A method of targeting lactylation modification to enhance nk cell effector function

Abstract

The application belongs to the technical field of immunotherapy, and particularly relates to a method for improving NK cell function by targeted inhibition of lactic acidification. The application provides a method for improving NK cell function by targeted inhibition of lactic acidification, wherein by targeted inhibition of an NK cell lactic acid transport carrier or inhibition of a modification enzyme in the lactic acidification process, the modification enzyme being an NK cell lactic acidification modification enzyme Writer or a lactic acid coenzyme A synthesis enzyme, lactic acidification modification of NK92MI is blocked, thereby reversing the functional exhaustion of NK cells, the NK cell function can be partially reversed by inhibiting lactic acidification modification, and the expression level of an effector molecule of NK92MI cells is recovered.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, specifically relating to a method for targeting and inhibiting lactation to enhance NK cell function. Background Technology

[0002] In recent years, immunotherapy has made groundbreaking progress in the field of cancer treatment, with treatment strategies such as immune checkpoint inhibitors, CAR-T cell therapy, cytokine therapy, and tumor vaccines significantly improving clinical efficacy. However, existing therapies still face many challenges, including low treatment response rates, drug resistance, significant toxic side effects, and limitations in the treatment of solid tumors.

[0003] The characteristic metabolic remodeling of the tumor microenvironment is a significant factor leading to immunosuppression. To adapt to the harsh environment of hypoxia and nutrient deprivation, tumor cells produce large amounts of lactate through aerobic glycolysis. This metabolite not only acidifies the microenvironment but also influences tumor progression through the epigenetic regulatory mechanism of lactation modification. Immune cells take up lactate from the tumor microenvironment via the monocarboxylic acid transporter MCT. Lactoyl-CoA synthase 2 (ACSS2), the succinyl-CoA synthase 2 (SCS) family (GTPSCS), and acetyltransferases ACAT1 / 2 can convert the catalytic site of lactate to lactoyl-CoA, subsequently undergoing lactation modification catalyzed by the lactation-modifying enzyme P300. As a newly discovered post-translational modification of proteins, lactation modification, with its substrate molecules and modifying enzymes continuously being identified, has become a novel target for cancer therapy.

[0004] NK cells participate in anti-tumor immune responses through multiple mechanisms, including cytokine secretion, antibody-dependent cell-mediated cytotoxicity (ADCC), dendritic cell (DC) recruitment and activation, and CD8+ T cell-assisted activation. However, in the tumor microenvironment, NK cells undergo lactation modification after taking up excessive lactate, leading to impaired cellular function. Currently, how to target and regulate NK cell lactation modification to reverse its functional exhaustion remains a major scientific challenge in tumor immunotherapy. Notably, targeted intervention strategies for NK cell lactation modification in solid tumors and their efficacy enhancement have not yet been clearly reported. Summary of the Invention

[0005] To address the problem of NK cell functional exhaustion in the tumor microenvironment, this invention provides a method for targeting and inhibiting lactation to enhance NK cell function.

[0006] A method for targeting and inhibiting lactation to enhance NK cell function, wherein the method involves targeting and inhibiting NK cell lactate transporters and inhibiting modifying enzymes in the lactation process; the modifying enzymes are NK cell lactation modifying enzyme Writer or lactate coenzyme A synthase.

[0007] Preferably, the targeted inhibition of NK cell lactate transporter is a monocarboxylate transporter (MCT), which targets and blocks NK cell lactate uptake, restoring NK cell effector function. The specific steps are as follows:

[0008] NK cells were diluted to 500,000 / ml and incubated for 48 h with 10 mM CHC (α-Cyano-4-hydroxycinnamic acid, CAS No. 28166-41-8), an inhibitor of MCT, in tumor supernatant or at a concentration of 15 mM sodium lactate.

[0009] Preferably, the lactation-modifying enzyme Writer is P300; the lactate-coenzyme A synthase is GTPSCS, ASCC2, or ACAT1 / 2; the specific steps for inhibiting the modifying enzyme during lactation and restoring NK cell effector function are as follows:

[0010] When the modifying enzyme is P300, NK cells are diluted to 500,000 / ml and incubated with NK92MI cells for 48 h in 15 mM sodium lactate with 20 μM of the P300 inhibitor C646 (CAS No.: 328968-36-1).

[0011] When the modifying enzyme is GTPSCS, NK cells are diluted to 500,000 / ml, and 100 mM sodium formate, an inhibitor of GTPSCS, is used in a 15 mM sodium lactate solution.

[0012] When the modifying enzyme is ASCC2, NK cells are diluted to 500,000 / ml, and 20 μM of the ASCC2 inhibitor ACSS2-IN-2 (CAS No.: 2332820-04-7) is used in a 15 mM sodium lactate solution.

