阅读说明：本技术 6-磷酸葡萄糖脱氢酶抑制剂的医药用途 (Medical application of 6-phosphoglucose dehydrogenase inhibitor ) 是由 王逸飞 郭传瑸 王琳 郭玉兴 李睿柳 于 2021-10-15 设计创作，主要内容包括：本发明公开了6-磷酸葡萄糖脱氢酶抑制剂改善患者个体抑制性肿瘤免疫微环境,以及作为PD-L1单抗疗效增强剂的用途。6-磷酸葡萄糖脱氢酶抑制剂通过多个方面改善患者个体抑制性肿瘤免疫微环境,提高个体对免疫治疗剂,特别是PD-L1单抗的响应,从而提高抗肿瘤效果。(The invention discloses a 6-phosphoglucose dehydrogenase inhibitor for improving an individual inhibitory tumor immune microenvironment of a patient and application of the inhibitor as a curative effect enhancer of a PD-L1 monoclonal antibody. The glucose-6-phosphate dehydrogenase inhibitor improves the tumor-inhibiting immune microenvironment of individual patients in multiple aspects, and improves the response of the individual to immunotherapeutic agents, particularly the PD-L1 monoclonal antibody, thereby improving the anti-tumor effect.)

Use of a glucose-6-phosphate dehydrogenase inhibitor for the manufacture of a medicament for improving the suppressive tumor immune microenvironment of a subject.

2. The use of claim 1, wherein said improvement in the inhibitory tumor immune microenvironment of the subject comprises one or more of an increase in the level of CD8+ T cells in the tumor infiltrating tissue, an increase in the proportion of CD8+/CD4+ T cells, an increase in the proportion of IFN- γ + cells in CD8+ T cells in the peripheral blood, a decrease in the proportion of CD25+ FOXP3+ cells in CD4+ T cells, and an inhibition of the expression of B7-H4 in the subject.

3. The use according to claim 1 or 2, wherein the glucose-6-phosphate dehydrogenase inhibitor is selected from one or more of DHEA, 6-AN, RRx-001.

4. Use of a glucose-6-phosphate dehydrogenase inhibitor for the manufacture of a medicament, wherein the medicament is for use in combination with PD-L1 mab in the treatment of a tumor.

5. The use of claim 4, wherein the glucose-6-phosphate dehydrogenase inhibitor is selected from one or more of DHEA, 6-AN, and RRx-001.

6. The use of claim 4, wherein the PD-L1 monoclonal antibody is selected from one or more of durvalumab, atezolizumab, and Avelumab.

7. The use of claim 4, wherein the glucose-6-phosphate dehydrogenase inhibitor is administered prior to or concurrently with the administration of PD-L1 monoclonal antibody.

8. A pharmaceutical combination comprising a therapeutically effective amount of a glucose-6-phosphate dehydrogenase inhibitor and PD-L1 mab, said glucose-6-phosphate dehydrogenase inhibitor and PD-L1 mab being used in combination for the treatment of a tumor.

9. The pharmaceutical combination according to claim 8, wherein the glucose-6-phosphate dehydrogenase inhibitor is selected from one or more of DHEA, 6-AN, RRx-001; DHEA is preferred.

10. The pharmaceutical combination according to claim 8, wherein the PD-L1 monoclonal antibody is selected from one or more of durvalumab, atezolizumab and Avelumab.

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 check point represented by PD-L1 is a hotspot in the research of tumor immunotherapy in recent years, and the monoclonal antibody taking the immune check point as a target achieves ideal effects in various malignant tumors, and particularly can obviously improve the prognosis of patients in the malignant tumors such as non-small cell lung cancer and the like. Nevertheless, in some tumor patients or tumor types, immunotherapy is not effective, mainly because of insufficient patient response to therapy.

Research shows that the tumor immune microenvironment has important influence on the response rate of immunotherapy, and the number and the function of CD8+ T cells in tumor tissues have important influence on the anti-tumor effect of the 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, particularly CD8+ T cells. Even if the effect between PD-L1 and PD-1 is blocked by a monoclonal antibody, the anti-tumor effect is still not exerted due to insufficient numbers or lack of function of effector cells, thereby causing no response of treatment, for tumor tissues with insufficient infiltration of CD8+ T cells or inhibited function. Therefore, the method for improving the tumor immune microenvironment characteristics is researched, and the method has important significance for improving the treatment effect of the PD-L1 monoclonal antibody.

