CN113769096B

CN113769096B - Medical application of glucose 6-phosphate dehydrogenase inhibitor

Info

Publication number: CN113769096B
Application number: CN202111201846.8A
Authority: CN
Inventors: 王逸飞; 郭传瑸; 王琳; 郭玉兴; 李睿柳
Original assignee: Peking University School of Stomatology
Current assignee: Peking University School of Stomatology
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2023-07-04
Anticipated expiration: 2041-10-15
Also published as: CN113769096A

Abstract

The invention discloses an application of a glucose 6-phosphate dehydrogenase inhibitor in improving the individual inhibitory tumor immune microenvironment of a patient and serving as a PD-L1 monoclonal antibody curative effect enhancer. The glucose 6-phosphate dehydrogenase inhibitor improves the inhibitory tumor immune microenvironment of a patient individual through various aspects, and improves the response of the individual to an immunotherapeutic agent, particularly PD-L1 mab, thereby improving the anti-tumor effect.

Description

Medical application of glucose 6-phosphate dehydrogenase inhibitor

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of a glucose 6-phosphate dehydrogenase inhibitor in enhancing an anti-tumor effect of a PD-L1 monoclonal antibody by improving an inhibitory tumor immune microenvironment.

Background

The immune checkpoints represented by PD-L1 are hot spots in recent tumor immunotherapy research, and monoclonal antibodies targeting the immune checkpoints have ideal effects in various malignant tumors, and particularly in malignant tumors such as non-small cell lung cancer and the like, and can remarkably improve the prognosis of patients. However, in some tumor patients or tumor types, immunotherapy is poorly effective, mainly due to the insufficient response rate of patients to treatment.

Research shows that the tumor immune microenvironment has an important influence on the response rate of immunotherapy, and the number and the function of CD8+ T cells in tumor tissues have an important influence on the anti-tumor effect of PD-L1 monoclonal antibody. Immune checkpoints represented by PD-L1 inhibit immune cell function by binding to the corresponding receptor PD-1 on the surface of T cells, in particular cd8+ T cells. For tumor tissues in which infiltration of cd8+ T cells is insufficient or function is inhibited, even if the action between PD-L1 and PD-1 is blocked by a monoclonal antibody, the anti-tumor action is not exerted due to insufficient number of effector cells or lack of function, thereby causing no response to treatment. Therefore, the method for exploring the characteristics of the tumor immunity microenvironment has important significance for improving the PD-L1 monoclonal antibody treatment effect.

Glucose 6-phosphate dehydrogenase (G6 PD) acts as a key rate-limiting enzyme in the pentose phosphate pathway of sugar metabolism, and its main physiological function is to regulate the production of 5-phosphoribosyl and NADPH, playing an important role in cell proliferation and against oxidative stress. Inhibition of G6PD increases the level of oxidative stress in tumor cells (manifested as elevated ROS levels), activates apoptotic pathways, inhibits their proliferation, migration, invasion, and the effect of inhibition of G6PD on tumor cell immune escape function is not clear. In addition, in immune cells, existing studies indicate that certain levels of ROS play an important role in T cell activation, but no related studies have been seen to explore how inhibition of G6 PD-mediated elevation of ROS levels has an impact on immune cell function.

Disclosure of Invention

Aiming at the problem of insufficient response rate of clinical patients to immunotherapy, the invention provides a novel medical application of the glucose-6-phosphate dehydrogenase inhibitor based on the discovery that the glucose-6-phosphate dehydrogenase inhibitor can improve the immune microenvironment of the inhibitory tumor.

First, a first aspect of the invention provides the use of a glucose 6-phosphate dehydrogenase inhibitor for the preparation of a medicament for improving the immune microenvironment of an inhibitory tumor in an individual of a patient.

