CN113069529A - Use of GSH in combination with PD-1/PD-L1 blockers for promoting CD8+Use of T cell function - Google Patents

Abstract

The invention belongs to the field of immunotherapy combined treatment research, and particularly relates to application of GSH combined with a PD-1/PD-L1 blocking agent in promotion of CD8⁺Use of T cell function. Specifically, the method is any one of the following four methods: 1) in preparation of medicine for promoting CD8⁺Use in a product for T cell proliferation; 2) in the preparation of medicine for inhibiting CD8⁺Use in a product of T cell apoptosis; 3) in preparation of medicine for promoting CD8⁺Use in a product of T cell competence, in particular the ability of a cell to secrete IFN- γ; 4) in the preparation of medicaments for treating CD8⁺Use in diseases associated with abnormal T cell development. The invention provides a new idea for improving the anti-tumor function of the antigen specific T cells in and out of a tumor patient.

Description

Use of GSH in combination with PD-1/PD-L1 blockers for promoting CD8+Use of T cell function

Technical Field

The invention belongs to the field of immunotherapy combined treatment research, and particularly relates to application of GSH combined with a PD-1/PD-L1 blocking agent in promotion of CD8⁺Use of T cell function.

Background

In the field of immunotherapy of tumors, adoptive cell therapy and immune checkpoint inhibitors have taken the stage of tumor therapy. Among them, chimeric antigen receptor T cells (CAR-T), as an emerging adoptive immunotherapy, can significantly improve the prognosis of B cell malignant tumor patients, but the therapeutic effect in solid tumors is poor, and immune checkpoint inhibitors represented by PD-1 monoclonal antibodies are approved for application to various malignant tumors, but some patients are unresponsive to therapy. On the one hand, the reason is that factors influencing the activation and the function of T cells exist in the tumor microenvironment; on the other hand, the proportion of reactive antigen-specific T cells induced by tumor antigens is low at the tumor site, and the reactive antigen-specific T cells are positively correlated with the survival time of tumor patients. Furthermore, tumor-infiltrating T cells tend to be hyporeactive, and how to increase the proportion and function of reactive antigen-specific T cells in a patient is one of the important directions in the field of immunotherapy research.

Antigen-induced enrichment of MAGE-A3-specific CD8⁺PD-1 is highly expressed on the surface of T cells, and specific CD8 can be increased to a certain extent after a PD-1/PD-L1 channel is blocked by using a small molecule peptide antagonist⁺The proliferative capacity and function of T cells, but to a limited extent. The search for new combination strategies to improve the response to the PD-1/PD-L1 blocker is a hotspot in the field of immunotherapy. PD-1 influences the metabolic reprogramming of T cells, namely inhibits glycolysis and amino acid metabolism, promotes fatty acid oxidation of the T cells, the fatty acid oxidation generates a large amount of Reactive Oxygen Species (ROS), and the PD-1 regulates the life span of the T cells through oxidative stress. Glutathione (GSH) is used as a scavenger of ROS, is a liver protection drug, is widely applied clinically at present, and has no obvious toxic or side effect. PD-1/PD-L1 blockers in combination with glutathione increase specificity CD8⁺The proliferation capacity and function of T cells have not been reported.

Disclosure of Invention

The invention aims to provide GSH combined with a PD-1/PD-L1 blocking agent for promoting CD8⁺Use of T cell function. The application is as follows:

GSH combined with PD-1/PD-L1 blocking agent for preparing medicine for promoting CD8⁺The application of the product for T cell proliferation, wherein the PD-1/PD-L1 blocking agent is^DppA-1, said promotion comprising in vivo promotion and in vitro promotion.

Further, the invention also provides a method for combining the GSH with a PD-1/PD-L1 blocking agentIn vitro promotion of CD8⁺Method for T cell proliferation by administering CD8 cultured in vitro⁺T cell GSH and^DppA-1, wherein the concentration of GSH is 40-45 μ M,^Dthe concentration of PPA-1 was 18-20. mu.g/mL.

GSH combined with PD-1/PD-L1 blocking agent for preparing medicine for inhibiting CD8⁺The application of the product of T cell apoptosis, wherein the PD-1/PD-L1 blocker is^DppA-1, said inhibition comprising in vivo inhibition and in vitro inhibition.

