Use of glutamine synthetase inhibitors for the preparation of antitumor medicaments

Abstract

The application discloses application of glutamine synthetase inhibitors in preparation of antitumor drugs and belongs to the field of biological medicines. The CRISPR-Cas9 technology is used to knock out glutamine synthetase (sgRNA), compared with shRNA, permanent gene knockout can be introduced, and the application has the potential as a drug for treating colorectal cancer. The application targets glutamine synthetase for treatment on the basis of cancer chemotherapy drugs, significantly improves the sensitivity of tumor cells to chemotherapy drugs, and reduces the cell survival rate. The application of the targeted glutamine synthetase and the neutralizing antibody of alphaPD-L1 combined treatment can significantly enhance the antitumor immune response and improve the treatment efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of glutamine synthetase inhibitors in preparation of anti-tumor drugs. BACKGROUND

[0002] As a key metabolic substrate, glutamine is massively taken up by tumor cells to provide carbon and nitrogen sources for the biosynthesis of nucleotides, fatty acids, and to maintain redox balance, supporting tumor growth. Meanwhile, glutamine metabolites (such as ammonia, glutamate) can acidify the microenvironment, promote immunosuppression (such as inhibiting T cell function, enhancing MDSC activity), and stimulate fibroblast activation, remodeling the extracellular matrix. In addition, competitive consumption of glutamine can lead to metabolic restriction of immune cells (such as T cells, macrophages), further helping tumor immune escape. These effects make it one of the core molecules of metabolic reprogramming of tumor microenvironment.

[0003] In tumor cells, the glutamine synthetic metabolic pathway mainly catalyzes glutamate and ammonia to generate glutamine through glutamine synthetase (GS), especially in a low glutamine environment to maintain intracellular glutamine pool. This pathway not only provides nitrogen and carbon skeleton required for tumor proliferation, but also supports bioenergy demand by regulating alpha-ketoglutarate to replenish the tricarboxylic acid cycle. In addition, overexpression of glutamine synthetase can help tumor cells adapt to oxidative stress and chemotherapy resistance, especially in GS high-activity tumors such as liver cancer and breast cancer, becoming a potential therapeutic target.

[0004] The invention patent with publication number CN115386579A shows that when glutamine is sufficient, glutamine synthetase regulates mitotic progression through non-metabolic function (binding with nuclear pore protein NUP88) to promote cancer cell proliferation. Knocking out GS can cause mitotic arrest, but inhibiting its enzyme activity has no such effect. However, the existing glutamine synthetase inhibitors (such as methionine sulfoximine MSO) in clinical practice only target metabolic function, and have limited effect on treating tumors in the case of sufficient glutamine. In addition, only reprogramming of glutamine metabolism in the tumor microenvironment may exacerbate immune escape.

[0005] Therefore, if through in-depth analysis of the tumor microenvironment, the key metabolic target of glutamine synthetase is found, and a new treatment strategy of combined immunotherapy is adopted for this target, not only can it open up new research ideas for cancer metabolic intervention, but also can significantly enhance anti-tumor immune response, improve treatment efficiency, reduce the toxic side effects of traditional therapy, and promote the wide application of personalized precision therapy in clinical oncology. SUMMARY

[0006] The application is directed to the core problem of tumor metabolism-immune escape and low response rate of immunotherapy in existing cancer treatment, and proposes an application of glutamine synthetase inhibitor in preparation of an anti-tumor drug, and an innovative therapy of targeting glutamine synthetase combined with immune checkpoint blockade. The technical scheme can inhibit the tumor metabolism pathway and activate the immune response synchronously, can destroy the energy supply of tumor cells, and can enhance the anti-tumor activity of T cells and macrophages, and realizes synergistic effect. The application solves the clinical problem that metabolic intervention and immunotherapy are difficult to cooperate, and provides a breakthrough solution for precise tumor treatment.