[0013] When the modifying enzyme is ACAT1 / 2, NK cells are diluted to 500,000 / ml and incubated with 20 μM of Avasimibe (CAS No. 166518-60-1), an inhibitor of ACAT1 / 2, for 48 h in 15 mM sodium lactate.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention targets lactation modification, a protein modification, and blocks NK92MI lactation modification to reverse NK cell functional exhaustion. By inhibiting lactation modification, it can partially reverse NK cell function and restore the expression level of effector molecules in NK92MI cells. Attached Figure Description

[0016] Figure 1 This refers to the lactate content within NK cells, of which... Figure 1 The lactate content in incubated NK92MI cells obtained in Example 1 and Comparative Examples 1-3, Figure 1 B represents the lactate content in NK92MI cells after incubation obtained in Example 2 and Comparative Example 4;

[0017] Figure 2 For the detection of NK cell toxicity-related indicators, among which Figure 2 A represents the killing ability of NK92MI cells after incubation against Huh-7 cells obtained in Example 1 and Comparative Examples 1-3; Figure 2 B represents the IFN-γ and TNF-α secretion levels in the supernatant of incubated NK92MI cells obtained in Example 2 (CHC concentration of 10 mM) and Comparative Example 4. Figure 2 C represents the killing ability of NK92MI cells against K562 cells obtained after incubation in Example 2 (CHC concentration of 10 mM) and Comparative Example 4. Figure 2 D represents the killing ability of NK92MI cells after incubation against Huh-7 cells obtained in Example 2 (CHC concentration of 10 mM) and Comparative Example 4.

[0018] Figure 3 This represents the overall lactation modification level of NK cells, among which, Figure 3 A represents the overall lactation modification level of NK92MI cells after incubation obtained in Example 1 and Comparative Examples 1-3; Figure 3 B represents the overall lactation modification level of NK92MI cells after incubation, obtained in Example 2 (CHC concentration of 10 mM) and Comparative Example 4.

[0019] Figure 4 The killing ability of incubated NK92MI cells against Huh-7 cells obtained in Example 3 and Comparative Example 4;

[0020] Figure 5 The changes in lactation levels of NK92MI cells after incubation obtained in Example 3 and Comparative Example 4 are shown, where A represents the overall lactation modification level of NK cells and B represents the H3K18 lactation modification level of NK cells.

[0021] Figure 6 For the detection of NK cell toxicity-related indicators, among which, Figure 6 A represents the killing ability of incubated NK92MI cells against Huh-7 cells obtained in Examples 4-6 and Comparative Example 4; Figure 6 B represents the expression level of the NK92MI cell effector molecule IFN-γ obtained after incubation in Examples 4-6 and Comparative Example 4; Figure 6C represents the expression level of the effector molecule TNF-α in NK92MI cells after incubation, obtained in Examples 4-6 and Comparative Example 4. Figure 6 D represents the expression level of perforin, the effector molecule of NK92MI cells obtained after incubation in Examples 4-6 and Comparative Example 4; Figure 6 E represents the expression level of granzyme-B, the effector molecule of NK92MI cells, obtained after incubation in Examples 4-6 and Comparative Example 4.

[0022] Figure 7 The changes in lactation modification levels in NK92MI cells after incubation obtained in Examples 4-6 and Comparative Example 4, wherein... Figure 7 A represents the overall lactation modification level of NK cells. Figure 7 B represents the level of H3K18 lactation modification in NK cells. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] NK cells were diluted to 500,000 / ml and incubated for 48 h with tumor supernatant from diffuse large B-cell lymphoma U2932, colorectal cancer HCT-15, and hepatocellular carcinoma Huh-7 lineage, along with 10 mM of the MCT inhibitor CHC (purchased from MedChemExpress).

[0026] Example 2

[0027] NK cells were diluted to 500,000 / ml and a high lactate environment was simulated using 15 mM sodium lactate. Subsequently, they were co-incubated with NK92MI cells at a concentration of 5 or 10 mM of CHC (purchased from MedChemExpress), an inhibitor of MCT, for 48 h.

[0028] Example 3

[0029] NK cells were diluted to 500,000 / ml, and a high lactate environment was simulated using 15 mM sodium lactate. NK92MI cells were then co-incubated with 20 μM of C646 (purchased from MedChemExpress), an inhibitor of P300, for 48 h.

[0030] Example 4

[0031] NK cells were diluted to 500,000 / ml, and a high lactate environment was simulated using 15 mM sodium lactate. Sodium formate (purchased from Beijing Solarbio Science & Technology Co., Ltd.), an inhibitor of GTPCS, was added to bring the final concentration to 100 mM. NK cells were then incubated for 48 h.

[0032] Example 5

[0033] NK cells were diluted to 500,000 / ml, and a high lactate environment was simulated using 15 mM sodium lactate. The ASCC2 inhibitor ACSS2-IN-2 (purchased from MedChemExpress) was added to bring the final concentration to 20 μM, and the NK cells were incubated for 48 h.