According to some embodiments of the invention, the improving the inhibitory tumor immune microenvironment in the subject includes one or more of increasing cd8+ T cell levels, increasing cd8+/cd4+ T cell ratios, increasing IFN- γ+ cell ratios in peripheral blood cd8+ T cells, decreasing cd25+ foxp3+ cell ratios in cd4+ T cells, and inhibiting B7-H4 expression in the subject.

The subject of the invention is a mammal. Preferably, the subject individual is a human patient.

The invention verifies that the combination of the glucose 6-phosphate dehydrogenase inhibitor and the anti-tumor immunotherapeutic agent can improve the effectiveness of immunotherapy. Such anti-tumor immunotherapeutic agents include, but are not limited to, cellular immunotherapeutic agents, PD-1 mAbs, PD-L1 mAbs, and the like.

A second aspect of the invention provides the use of a glucose 6-phosphate dehydrogenase inhibitor for the preparation of a medicament for use in combination with PD-L1 mab in the treatment of a tumor.

According to experimental study, the invention discovers that the combined use of the 6-phosphoglucose dehydrogenase inhibitor (especially DHEA) and the PD-L1 monoclonal antibody has better anti-tumor effect than the single use of the 6-phosphoglucose dehydrogenase inhibitor or the PD-L1 monoclonal antibody, and the 6-phosphoglucose dehydrogenase inhibitor plays a role in synergism.

A third aspect of the invention provides a pharmaceutical combination comprising a glucose-6-phosphate dehydrogenase inhibitor and PD-L1 mab, in combination for use in tumor therapy.

In the first to third embodiments described above, the glucose 6-phosphate dehydrogenase inhibitor is one or more selected from DHEA, 6-AN, RRx-001.

Further, in the first to third embodiments described above, the glucose 6-phosphate dehydrogenase inhibitor is DHEA.

DHEA, dehydroepiandrosterone, CAS number 53-43-0.

Further, in the second to third schemes described above, the PD-L1 mab is selected from one or more of durvalumab, atezolizumab and Avelumab.

Further, in the second to third embodiments described above, the glucose 6-phosphate dehydrogenase inhibitor is used before or simultaneously with the use of the PD-L1 mab.

According to the research of the invention, the glucose 6-phosphate dehydrogenase inhibitor, especially DHEA, can improve the CD8+/CD4+ T cell proportion of tumor infiltration tissues, improve the IFN-gamma+ cell proportion in peripheral blood CD8+ T cells, reduce the CD25+ FOXP3+ cell proportion in CD4+ T cells and inhibit B7-H4, thereby improving the inhibitory immune microenvironment of tumor tissues. The glucose 6-phosphate dehydrogenase inhibitor and the PD-L1 monoclonal antibody are combined, so that the inhibition effect on tumor growth is obviously stronger than that of the two monoclonal antibodies which are singly used, and the synergistic effect is realized. The method has important significance for patients with insufficient response and poor curative effect in clinical use of PD-L1 monoclonal antibody.

Drawings

FIG. 1 effect of DHEA on tumor growth and tumor-infiltrating lymphocyte typing in a model of a fully immunized mouse engrafting tumor for malignant melanoma. Wherein, (A) DHEA can obviously inhibit the tumor growth of a full-immune mouse transplantation tumor model; (B) The proportion of CD4+ cells in tumor-infiltrating T cells of the DHEA-treated mice is reduced; (C) increased CD8+ cell fraction; (D) the CD8+/CD4+ T cell ratio was significantly increased.

FIG. 2 effect of DHEA in combination with PD-L1 monoclonal antibody treatment on tumor growth and tumor-infiltrating lymphocyte typing in a model of a fully immunized mouse engrafted tumor with malignant melanoma. Wherein (A) the DHEA and the PD-L1 monoclonal antibodies can inhibit the tumor growth of a full-immune mouse transplanted tumor model, and the inhibition effect is more obvious when the DHEA and the PD-L1 monoclonal antibodies are combined; (B) The combination of DHEA and PD-L1 monoclonal antibody can significantly improve the proportion of cells secreting IFN-gamma in the peripheral blood CD8+ T cells of the mice; (C) The ratio of Treg cells (CD4+CD25+FOXP3+) in peripheral blood CD4+T cells can be reduced by both DHEA and PD-L1 monoclonal antibodies, and the combined effect of the DHEA and the PD-L1 monoclonal antibodies is more remarkable.