[0007] In order to achieve the above object, the application adopts the following technical scheme:

[0008] In the first aspect, the application provides an application of glutamine synthetase inhibitor in preparation of an anti-tumor drug.

[0009] Specifically, the glutamine synthetase inhibitor targets to knock down or knock out the expression of glutamine synthetase of tumor cells.

[0010] Further, the glutamine synthetase inhibitor is a CRISPR / Cas9 preparation for inhibiting the expression of glutamine synthetase.

[0011] Preferably, the CRISPR / Cas9 preparation comprises sgRNA; the sgRNA sequence is shown in SEQ ID NO. 1-3.

[0012] More preferably, the sgRNA sequence is shown in SEQ ID NO. 1, wherein the sgRNA knock-out efficiency of the sequence of SEQ ID NO. 1 is the best.

[0013] Preferably, the tumor is colorectal cancer.

[0014] Further, the anti-tumor drug further comprises an anti-PD-1 / anti-PD-L1 antibody component.

[0015] Further, the anti-tumor drug further comprises a chemotherapeutic drug component; the chemotherapeutic drug is 5-fluorouracil or oxaliplatin.

[0016] In the second aspect, the application further provides an anti-tumor drug comprising a glutamine synthetase inhibitor targeting glutamine synthetase of tumor cells.

[0017] The glutamine synthetase inhibitor is a CRISPR / Cas9 preparation for inhibiting the expression of glutamine synthetase, and the CRISPR / Cas9 preparation comprises sgRNA; the sgRNA sequence is shown in SEQ ID NO. 1-3.

[0018] Further, the anti-tumor drug of the present application further comprises an anti-PD-1 / anti-PD-L1 antibody.

[0019] Further, the anti-tumor drug of the present application further comprises a chemotherapeutic drug component; the chemotherapeutic drug is 5-fluorouracil or oxaliplatin.

[0020] In a third aspect, the present application further provides a use of a glutamine synthetase inhibitor targeting glutamine synthetase in tumor cells in combination with a chemotherapeutic drug or an anti-PD-1 / anti-PD-L1 antibody in the preparation of a drug for resisting colorectal cancer.

[0021] In a fourth aspect, the present application further provides a use of a glutamine synthetase inhibitor targeting glutamine synthetase in tumor cells in the preparation of a drug for enhancing the immune response of an anti-PD-1 / anti-PD-L1 antibody against colorectal cancer.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] 1. The present application uses CRISPR-Cas9 technology to knock out glutamine synthetase (sgRNA), which can introduce permanent gene knockout compared with shRNA, and has the potential as a drug for resisting colorectal cancer.

[0024] 2. The present application targets glutamine synthetase for treatment on the basis of cancer chemotherapeutic drugs, significantly improves the sensitivity of tumor cells to chemotherapeutic drugs, and reduces cell survival rate.

[0025] 3. The present application can significantly enhance the anti-tumor immune response and improve the treatment efficiency by combining the treatment of targeting glutamine synthetase with the neutralization of αPD-L1 antibody. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Expression characteristics of glutamine synthetase in colorectal cancer progression; Figure 1 A: Signal intensity and percentage of glutamine synthetase in paracancerous tissue and tumor tissue, data analysis according to mean±SD, N≥3, P=0.0034; Figure 1 B: Survival curve of patients in high and low glutamine synthetase expression groups in tumor tissue, data analysis according to mean±SD, N≥3, P=0.0440.

[0027] Figure 2 Glutamine synthetase knockout efficiency detection in CT26 cells and MC38 cells.

[0028] Figure 3 Glutamine synthetase knockout enhances the sensitivity of colorectal cancer chemotherapeutic drugs; Figure 3 A and Figure 3B is oxaliplatin treatment of CT26 and MC38 cell lines, respectively, with oxaliplatin concentration of 0.5 μM, data analysis according to mean ± SD, N≥3, P values are <0.0001 and 0.0434, respectively; Figure 3 C and Figure 3 D is 5-fluorouracil treatment of CT26 and MC38 cell lines, respectively, with 5-fluorouracil concentration of 0.5 μM, data analysis according to mean ± SD, N≥3, P values are 0.1120 and 0.2894, respectively.

