become one of the directions for developing tumor treatment strategies.

[0003] Natural killer cells (NK cells) enhance anti-tumor immune responses by secreting cytokines, ADCC effect, recruitment and maturation of DC cells, and assistance of 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

[0004] In order to solve the problem of NK cell function exhaustion in the tumor microenvironment, the application provides a method for improving the effector function of NK cells.

[0005] A method for improving the effector function of NK cells, the method is to construct a point mutation vector of PKM2 gene, affect the glycolysis metabolic process, reduce the production of lactic acid and thereby inhibit the level of lactylation. The mutation vector is PKM2-322K-R (the 322th lysine is mutated to arginine) and PKM2-433K-R (the 433th lysine is mutated to arginine). The coding nucleic acid of PKM2-322K-R is SEQ ID NO: 1, and the coding nucleic acid of PKM2-433K-R is SEQ ID NO: 2.

[0006] A method for constructing NK92MI cells of PKM2 protein, comprising the following steps: (1) Constructing a bacterial strain containing a point mutation vector PKM2-322K-R or PKM2-433K-R plasmid of the PKM2 gene according to claim 1; (2) Extracting the plasmid: inoculating the bacterial strain containing the PKM2-322K-R or PKM2-433K-R plasmid into an ampicillin-containing solid culture medium, culturing in a 37°C incubator for 12 h, picking a single colony, transferring it into LB liquid medium, and then placing it on a shaker for shaking to obtain a bacterial solution, and then using an endotoxin-free plasmid extraction kit to extract the plasmid and determine the plasmid purity and concentration; (3) Packaging and concentrating the virus: using 293T cells to package the PKM2-322K-R or PKM2-433K-R plasmid obtained in step (2), filtering the culture supernatant of the 293T cells, then adding 4×PEG8000, shaking on a shaker, centrifuging, resuspending the lower precipitate with 1×PBS, and adding 1000×protamine and BX795 to obtain concentrated virus; (4) Transfecting the target cells: adding the concentrated virus obtained in (3) to the NK92MI cell culture supernatant (the volume of the virus is 1 / 100 of the culture supernatant); 8 h later, change the liquid to obtain NK92MI cells transfected with the target plasmid.

[0007] Preferably, the shaking temperature in step (2) is 37°C, the rotation speed is 2000 rpm, and the shaking time is 12 h.

[0008] Preferably, the plasmid purity and concentration requirement in step (2) is that the absorbance 260nm: 280nm is within the range 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.

[0009] Preferably, the filter used in step (3) is a filter with a pore size of 0.45 μm.

[0010] Preferably, the shaking temperature of the shaker in step (3) is 4°C, and the shaking and overturning time is 12 h.

[0011] Preferably, the centrifugal force of the centrifugation in step (3) is 1600 g, and the centrifugation time is 1 h.

[0012] The beneficial effects of the present application are: The present invention targets the protein modification of lactylation, blocks lactylation modification, and thus reverses the functional exhaustion of NK cells. Delactation of PKM2 through point mutation can partially reverse NK cell function and restore the expression level of effector molecules of NK92MI cells. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Schematic diagram of the target fragment structure of the PKM2 target fragment modified by mutating lysine 322 / 433 to arginine to simulate delactation; Figure 2 This is the GFP fluorescence image after the virus solution was transfected into 293T cells; Figure 3 A is the expression level of TNF-α, an effector molecule of GFP+ NK cells; Figure 3 B is the expression level of GFP+ NK cell effector molecule granzyme B. DETAILED DESCRIPTION

[0013] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely further describe the technical solutions in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0014] Example 1 A method for constructing NK92MI cells expressing PKM2 protein comprises the following steps: (1) Construction of the bacterial strain of the PKM2 gene point mutation vector PKM2-322K-R plasmid. The schematic diagram of the vector target fragment structure is as follows: Figure 1 As shown; (2) Plasmid extraction: The strain containing the PKM2-322K-R plasmid was inoculated into a solid culture medium containing ampicillin resistance and cultured in a 37°C incubator for 12 hours. A single clone colony was picked and transferred to LB liquid culture medium, which was then placed on a shaker for shaking to obtain a bacterial solution. The plasmid was then extracted using the TIANGEN endotoxin-free plasmid extraction kit, and the plasmid purity and concentration were determined. The shaking temperature was 37°C, the speed was 2000 rpm, and the shaking time was 12 hours. The plasmid purity and concentration requirements were: absorbance at 260nm:280nm was in the range of 1.8-2.0, and absorbance at 260nm:230nm was between 1.8-2.0; the plasmid concentration was greater than 2 μg / μl; (3) Packaging and concentrating virus: the PKM2-322K-R plasmid obtained in step (2) is packaged using 293T cells, filtered using a filter with a pore size of 0.45 μm, then 1 / 3 of the volume of PEG8000 is added, and the mixture is shaken at 4°C for 12 h, and centrifuged at 1600 g for 1 h; the lower precipitate is resuspended using 150 μL of 1×PBS, and 0.15 μL of protamine and BX795 are added to obtain the concentrated virus; (4) Transfecting target cells: 50 μL of the concentrated virus obtained in step (3) is added to the NK92MI cell culture supernatant (1 million / 1 ml), and the volume of the virus is 1 / 100 of the volume of the culture supernatant; the medium is changed after 8 h to obtain NK92MI cells transfected with the target plasmid.