[0034] Example 6

[0035] NK cells were diluted to 500,000 / ml, and a high lactate environment was simulated using 15 mM sodium lactate. 20 μM of Avasimibe (purchased from TargetMol), an inhibitor of ACAT1 / 2, was added to bring the final concentration to 20 μM. NK cells were then incubated for 48 h.

[0036] Comparative Example 1

[0037] NK cells were diluted to 500,000 / ml and incubated with diffuse large B-cell lymphoma U2932 for 48 h.

[0038] Comparative Example 2

[0039] NK cells were diluted to 500,000 / ml and used as HCT-15 tumor supernatant for 48 h.

[0040] Comparative Example 3

[0041] NK cells were diluted to 500,000 / ml and incubated with the supernatant of Huh-7 lineage liver cancer cells for 48 h.

[0042] Comparative Example 4

[0043] NK cells were diluted to 500,000 / ml and incubated for 48 hours using 15 mM sodium lactate to simulate a high lactate environment.

[0044] Experimental Example 1

[0045] like Figure 1 As shown, the lactate content in NK92MI cells after incubation in Example 1 was detected. It was found that after treatment with the tumor supernatant of diffuse large B-lymphoma, the L-lactate content in NK92MI cells was significantly increased, while the L-lactate content in NK92MI cells was significantly inhibited after the use of MCT inhibitors. Figure 1 A, left), in the supernatant of colorectal cancer ( Figure 1A, middle) and liver cancer cell supernatant ( Figure 1 The right group (A) was also suppressed.

[0046] The L-lactic acid content of NK92MI cells after incubation in Example 2 was detected. Figure 1 B), sodium lactate treatment significantly increased the concentration of L-lactic acid in NK92MI cells; however, with increasing CHC concentration, the accumulation of L-lactic acid in NK92MI cells significantly decreased. Figure 1 B). This indicates that MCT inhibitors can effectively block the transmembrane transport of lactate.

[0047] Experimental Example 2

[0048] The NK92MI cells obtained in Example 1, after incubation, were used to kill Huh-7 cells at an effector-to-target ratio of 5:1. Tumor cell adhesion was monitored in real time using a Brochure-Smart Cell Real-Time Monitor. Figure 2 As shown, the horizontal axis represents time, and the vertical axis represents electrical resistance. Huh-7 cells were added to the wells at a ratio of 10,000 cells / 100 μl / well. After 12 hours of plating, treated NK92MI cells were added to the wells at a ratio of 50,000 cells / 100 μl / well. Compared to Comparative Example 1, the killing effect of NK92MI cells on Huh-7 cells was significantly reduced after treatment with diffuse large B-cell lymphoma tumor supernatant. However, the killing ability of NK92MI cells was significantly restored after the use of an MCT inhibitor. Figure 2 A, left); Compared with Comparative Example 2, the killing effect of NK92MI cells on Huh-7 cells was significantly reduced after treatment with HCT-15 tumor supernatant, while the killing ability of NK92MI cells did not change significantly after treatment with MCT inhibitors ( Figure 2 (A, Middle); Compared with Comparative Example 3, the killing effect of NK92MI cells on Huh-7 cells was significantly reduced after treatment with Huh-7 tumor supernatant, while the killing ability of NK92MI cells was significantly restored after treatment with MCT inhibitors. Figure 2 A, right).

[0049] The NK92MI cells obtained in Example 2 after incubation were collected, and the cell culture supernatant was then centrifuged at 1000 g for 10 min. The precipitate was discarded, and the supernatant was used for ELISA to detect the content of IFN-γ and TNF-α in the cell culture supernatant. After exogenous sodium lactate treatment, the secretion of IFN-γ and TNF-α by NK92MI cells was significantly reduced. This phenomenon was reversed after the use of MCT inhibitors, that is, the content of IFN-γ and TNF-α secreted by NK92MI cells was significantly increased. Figure 2 B). The cytotoxicity assay kit was used to analyze the effect of NK cells on K562 cells after co-incubation with sodium lactate 15 mM and CHC 10 mM for 48 h. Figure 2 C) and Huh-7 Figure 2 Regarding the changes in killing ability (D), when K562 cells were used as target cells, the NK cell killing efficiency in the sodium lactate group was significantly reduced compared to the untreated group at an effector-to-target ratio of 10:1. However, the use of MCT inhibitors could effectively reverse this phenomenon. In addition, when Huh-7 cells were used as target cells, MCT inhibitors could also restore the killing function of NK92MI cells to a certain extent at effector-to-target ratios of 5:1 and 2.5:1.