Glucose-6-phosphate dehydrogenase (G6PD), a key rate-limiting enzyme in the pentose phosphate pathway of sugar metabolism, has a major physiological function of regulating the production of ribose-5-phosphate and NADPH, and plays an important role in cell proliferation and in fighting oxidative stress. Inhibition of G6PD increases oxidative stress levels (manifested as elevated ROS levels) in tumor cells, activates apoptotic pathways, inhibits their proliferation, migration, and invasion, while inhibition of G6PD has no clear effect on tumor cell immune escape function. In addition, in immune cells, existing research shows that certain levels of ROS play an important role in T cell activation, but no relevant research is found for exploring the influence of inhibiting the increase of ROS level mediated by G6PD on the function of immune cells.

Disclosure of Invention

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

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

According to some embodiments of the invention, in the above use, the improvement of the suppressive tumor immune microenvironment of the subject includes one or more of increasing CD8+ T cell level, increasing CD8+/CD4+ T cell ratio, increasing IFN- γ + cell ratio in peripheral blood CD8+ T cells, decreasing CD25+ FOXP3+ cell ratio in CD4+ T cells, and suppressing B7-H4 expression.

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

The invention proves that the combination of the glucose-6-phosphate dehydrogenase inhibitor and the anti-tumor immunotherapy agent can improve the effectiveness of immunotherapy. The anti-tumor immunotherapy agent comprises but is not limited to a cellular immunotherapy agent, PD-1 monoclonal antibody, PD-L1 monoclonal antibody and the like.

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

According to experimental research, the 6-phosphoglucose dehydrogenase inhibitor (especially DHEA) and the PD-L1 monoclonal antibody are combined, so that the anti-tumor effect of the inhibitor is better than that of the inhibitor of the 6-phosphoglucose dehydrogenase or the PD-L1 monoclonal antibody, and the inhibitor of the 6-phosphoglucose dehydrogenase plays a synergistic role.

The third aspect of the present invention provides a pharmaceutical composition comprising a glucose-6-phosphate dehydrogenase inhibitor and PD-L1 monoclonal antibody, in combination for use in the treatment of tumors.

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

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

DHEA is dehydroepiandrosterone with CAS number 53-43-0.

Further, in the second to third embodiments described above, the PD-L1 monoclonal antibody is one or more selected from the group consisting 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 PD-L1 monoclonal antibody.

The research of the invention finds that the glucose dehydrogenase-6-phosphate inhibitor, especially DHEA, can improve the proportion of CD8+/CD4+ T cells in a tumor infiltration tissue, improve the proportion of IFN-gamma + cells in CD8+ T cells in peripheral blood, reduce the proportion of CD25+ FOXP3+ cells in CD4+ T cells, and inhibit B7-H4, thereby improving the inhibitory immune microenvironment of the tumor tissue. The 6-phosphoglucose dehydrogenase inhibitor and the PD-L1 monoclonal antibody are combined, the inhibition effect on the tumor growth is obviously stronger than that of the single use of the two, and the synergistic interaction is realized. The method has important significance for patients who have insufficient response and poor curative effect when the PD-L1 monoclonal antibody is clinically used.

Drawings

FIG. 1 Effect of DHEA on tumor growth and tumor-infiltrating lymphocyte typing in a malignant melanoma pan-immune mouse graft tumor model. Wherein (A) DHEA can significantly inhibit tumor growth in a fully-immunized mouse graft tumor model; (B) the proportion of CD4+ cells in tumor infiltrating T cells of mice in the DHEA treatment group is reduced; (C) increased proportion of CD8+ cells; (D) the ratio of CD8+/CD4+ T cells is 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 malignant melanoma whole immune mouse transplant tumor model. The DHEA and the PD-L1 monoclonal antibody can inhibit the tumor growth of a full-immune mouse transplanted tumor model, and the combination of the DHEA and the PD-L1 monoclonal antibody has more obvious inhibition effect; (B) the combination of DHEA and PD-L1 monoclonal antibody can obviously improve the proportion of cells secreting IFN-gamma in mouse peripheral blood CD8+ T cells; (C) both DHEA and PD-L1 monoclonal antibodies can reduce the proportion of Treg cells (CD4+ CD25+ FOXP3+) in peripheral blood CD4+ T cells, and the combined effect of the DHEA and the PD-L1 monoclonal antibodies is more obvious.

FIG. 3 Effect of DHEA treatment in vitro on tumor cell lines expression of immune co-suppressor molecules. Wherein (A-B) DHEA significantly reduces the 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 CAL27 and A375 cell line B7-H4.