FIG. 3 effect of DHEA in vitro treatment of tumor cell lines on the expression of their immunosuppressive molecules. Wherein, (a-B) DHEA significantly reduces B7-H4 expression of human tongue squamous carcinoma cell line CAL27 (a) and human malignant melanoma cell line a375 (B); (C) Western blot experiments prove that DHEA can inhibit the expression of B7-H4 of CAL27 and A375 cell lines.

FIG. 4 influence of in vitro treatment of mouse CD8+ and CD4+ T cells on their immune function by DHEA. (A) DHEA treated cd8+ T cells secrete increased cell proportions of IFN- γ and TNF- α; (B) DHEA treated cd4+ T cells showed a reduced proportion of cd25+ foxp3+ Treg cells.

Terminology

The "glucose-6-phosphate dehydrogenase inhibitor" of the present invention refers to a substance capable of producing a physiological and biological inhibitory effect on glucose-6-phosphate dehydrogenase, and generally a small molecule substance, including but not limited to DHEA, 6-AN, RRx-001, etc.

The term "PD-L1 mab" as used herein, unless specifically indicated, refers to monoclonal antibodies that target the human PD-L1 protein, including but not limited to durvalumab, atezolizumab and Avelumab, and the like.

The "neoplasm" of the invention is also referred to as cancer or malignancy, including, but not limited to, renal cell carcinoma, prostate cancer, bladder cancer, adenocarcinoma, fibrosarcoma, chondrosarcoma, osteosarcoma, liposarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, myelogenous leukemia, erythroleukemia, multiple myeloma, glioma, human tongue squamous cell carcinoma, meningioma, she Zhuangnang sarcoma, nephroblastoma, teratocarcinoma, choriocarcinoma, cutaneous T-cell lymphoma (CTCL), skin tumors primarily directed against the skin (e.g., basal cell carcinoma, squamous cell carcinoma, melanoma, and brown's disease), mastoma, posi's cancer, and pre-malignant diseases of mucosal tissue, including oral, bladder, and rectal diseases, central nervous system tumors (glioblastoma), meningioma, astrocytoma, and the like.

Detailed Description

DHEA in the following test examples was purchased from Selleck corporation, usa; PD-L1 mab is mouse PD-L1 mab InVivoMAb anti-mouse PD-L1 (B7-H1) (RRID: AB_ 10949073) purchased from Bio X cell company, USA.

In clinical applications, PD-L1 mab may correspond to durvalumab, atezolizumab and Avelumab et al monoclonal antibodies targeting human PD-L1 protein.

Unless otherwise specified, the test conditions, test methods, etc. in the test examples of the present invention are those conventionally used in the art.

Test example one test of the inhibitory immune microenvironment Effect of DHEA on fully immunized mice with malignant melanoma and test of the anti-tumor Effect of DHEA on PD-L1

1.1

10 female C57BL/6J mice (Experimental animal technologies Co., ltd., beijing, violet) of 7-8 weeks old were randomly divided into DHEA group and control group. The B16 cell line (national biomedical experiment cell bank) is injected into the back of the mouse subcutaneously to construct a malignant melanoma total immune mouse transplantation tumor model. After the 8 th day of cell injection, DHEA is dissolved in animal experiment drug carrier (2% DMSO+30% PEG300+5% Tween-80+distilled water) according to the instruction, and the drug is administered once per day at fixed time by intraperitoneal injection according to the dosage of 25mg/kg body weight, and the control group adopts the drug carrier and simultaneously measures the tumor volume by vernier caliper (the calculation formula of tumor volume is: long diameter x short diameter 2/2), so as to construct a tumor growth curve.