[0050] Experimental Example 3

[0051] Proteins were extracted from the incubated NK92MI cells obtained in Examples 1 and 2, and then detected by Western blotting using a lactated pan-antibody. Figure 6 As shown, after treatment with tumor supernatants from diffuse large B-cell lymphoma, colorectal cancer, and liver cancer cells, the overall lactation level of NK92MI cells significantly increased. Combined with MCT inhibition, the lactation level induced by the tumor supernatant was reversed. Figure 3 Similarly, after sodium lactate treatment led to an increase in the overall lactation level of NK92MI cells, the combined use of an MCT inhibitor significantly suppressed the overall lactation level of NK92MI cells. Figure 3 B). Combined with the results of experiments 1 and 2, MCT inhibition can effectively block lactate uptake, reduce lactation modification levels, and reverse NK cell functional exhaustion.

[0052] Test Example 4

[0053] The NK92MI cells obtained in Example 3, after incubation, were used to kill Huh-7 cells at an effector-to-target ratio of 5:1. Tumor cell adhesion was monitored in real time using a Brochure-Smart Cell Real-Time Monitor. Figure 4 As shown, the horizontal axis represents time, and the vertical axis represents electrical resistance. Huh-7 cells were added to the wells at a ratio of 10,000 cells / 100 μl / well. After 12 hours of plating, pre-treated NK92MI cells were added to the wells at a ratio of 50,000 cells / 100 μl / well. Figure 4 As shown, sodium lactate treatment significantly reduced the killing efficiency of NK92MI cells; however, further treatment with P300 inhibitors effectively restored the killing ability of NK92MI cells against target cells.

[0054] Experimental Example 5

[0055] Proteins were extracted from the incubated NK92MI cells obtained in Example 3, and immunoblotting analysis was performed using lactated pan-antibody and H3K18 lactated antibody. Figure 5As shown, compared with Comparative Example 4, the overall lactation modification level of NK cells was significantly increased after sodium lactate treatment, while the overall level of NK92MI cells was reduced to some extent after the addition of P300 inhibitor. Combined with the results of Experiment 4, it can be seen that the inhibition of P300 can weaken the lactation modification level and reverse the functional exhaustion of NK cells.

[0056] Experimental Example 6

[0057] The NK92MI cells obtained in Examples 4, 5, and 6 were incubated and then used to kill liver cancer cells for 5 hours at effector-to-target ratios of 10:1, 5:1, and 2.5:1. The killing efficiency was then calculated using a lactate dehydrogenase assay kit. Figure 6 As shown, sodium lactate treatment significantly inhibited the killing efficiency of NK92MI cells, while the three lactate-coenzyme A synthase inhibitors could improve the killing efficiency of NK92MI cells to some extent (6A). Flow cytometry was used to detect the expression of IFN-γ (6B), TNF-α (6C), perforin (6D), and granzyme B (6E) in NK92MI cells after treatment with sodium lactate, GTPCS inhibitor, ASCC2 inhibitor, and ACAT1 / 2 inhibitor for 48 h. The results showed that sodium lactate treatment significantly decreased the levels of the above effector molecules: after GTPCS, ACSS2, and ACAT1 / 2 inhibition, there was no significant difference in the IFN-γ positive rate in NK92MI cells, but the mean fluorescence intensity was significantly increased (6B); at the same time, the TNF-α positive rate and the mean fluorescence intensity were significantly increased (6C); the perforin positive rate was significantly increased, but the mean fluorescence intensity did not change significantly (6B). Figure 6 D); The proportion of granzyme B positive and the average fluorescence intensity were both significantly increased (6E).

[0058] Experimental Example 7

[0059] The NK92MI cells obtained in Examples 4, 5, and 6 after incubation were analyzed using Western blotting to detect the overall level and H3K18 lactation level, as follows: Figure 7 As shown, sodium lactate treatment increased the overall lactation level of NK92MI cells, while treatment with ACSS2 and ACAT1 / 2 inhibitors significantly reduced the overall lactation level (7A). Similarly, sodium lactate treatment increased the H3K18 lactation level in NK92MI cells, and GTPCS, ACSS2, and ACAT1 / 2 inhibitors all reduced histone H3K18 lactation levels (7B). This indicates that the inhibition of these three lactate-coenzyme A synthases can effectively block lactate-coenzyme A synthesis, thereby inhibiting the lactation level of NK cells and reversing NK cell functional exhaustion.

Claims

1. A method of targeted inhibition of lactate oxidation to enhance NK cell function for non-therapeutic purposes, characterized in that, The method is to target inhibit the modified enzyme in the process of lactate; the modified enzyme is lactate coenzyme A synthetase GTPSCS; the specific steps of inhibiting the modified enzyme in the process of lactate are as follows: Dilute NK cells to 500,000 / ml, 15 mM sodium lactate concentration, use formic acid sodium 100 mM inhibitor of GTPSCS.

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