Further, the invention also provides the in vitro inhibition of CD8 by using the GSH in combination with a PD-1/PD-L1 blocking agent⁺Method of T cell apoptosis by administration of CD8 cultured in vitro⁺T cell GSH and^DppA-1, wherein the concentration of GSH is 40-45 μ M,^Dthe concentration of PPA-1 was 18-20. mu.g/mL.

GSH combined with PD-1/PD-L1 blocking agent for preparing medicine for promoting CD8⁺The application of the product with T cell capacity, wherein the PD-1/PD-L1 blocker is^DppA-1, said function referring to the ability to secrete IFN- γ, said promotion including in vivo promotion and in vitro promotion.

Further, the invention also provides the application of the GSH combined with a PD-1/PD-L1 blocking agent in promoting CD8 in vitro⁺Method for the secretion of IFN-gamma by T cells, administration of CD8 cultured in vitro⁺T cell GSH and^DppA-1, wherein the concentration of GSH is 40-45 μ M,^Dthe concentration of PPA-1 was 18-20. mu.g/mL.

GSH combined with PD-1/PD-L1 blocker for preparing medicine for treating and preventing CD8⁺Use of a product for the treatment of a disease associated with T cell dysfunction, said dysfunction being CD8⁺T cells have reduced ability to proliferate and secrete IFN-gamma and increased apoptosis.

Further, the disease includes the disease including but not limited to esophageal cancer.

Compared with the prior art, the invention has the following beneficial effects:

experiments prove that GSH combined with PD-1/PD-L1 blocking agent (specifically shown as^DPPPA-1) is capable of promoting CD8⁺T cells' ability to proliferate and secrete IFN-gamma, and can reduce CD8⁺Of T cellsApoptosis and animal experiments prove that the GSH combined with the PD-1/PD-L1 blocking agent can obviously slow down the growth of tumors, and the invention provides a new idea for improving the anti-tumor function of in-vivo and in-vitro antigen specific T cells of tumor patients.

Drawings

FIG. 1 shows the peripheral blood MAGE-A3-specific CD8 of patients with esophageal cancer⁺The proportion of T cells in different culture systems, panel A is the detection of MAGE-A3-specific CD8 by flow cytometry⁺The proportion of T cells in different culture systems; panel B shows peripheral blood MAGE-A3 specific CD8 of patients with esophageal cancer⁺T cell ratio expansion ability in different culture systems (P)<0.05). t test, P < 0.05, P < 0.01, P < 0.001.

FIG. 2 shows the detection of CD8 by flow cytometry under different treatment groups⁺Apoptosis ratio of T cells, in the figure, a: control, b:^Dgroup ppA-1, c: GSH group, d: GGSH +^DGroup PPA-1.

FIG. 3 shows the detection of CD8 by flow cytometry under different treatment groups⁺The ability of T cells to secrete IFN- γ, in the figure, a: control, b:^Dgroup ppA-1, c: GSH group, d: GGSH +^DGroup PPA-1.

FIG. 4 shows PD-1 in peripheral blood of chemotherapy groups before and after chemotherapy and chemotherapy + GSH groups⁺CD8⁺And comparing the apoptosis ratio of the T cells.

FIG. 5 shows CD8 of two groups of patients before and after chemotherapy and chemotherapy + GSH group⁺Comparison of the ability of T cells to secrete IFN- γ, in the figure, a: before chemotherapy, b: after chemotherapy, c: chemotherapy + GSH pre, d: after chemotherapy + GSH.

FIG. 6 shows CD8 of two groups of patients before and after chemotherapy and chemotherapy + GSH group⁺The ability of T cells to secrete TNF- α, in the figure, a: before chemotherapy, b: after chemotherapy, c: chemotherapy + GSH pre, d: after chemotherapy + GSH.

FIG. 7 is a comparison of the tumor suppression effect of MAGE-A3 specific CD8+ T cells from different treatment groups, a: blank control, b: t cell infusion group, c: t cell infusion +^DPPA-1 group, d: t cell infusion + GSH group, e: t cell infusion +^DGroup PPA-1+ GSH.