On day 12 after cell injection, mice were sacrificed by cervical removal, tumor tissues were excised, single cell suspensions were prepared by tissue single cell preparation consumables (magic filter pestle MagicVajra kit) purchased from zhejiang bozhen biotechnology limited, flow cytometry was performed, single cell suspensions were added to PE-CD3, FITC-CD4, APC-CD8 flow antibodies (1:100 dilution, flow antibodies were all purchased from Biolegend, usa, the same applies below), incubation for 30min at room temperature in the absence of light, centrifugation for 5min, supernatant removal, PBS resuspension of 500 μl, and on-machine detection.

The results are shown in FIG. 1: according to the tumor growth volume change graph and the curve thereof, the DHEA can obviously inhibit the growth of malignant melanoma; from the plot of the change in the ratio of CD8+ to CD4+ cells in T lymphocytes, the increase in the proportion of CD8+ T cells in tumor-infiltrated T lymphocytes (CD3+) in the DHEA-treated group, the decrease in the proportion of CD4+ T cells, and the significant increase in the CD8+/CD4+ T cell ratio, indicate that DHEA can improve the inhibitory immune microenvironment of the tumor tissue.

1.2

The method 1.1 is referred to for constructing a model of the malignant melanoma total immune mice transplantation tumor, the model is divided into a DHEA group, a PD-L1 monoclonal antibody group, a DHEA combined PD-L1 monoclonal antibody group and a blank control group, wherein the DHEA group is given with DHEA (administration mode, dose equivalent to 1.1), the PD-L1 monoclonal antibody group is given with PD-L1 monoclonal antibody (diluted to 1mg/ml by physiological saline, 100 μl is intraperitoneally injected every two days of each mouse), the DHEA combined PD-L1 monoclonal antibody group is given with the DHEA and the PD-L1 monoclonal antibody at the same dose as the single drug group, and the blank control group adopts a drug carrier (administration mode, dose equivalent to 1.1). Tumor volume was measured every two days (fixed time) by vernier calipers and tumor growth curves were drawn.

The single cell suspension prepared from the transplanted tumor tissue of the mice is taken at the 14 th day after the cell injection for flow cytometry detection (the method is the same as 1.1). Meanwhile, taking peripheral blood of a Mouse, separating peripheral blood lymphocytes of the Mouse through a peripheral blood lymphocyte separation kit (Beijing Soy Bao Co.), adding FITC-CD4, perCP-CD8 and APC-CD25 streaming antibodies, incubating for 30min at room temperature in a dark place, centrifuging to remove redundant antibodies, fixing and rupture membranes by adopting a Mouse Foxp3 Buffer Set of BD company in the United states, adding APC/Cy7-IFN-gama and PE-FOXP3, incubating for 30min at room temperature in a dark place, centrifuging for 5min, removing supernatant, re-suspending with 500 mu l PBS, and detecting on a machine.

The results are shown in FIG. 2: 1. the inhibition effect of DHEA combined with PD-L1 monoclonal antibody on tumor growth is obviously stronger than that of the two monoclonal antibodies which are singly used, and the combination of the invention has the synergistic effect; 2. after DHEA combined with PD-L1 monoclonal antibody treatment, the proportion of CD8+ T cells in tumor-infiltrated T lymphocytes (CD3+) is increased, and the proportion of CD4+ T cells is reduced; the increase in the proportion of IFN-gamma+ cells in peripheral blood CD8+ T cells and the decrease in the proportion of CD25+ FOXP3+ cells in CD4+ T cells indicate that the inhibitory immune microenvironment in tumor tissue is improved.

Test example two influence of DHEA on the expression of B7-H4 in tumor cells

The effect of DHEA on tumor cell immune escape capacity was examined by in vitro cell culture.