[0029] Figure 4 Effect of glutamine synthetase knockout on tumor progression of colorectal cancer; Figure 4 A to Figure 4 C is the in vivo tumorigenesis of control and glutamine synthetase knockout groups: wherein, Figure 4 A is the gross specimen observation of subcutaneous tumor; Figure 4 B is the tumor volume measurement statistics, P = 0.2221; Figure 4 C is the tumor weight statistics on day 28, P = 0.3588.

[0030] Figure 5 Effect of glutamine synthetase knockout on enhancing the sensitivity of colorectal cancer to immunotherapy drugs; Figure 5 A to Figure 5 C is the in vivo tumorigenesis of control and glutamine synthetase knockout groups under immunotherapy: wherein, Figure 5 A is the gross specimen observation of subcutaneous tumor; Figure 5 B is the tumor volume measurement statistics, P = 0.1450; Figure 5 C is the tumor weight statistics on day 28, P = 0.3056; data analysis according to mean ± SD, N≥3.

[0031] Figure 6 Effect of glutamine synthetase knockout on changing the immune microenvironment; Figure 6 A to Figure 6 C is the immunohistochemical results of tumor tissue after glutamine synthetase knockout combined with αPD-L1 immunotherapy: wherein, Figure 6 A is T cell marker (CD8); Figure 6 B is M1 cell marker (CD86); Figure 6 C is M2 cell marker (CD206); scale bar is 50 μm. DETAILED DESCRIPTION

[0032] The present application is further described in the following examples. These examples are intended to be illustrative only and are not intended to limit the scope of the present application. The present application is also not limited to the specific embodiments described in these examples.

[0033] Cancer tissue chips were provided by the First People's Hospital of Hangzhou. All patients provided written informed consent for sample collection and analysis. The experimental animals used in the following example and results section were cared for in accordance with the guidelines approved by Zhejiang University and met the requirements of the animal ethics code.

[0034] The cell lines used in the following example and results section were obtained from ATCC. Unless otherwise specified, all cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin and 100 U / ml streptomycin at 37°C in a 5% CO2 incubator.

[0035] Example 1: Expression level of glutamine synthetase in tumor tissue affects postoperative survival rate of patients

[0036] With the approval of the Ethics Committee of the First People's Hospital of Hangzhou, tumor tissues and para-cancer tissues from 79 colorectal cancer patients after surgery were collected to make chips, and their clinical stage information (postoperative survival, etc.) was collected. After the chip samples were deparaffinized and immersed in water, they were placed in sodium citrate antigen repair solution for antigen repair, followed by incubation with 3% hydrogen peroxide-PBS at room temperature in the dark to remove endogenous peroxidase. After washing with PBS twice, blocking solution was added and incubated at room temperature in the dark for 1 h. The blocked sections were incubated with glutamine synthetase antibody and incubated overnight in a dark humidified box at 4°C. After washing with PBST (0.05% Tween 20) at room temperature for 4 times, SABC incubation and DAB color development were performed. The sections were scanned and photographed, and the staining results were interpreted. As shown in Figure 1 , compared with para-cancer tissues, the expression of glutamine synthetase in tumor cells was significantly increased (Fig. A). In addition, the survival information obtained by following up the patients and the immunohistochemical results were jointly analyzed, and it was found that patients with high expression of glutamine synthetase in tumor tissues had a higher survival rate than the low expression group (Fig. B). Figure 1 Figure 1

[0037] Example 2: Construction of glutamine synthetase knockout colorectal cancer cell line

[0038] According to the mouse glutamine synthetase gene (Gene ID: 14645), the sgRNA sequence of the glutamine synthetase gene was synthesized, and the coding sequence was as follows:

[0039] sgGlul-1: 5'-GTATGCTGGAGTCAAGATTACGG-3';

[0040] sgGlul-2: 5'-TCGCGCCTACGATCCCAAGGGGG-3';

[0041] ​​sgGlul-3: 5'-TTCGCGCCTACGATCCCAAGGGG-3';

[0042] Control sgScr: 5'-GCACTACCAGAGCTAACTCA-3'.