FIG. 8 shows a tumor-bearing zoneInfiltration of T cells in mouse tumor tissue, in the figure, a: t cell infusion group, b: t cell infusion +^DPPA-1 group, c: t cell infusion + GSH group, d: t cell infusion +^DGroup PPA-1+ GSH.

FIG. 9 is a graph showing the function of T cells in tumor tissues of tumor-bearing mice, wherein a: t cell infusion group, b: t cell infusion +^DPPA-1 group, c: t cell infusion + GSH group, d: t cell infusion +^DGroup PPA-1+ GSH.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

Study object and specimen acquisition

The invention has been examined by the Zhengzhou university Life sciences ethical examination committee, and meets the medical ethical requirements.

And (3) inclusion standard: esophageal cancer patients of thoracic surgery, oncology and digestive department of the first subsidiary hospital of Zhengzhou university were selected as subjects, and the age range of the patients was limited to 40-70 years.

Exclusion criteria: those with combined immune system diseases; the pathological diagnosis is not clear.

Collecting peripheral blood samples: collecting peripheral blood of a patient under an aseptic condition, performing heparin anticoagulation, and separating PBMCs in a laboratory for flow cytometry detection and lymphocyte separation and culture.

Acquisition of esophageal cancer tissue and normal tissue specimens: immediately putting a part of tumor tissue and normal tissue into liquid nitrogen, and then putting the part of the tumor tissue and the normal tissue into an ultra-low temperature refrigerator for storing for reverse transcription PCR detection; the other part of fresh specimen is required to be put into a sterile culture medium and sent back to a laboratory to be prepared into single cell suspension for flow cytometry detection and cell separation and culture.

Flow cytometry to detect expression of HLA-a 2: obtaining PBMCs from the patient, light-shielding labeled flow antibody: anti-human CD3 antibody and anti-human HLA-A2 antibody, and detecting the expression of HLA-A2 by flow cytometry.

RT-PCR detection of MAGE-A3 expression in tumor tissues: RNA of fresh tumor tissue is extracted, reverse transcription is carried out to obtain cDNA, and expression of MAGE-A3 in the tumor tissue is detected by an RT-PCR method.

According to the expression of HLA-A2 and MAGE-A3, a specimen which is positive to both HLA-A2 and MAGE-A3 is selected as a research object.

Example 1 GSH in combination with a PD-1/PD-L1 blocker on CD8⁺Effect of T cell apoptosis, proliferation and function

1. Isolation of Peripheral Blood Mononuclear Cells (PBMC)

The method specifically comprises the following steps:

1) labeling 1-2 negative pressure heparin anticoagulation tubes with 5mL, extracting 5-10mL peripheral venous blood of a patient, and storing for a short time at 4 ℃ (<6 hours) or directly operating in the next step.

2) Separating serum by a centrifuge: placing the blood collection tube in a centrifuge for centrifugation, wherein the centrifugation conditions are as follows: 9ACC, 9DEC, 1500rpm/min, 10 min.

3) After centrifugation is finished, the blood collection tube is taken out, placed in a super clean bench, an alcohol lamp is ignited, positive pressure ventilation is carried out, a pipette is used for transferring the serum on the upper layer of the blood collection tube into a 4mL sterile centrifuge tube to be frozen at the temperature of 80 ℃, the pipette is used for transferring the blood cells on the lower layer of the blood collection tube into a 50mL sterile centrifuge tube, and the volume ratio of normal saline to the blood cells is 2:1 for dilution.

4) 15mL of lymphocyte separation solution was taken into a 50mL centrifuge tube, the tube was tilted at 45 ℃ and the blood mixture was pipetted by a sterile pipette, slowly superimposed over the separation solution surface along the tube wall, taking care not to disturb the interface.

5) Placing the centrifugal tube into a centrifuge for centrifugation, and centrifuging: 5ACC, 5DEC, 2500rpm/min, 25 min. Taking out the centrifugal tube after centrifugation, putting the centrifugal tube into a super-clean bench, wherein the liquid in the tube is divided into three layers, the upper layer is plasma and normal saline, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separation liquid, a white cloud layer narrow band mainly comprising mononuclear cells is arranged at the interface of the upper layer and the middle layer, and the mononuclear cells comprise the lymphocytes and the mononuclear cells.