FIG. 4 Effect of DHEA treatment in vitro on mouse CD8+ and CD4+ T cells on their immune function. (A) The proportion of cells secreting IFN-gamma and TNF-alpha from DHEA treated CD8+ T cells is increased; (B) the proportion of Treg cells of DHEA-treated CD4+ T cells CD25+ FOXP3+ was reduced.

Term(s) for

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

Unless otherwise specified, "PD-L1 monoclonal antibody" of the present invention refers to monoclonal antibodies targeting human PD-L1 protein, including but not limited to Durvalumab, atezolizumab, Avelumab and the like.

The "tumor" of the present invention, also referred to as cancer or malignant tumor, includes, but is not limited to, renal cell carcinoma, prostate cancer, bladder cancer, adenocarcinoma, fibrosarcoma, chondrosarcoma, osteosarcoma, liposarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, myelocytic leukemia, erythroleukemia, multiple myeloma, gliomas, human tongue squamous cell carcinoma, meningeal sarcoma, phyllocytic sarcoma, nephroblastoma, teratoma, choriocarcinoma, cutaneous T-cell lymphoma (CTCL), cutaneous tumors mainly directed against the skin (e.g., basal cell carcinoma, squamous cell carcinoma, melanoma, and Brower's disease), breast tumors, boji's cancer, and premalignant and malignant diseases of mucosal tissues, including oral, bladder, and rectal diseases, central nervous system tumors (gliomas), meningiomas, and astrocytomas, among others.

Detailed Description

DHEA in the following test examples was purchased from seleck, usa; PD-L1 monoclonal antibody was obtained from the mouse purchased from Bio X cell, USA using PD-L1 monoclonal antibody InVivoMAb anti-mouse PD-L1(B7-H1) (RRID: AB-10949073).

In clinical application, the PD-L1 monoclonal antibody can be corresponding to monoclonal antibodies targeting human PD-L1 protein such as durvalumab, atezolizumab and Avelumab.

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

Test example A test of inhibitory immune microenvironment effect of DHEA on malignant melanoma pan-immunized mice and an anti-tumor effect test of DHEA on PD-L1

1.1

10 female C57BL/6J mice (Beijing Wittingle laboratory animal technology Co., Ltd.) aged 7-8 weeks were selected and randomly divided into DHEA group and control group. B16 cell line (national biomedical experimental cell bank) is injected into the subcutaneous back of the mouse to construct a malignant melanoma full immune mouse transplantation tumor model. When the administration starts on the 8 th day after cell injection, DHEA is dissolved in animal experiment drug carrier (2% DMSO + 30% PEG300+ 5% Tween-80+ distilled water) according to the instruction, the DHEA is injected into the abdominal cavity according to the dose of 25mg/kg body weight, the administration is carried out once every day for a fixed time, the drug carrier is adopted in a control group, and the tumor volume is measured by a vernier caliper (the tumor volume calculation formula is: long diameter multiplied by short diameter multiplied by 2 divided by 2), so that a tumor growth curve is constructed.

On day 12 after cell injection, the mice were sacrificed by decapitation, tumor tissues were excised, single cell suspensions were prepared from tissue single cell preparation consumables (magic Filter magic pestle magic Vajra kit) purchased from Bozhen, Zhejiang Biotech, Inc., and subjected to flow cytometry, PE-CD3, FITC-CD4, and APC-CD8 flow antibodies (1:100 dilution, flow antibodies purchased from Biolegend, USA, same below) were added to the single cell suspensions, incubated at room temperature in the dark for 30min, centrifuged at 300g for 5min, supernatants were removed, resuspended in PBS at 500. mu.l, and subjected to on-machine detection.

The results are shown in FIG. 1: according to the change diagram and the curve of the tumor growth volume, DHEA can obviously inhibit the growth of malignant melanoma; according to the proportion change chart of CD8+ and CD4+ cells in T lymphocytes, the proportion of CD8+ T cells in T lymphocytes infiltrated by the DHEA treatment group (CD3+) is increased, the proportion of CD4+ T cells is reduced, and the proportion of CD8+/CD4+ T cells is obviously increased, which indicates that DHEA can improve the inhibitory immune microenvironment of tumor tissues.

1.2

A malignant melanoma total immune mouse transplanted tumor model is constructed by referring to the method 1.1, 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, the DHEA group is given DHEA (the administration mode and the dosage are equal to 1.1), the PD-L1 monoclonal antibody group is given PD-L1 monoclonal antibody (diluted to 1mg/ml by normal saline, 100 mu L of DHEA is injected into the abdominal cavity of each mouse every two days) for treatment, the DHEA combined PD-L1 monoclonal antibody group is given DHEA and PD-L1 monoclonal antibodies with the same dosage as a single medicine group at the same time, and the blank control group adopts a medicine carrier (the administration mode and the dosage are equal to 1.1). Tumor volume was measured every two days (fixed time) by a vernier caliper and tumor growth curves were plotted.