[0043] The sgRNA sequence of glutamine synthetase was inserted into the CRISPR / CAS9 vector, and the vector and the packaging plasmid (pSPAX2, Addgene 12260) and helper plasmid (pMD2.G, Addgene 12259) of lentivirus were co-transfected into 293T cells to package lentivirus. With the help of 10 μg / ml polybrene, the virus was added to the CT26 and MC38 cell lines at a multiplicity of infection of 1. After 12 h of cell infection, the medium was replaced with fresh medium, and 48 h later, the medium containing 4 μg / ml puromycin was replaced for screening. The cells with puromycin resistance were successfully constructed cell lines. In order to verify the knockout efficiency of glutamine synthetase, 1 x 10 5 cells were collected from each group, and the protein was extracted with RIPA lysis buffer. After separation of the protein by 10% SDS-PAGE gel, the protein was transferred to a PVDF membrane, and immunoblotting was performed with the specified antibodies to visualize the amount of glutamine synthetase protein. As shown in FIG. 2A, the glutamine synthetase knockout CT26 and MC38 cell lines have been successfully constructed. Figure 2

[0044] Example 3: Knockout of glutamine synthetase can improve the sensitivity of tumor cells to chemotherapeutic drugs

[0045] Chemotherapeutic drugs (0.5 μM oxaliplatin or 5-fluorouracil) were added to tumor cells with knockout of glutamine synthetase and its control for in vitro culture, and the solvent group with the addition of DMSO was used as a control. On the 3rd day, 10 μL of CCK-8 solution was added, and incubated in the incubator for 1 h, followed by detection of the absorbance at 450 nm by an enzyme marker, and the results were processed and analyzed by Graphpad Prism. As shown in FIG. 3A and FIG. 3B, the knockout of glutamine synthetase in CT26 and MC38 cell lines improved the sensitivity of cells to two chemotherapeutic drugs, resulting in a decrease in cell survival rate. Figure 3 For oxaliplatin treatment, targeting glutamine synthetase knockout significantly reduced the survival rate of tumor cells (A in FIG. 3A and B in FIG. 3B); and for 5-fluorouracil treatment, targeting glutamine synthetase knockout also reduced the survival rate of tumor cells to some extent (C in FIG. 3A and D in FIG. 3B). Figure 3 Figure 3 Figure 3 Figure 3

[0046] ​​​​​Example 4: Targeting glutamine synthetase in combination with aPD-L1 neutralizing antibody treatment can reduce tumor volume

[0047] By screening, a cell line stably knocking out glutamine synthetase was obtained, and 5x10 5 MC38 cells were injected subcutaneously into the back of C57BL / 6 mice on both sides, and unknocked cells were used as controls, with 12 mice in each group. When the tumor volume of the mice was 100-200 mm 3 The first injection of aPD-L1 neutralizing antibody was 200 μg per mouse, and the injection was performed every 3 days for a total of 3 injections. The solvent PBS was used as a control group, with 6-7 mice in each group. On days 7, 10, 13, 16, 19, 22, 25, and 28 after injection, the subcutaneous tumor volume of the mice was measured, and the mice were photographed and weighed on day 28. The tumor tissue was stained for T cell and macrophage markers to evaluate the changes in immune cells in the tumor microenvironment.

[0048] As can be seen from Figure 4 and Figure 5 , the injection of aPD-L1 neutralizing antibody reduced the tumor volume of MC38, and the knockout of glutamine synthetase further reduced the volume. As shown in Figure 6 the results of immunohistochemical staining of tumor tissue sections, after the knockout of glutamine synthetase and the combination of aPD-L1 neutralizing antibody treatment, the T cells and M1 macrophages in the tumor microenvironment increased, while the M2 macrophages decreased, significantly enhancing the anti-tumor immune response.