[0015] Preferably, the plasmid obtained in step (2) has a purity and concentration requirement that the absorbance at 260 nm: 280 nm is in the range of 1.8-2.0, and the absorbance at 260 nm: 230 nm is in the range of 1.8-2.0, and the plasmid concentration is greater than 2 μg / μl.

[0016] Example 2 The point mutation vector of the PKM2 gene is PKM2-433K-R, and the other steps are the same as in Example 1.

[0017] Comparative Example 1 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.

[0018] Test Example 1 50 μL of the concentrated virus obtained in Examples 1-2 and Comparative Example 1 is added to the 293T cell culture supernatant (2 million / 1 ml); the medium is changed after 8 h to obtain 293T cells transfected with the target plasmid, and the GFP fluorescence is observed using an immunofluorescence microscope. After the virus solution transfects the 293T cells, green fluorescence can be observed under a fluorescence microscope, as shown in Figure 2 , indicating that the plasmid transfection is successful.

[0019] Test Example 2 The NK92MI cells transfected with the target plasmid obtained in Examples 1-2 and Comparative Example 1 are treated with 15 mM sodium lactate for 48 h, and the proportion and mean fluorescence intensity of TNF-α (A) and granzyme B (B) positive cells in the NK cells are analyzed by flow cytometry.

[0020] As shown in Figure 3 , it is found by flow cytometry that the de-lactation mutation of 322 / 433 restores the expression levels of TNF-α and granzyme B under the high-lactate background to different degrees.

Claims

1. A method of enhancing NK cell effector function, characterized by, The method is to construct a point mutation vector of a PKM2 gene; the mutation vector is PKM2-322K-R (a lysine at the 322nd position is mutated into an arginine) and PKM2-433K-R (a lysine at the 433rd position is mutated into an arginine); the coding nucleic acid of the PKM2-322K-R is SEQ ID NO: 1; and the coding nucleic acid of the PKM2-433K-R is SEQ ID NO:

2.

2. A method of constructing NK92MI cells of PKM2 protein, characterized in that, The construction method comprises the following steps: (1) constructing a bacterial strain containing the point mutation vector PKM2-322K-R or PKM2-433K-R plasmid of the PKM2 gene according to claim 1; (2) extracting the plasmid: inoculating the bacterial strain containing the PKM2-322K-R or PKM2-433K-R plasmid into a solid culture medium containing ampicillin resistance, and culturing in a 37°C incubator for 12 hours; picking up a single colony, transferring it into LB liquid medium, and placing it on a shaker for shaking to obtain a bacterial solution; then using an endotoxin-free plasmid extraction kit to extract the plasmid, and determining the plasmid purity and concentration; (3) packaging and concentrating the virus: using 293T cells to package the PKM2-322K-R or PKM2-433K-R plasmid obtained in step (2), filtering the culture supernatant of the 293T cells, then adding 4×PEG8000, and shaking on a shaker, centrifuging, resuspending the lower precipitate with 1×PBS, and adding 1000×protamine and BX795 to obtain concentrated virus; (4) transfecting the target cells: adding 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); 8 hours later, changing the liquid to obtain NK92MI cells transfected with the target plasmid.

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

4. The construction method of claim 2, wherein, The plasmid purity requirement in step (2) is that the absorbance at 260 nm: 280 nm is within the range of 1.8-2.0, and the absorbance at 260 nm: 230 nm is between 2.0-2.

2.

5. The method of construction of claim 2, wherein, The plasmid concentration in step (2) is greater than 2 μg / μl.

6. The construction method of claim 2, wherein, In step (3), the filter screen with a pore size of 0.45 μm is used for filtering.

7. The construction method of claim 2, wherein, The shaking temperature in step (3) is 4°C, and the shaking and overturning time is 12 hours.

8. The construction method of claim 2, wherein, The centrifugal force in step (3) is 1600g, and the centrifugation time is 1 hour.

9. NK92MI cells containing the PKM2 protein obtained by the construction method according to any one of claims 2-8.

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