Human malignant melanoma cell line A375 (national biomedical laboratory cell bank), human tongue squamous cell carcinoma cell line CAL27 (ATCC) was cultured in vitro. The method comprises the steps of dividing the administration group into a control group, wherein 50 mu M DHEA (solid DHEA is dissolved in DMSO, 50mM storage solution is prepared, and the culture solution is diluted by DMEM cell culture medium when in use), adding the same volume of DMSO into the DMEM culture medium in the control group, performing in vitro treatment for 24 hours, extracting RNA by using a Trizol reagent (Thermo Fisher), obtaining cDNA by using a reverse transcription kit of Promega company, performing real-time quantitative fluorescence PCR (SYRB green (BD company)) to detect the expression of related immune co-suppression molecules, and obtaining related primer sequences by inquiring a Primerbank database (https:// pga.mgh. Harvard. Edu/Primerbank /), and synthesizing the related primer sequences by Shanghai biological engineering Co.

The total protein of both DHEA-treated cells was extracted by RIPA (Beijing Xingzhu) and the expression of B7-H4 was detected by Western blot experiments (B7-H4 primary antibody was purchased from Eboltag, inc. of Wuhan, dilution ratio 1:1000; secondary antibody was purchased from CST, inc. of America, dilution ratio 1:10000).

The results are shown in FIG. 3: a, B shows that there was no significant change in PD-L1 expression in both the dosing and control groups, whereas B7-H4 expression was evident in both cell linesAnd significantly reduced. Panel C further demonstrates that DHEA can reduce tumor cell B7-H4 expression. Current research ^[1,2] The experiment shows that the B7-H4 can inhibit the anti-tumor immunity function by promoting the differentiation of Treg cells, and the experiment shows that the DHEA acts on tumor cells to possibly improve the tumor immunity microenvironment by inhibiting the expression of the B7-H4.

[1]Kryczek I,Wei S,Zhu G,et al.Relationship between B7-H4,regulatory T cells,and patient outcome in human ovarian carcinoma.Cancer Res.2007；67(18):8900-8905.doi:10.1158/0008-5472.CAN-07-1866.

[2]Kryczek I,Wei S,Zhu G,et al.Relationship between B7-H4,regulatory T cells,and patient outcome in human ovarian carcinoma.Cancer Res.2007；67(18):8900-8905.doi:10.1158/0008-5472.CAN-07-1866.

Test example three effects of DHEA on IFN-gamma and TNF-alpha secreting cell ratios in CD8+ T cells

The 7-8 week old C57BL/6J mice were removed from the neck, the spleens were removed after neck removal, single cell suspensions were prepared by C-tube (Methaolium, germany) followed by isolation of CD4+ and CD8+ T cells using CD4+ and CD8+ magnetic bead sorting kit (both from Methaolium, germany) and in vitro culture in RPMI1640 medium (containing 10% FBS,1% penicillin-streptomycin, both from ThermoFisher, USA). Cells were divided into dosing and control groups, drug concentration and treatment methods were the same as in test example two, and flow cytometry (detailed experimental methods were 1.1) was used to detect changes in immune cell type.

The results are shown in FIG. 4: it was found that CD25+ FOXP3+ cells (Treg cells) were decreased in CD4+ T cells and IFN-. Gamma., TNF-. Alpha. (PE/Cy 7-labeled) secreting cells (CTL cells) were increased in CD8+ T cells, consistent with the experimental results of mouse transplantation tumor.

Claims

1. Use of a glucose 6-phosphate dehydrogenase inhibitor in the preparation of a medicament, wherein the medicament is used in combination with PD-L1 mab for the treatment of a tumor, the glucose 6-phosphate dehydrogenase inhibitor is selected from DHEA, the PD-L1 mab is selected from one or more of durvalumab, atezolizumab and Avelumab, and the tumor is melanoma.

2. The use according to claim 1, wherein the glucose 6-phosphate dehydrogenase inhibitor is used simultaneously with PD-L1 mab.