[0049] In summary, high expression of glutamine synthetase is positively correlated with the survival rate of patients with colorectal cancer after surgery, and its high expression may indicate a better survival rate, which can be used as a potential biomarker for colorectal cancer.

[0050] CRISPR / Cas9 technology successfully constructed a glutamine synthetase knockout cell line, which provides an important tool for subsequent research on the role of glutamine synthetase in tumor metabolism, drug sensitivity, and immune regulation.

[0051] In GS-knocked CT26 and MC38 cells, the killing effect of oxaliplatin and 5-fluorouracil was significantly enhanced, and the cell survival rate was reduced. Therefore, inhibition of glutamine synthetase may improve the efficacy of existing chemotherapy drugs and provide a new strategy to overcome drug resistance.

[0052] Glutamine synthetase knockout combined with aPD-L1 antibody treatment significantly inhibited tumor growth and remodeled the immune microenvironment. This suggests that glutamine synthetase may affect immunotherapy response by regulating the tumor microenvironment, providing a new idea for combination therapy.

Claims

1. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in combination with a chemotherapeutic drug or a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in combination with an anti-PD-Ll antibody in the preparation of an anti-colorectal cancer drug, wherein the glutamine synthetase inhibitor is a CRISPR / Cas9 preparation comprising an sgRNA sequence as shown in SEQ ID NO. 1; and the chemotherapeutic drug is 5-fluorouracil or oxaliplatin.

2. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in combination with a chemotherapeutic drug or a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in combination with an anti-PD-Ll antibody in the preparation of an anti-colorectal cancer drug, wherein the glutamine synthetase inhibitor comprises a CRISPR / Cas9 preparation; and the chemotherapeutic drug is 5-fluorouracil or oxaliplatin.

2. An anti-colorectal cancer medicament, characterized by comprising the compound of claim 1.

3. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in the preparation of a drug for enhancing the immune response of an anti-PD-Ll antibody against colorectal cancer, wherein the glutamine synthetase inhibitor is a CRISPR / Cas9 preparation comprising an sgRNA sequence as shown in SEQ ID NO.

1.

4. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in the preparation of a drug for enhancing the immune response of an anti-PD-Ll antibody against colorectal cancer, wherein the glutamine synthetase inhibitor comprises a CRISPR / Cas9 preparation.

5. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in the preparation of a drug for enhancing the immune response of an anti-PD-Ll antibody against colorectal cancer, wherein the glutamine synthetase inhibitor comprises a CRISPR / Cas9 preparation; and the anti-PD-Ll antibody is an anti-PD-Ll antibody.

6. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in the preparation of a drug for enhancing the immune response of an anti-PD-Ll antibody against colorectal cancer, wherein the glutamine synthetase inhibitor comprises a CRISPR / Cas9 preparation; and the anti-PD-Ll antibody is an anti-PD-Ll antibody.

7. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in the preparation of a drug for enhancing the immune response of an anti-PD-Ll antibody against colorectal cancer, wherein the glutamine synthetase inhibitor comprises a CRISPR / Cas9 preparation; and the anti-PD-Ll antibody is an anti-PD-Ll antibody.

8. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in the preparation of a drug for enhancing the immune response of an anti-PD-Ll antibody against colorectal cancer, wherein the glutamine synthetase inhibitor comprises a CRISPR / Cas9 preparation; and the anti-PD-Ll antibody is an anti-PD-Ll antibody.

9. The use of a glutamine synthetase inhibitor targeting tumor cell glutamine synthetase in the preparation of a drug for enhancing the immune response of an anti-PD-Ll antibody against colorectal cancer, wherein the glutamine synthetase inhibitor comprises a CRISPR / Cas9 preparation; and the anti-PD-Ll antibody is an anti-PD-Ll antibody.

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