On the 14 th day after cell injection, mouse transplanted tumor tissues were taken to prepare single cell suspensions for flow cytometry detection (method 1.1). Meanwhile, taking peripheral blood of a Mouse, separating peripheral blood lymphocytes of the Mouse by using a peripheral blood lymphocyte separation kit (Beijing Solebao company), adding FITC-CD4, PerCP-CD8 and APC-CD25 flow antibodies, incubating for 30min at room temperature in a dark place, centrifuging to remove redundant antibodies, fixing and breaking a membrane by using a Mouse Foxp3 Buffer Set of American BD company, adding APC/Cy7-IFN-gama and PE-FOXP3, incubating for 30min at room temperature in a dark place, centrifuging for 5min at 300g, removing supernatant, resuspending 500 mu l of PBS, and detecting by using an on-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, and the combination of the DHEA and the PD-L1 monoclonal antibody plays a role in synergy; 2. after the DHEA is combined with the PD-L1 monoclonal antibody for 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 proportion of IFN-gamma + cells in the peripheral blood CD8+ T cells is increased, and the proportion of CD25+ FOXP3+ cells in the CD4+ T cells is reduced, which indicates that the inhibitory immune microenvironment in the tumor tissues is improved.

Experimental example Effect of DHEA on tumor cell B7-H4 expression

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

Human malignant melanoma cell line A375 (national biomedical laboratory cell bank) and human tongue squamous cell carcinoma cell line CAL27(ATCC) were cultured in vitro. The administration group was divided into an administration group to which 50. mu.M DHEA (solid DHEA in DMSO, 50mM stock solution prepared, diluted with DMEM cell culture medium when used) was administered, and a control group to which the same volume of DMSO was added into DMEM medium, treated in vitro for 24 hours, RNA was extracted by Trizol reagent (Thermo Fisher), cDNA was obtained using a reverse transcription kit from Promega corporation, expression of the relevant immune co-inhibitory molecules was detected by real-time quantitative fluorescent PCR using SYRB green (BD Co.), and the relevant primer sequences were obtained by Primerbank database (https:// pgpa.mgh.harvard.edu/Primerbank /) query and synthesized by Shanghai Bioengineering Co., Ltd.

Total proteins of the two DHEA-treated cells were extracted by RIPA (Beijing Huaxingbobo) and the expression of B7-H4 was examined by Western blot assay (B7-H4 primary antibody was purchased from Mactak, Wuhan Egypti, Inc. at a dilution ratio of 1: 1000; secondary antibody was purchased from CST, USA at a dilution ratio of 1: 10000).

The results are shown in FIG. 3: a, B in the figure shows that there was no significant change in the expression of PD-L1 in the administered and control groups, whereas the expression of B7-H4 was significantly reduced in both cell lines. Panel C further demonstrates that DHEA reduces tumor cell B7-H4 expression. Prior study^[1,2]The experiments show that the B7-H4 inhibits the anti-tumor immune function by promoting the differentiation of Treg cells, and the experiments indicate that the DHEA acts on the tumor cells and possibly improves the tumor immune microenvironment by inhibiting the expression of 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 influence of DHEA on the proportion of IFN-. gamma.and TNF-. alpha.secreting cells in CD8+ T cells

7-8 week old C57BL/6J mice were removed from their necks and spleens were removed from the mice and sacrificed to prepare single cell suspensions by C-tube (Meitian whirlpool, Germany) followed by isolation of CD4+ and CD8+ T cells using CD4+ and CD8+ cell magnetic bead sorting kits (both from Meitian whirlpool, Germany) and in vitro culture in RP1640 MI medium (containing 10% FBS, 1% penicillin-streptomycin, both from Thermo Fisher, USA). The cells were divided into an administration group and a control group, the drug concentration and the treatment method were the same as those in test example two, and the change in the typing of the immune cells was detected by flow cytometry (detailed experimental method was the same as 1.1).

The results are shown in FIG. 4: it was found that the proportion of CD25+ FOXP3+ cells (Treg cells) in CD4+ T cells was decreased, and the proportion of cells (CTL cells) secreting IFN-. gamma.and TNF-. alpha.labeled by PE/Cy7 in CD8+ T cells was increased, which was consistent with the experimental results of mouse transplantable tumors.