6) Carefully absorbing the white cloud layer at the interface of the upper layer and the middle layer by using a pipette, transferring the white cloud layer into another 50mL sterile centrifuge tube, adding physiological saline to 50mL, and washing PBMC.

7) Centrifuging: 9ACC, 9DEC, 1500rpm/min, 10min, centrifugation. And taking out the centrifuge tube after the centrifugation is finished, putting the centrifuge tube into a super clean bench, and discarding the supernatant. Adding 50mL of physiological saline for secondary washing, and centrifuging: 9ACC, 9DEC, 1200rpm/min, 8 min. The supernatant was discarded to obtain PBMC.

8) PBMC counts (10mL cells +10mL dolichol blue), one portion was cryopreserved: frozen stocks (90% FBS + 10% DMSO) were frozen at 2X 106/tube.

2. Cell culture and flow assay

(1) The resulting MAGE-A3-specific CD8⁺T cells

Screening HLA-A2/MAGE-A3 co-positive patients, separating peripheral blood mononuclear cells, activating with 10% human AB serum 1640(Hyclone) culture medium, IL2(50u/mL), CD3/CD28 Dynabeads (10 u/mL)⁶2uL), 10ug/mL of MAGE-A3 peptide fragment, divided into four groups, a: control group (MAGE-A3-specific CD 8)⁺T cell culture alone), b: PD-1/PD-L1 blocker group (also referred to as^DppA-1 group, 20. mu.g/mL), c: glutathione group (also denoted GSH group, 40 μ M), d: GSH (40 μ M) in combination with PD-1/PD-L1 blocker group (also denoted as GSH +^DGroup PPA-1, GSH: the content of the active carbon is 40 mu M,^DppA-1: 20. mu.g/mL), after 7 days of culture, MAGE-A3-specific CD8 was detected by flow cytometry⁺The proportion of T cells varied.

(2) The expression of PD-1 on the surface of MAGE-A3 specific CD8+ T cells was analyzed by flow analysis and classified according to the expression level of PD-1 as: PD-1^high、PD-1^lowAnd PD-1^-CD8⁺T cells, comparative control group,^DppA-1 group (20 μ g/mL), GSH group (40 μ M), GGSH +^DThe ppA-1 group (GSH: 40 μ M,^DppA-1: 20. mu.g/mL L), 7 days after incubation, MAGE-A3-specific CD8⁺The apoptotic rate of T cells and the ability to secrete IFN- γ.

The results show that GSH can increase the specificity of the PD-1/PD-L1 blocker on MAGE-A3 specific CD8⁺Expansion capacity of T cells (fig. 1). The PD-1/PD-L1 blocking agent after application is analyzed^highSame as PD-1^lowCD8 (1)⁺Apoptosis ratio of T cells, PD-1 was found^highCD8⁺The apoptosis rate of T cells remains high; based on PD-1^highCD8⁺T-cell ROS levels are increased, and GSH acts as a scavenger of ROS, and is found to reduce PD-1 by treating peripheral blood mononuclear cells with GSH^highCD8⁺Apoptosis of T cells, GSH in combination with PD-1/PD-L1 blocker group PD-1^highCD8⁺T cell apoptosis ratio was lowest (FIG. 2)

PD-1^highSame as PD-1^lowCD8⁺PD-1 in comparison with T cells^highCD8 (1)⁺The IFN-gamma secretion capability of T cells is not obviously recovered after the T cells are treated by the PD-1/PD-L1 blocking agent, and the CD8 caused by GSH is detected⁺Effect of T cell function, GSH was found to upregulate CD8⁺The ability of T cells to secrete IFN-gamma, and the ability of GSH to synergistically enhance CD8 in combination with a PD-1/PD-L1 blocker⁺The ability of T cells to secrete IFN- γ (FIG. 3).

Example 2

GSH-CD 8 in chemotherapy patients⁺Effect of T cells and synergistic enhancement of MAGE-A3 specific CD8 in combination with PD-1/PD-L1 blockers⁺Anti-tumor effect of T cells

1. The experimental method comprises the following steps: selecting two groups of patients of chemotherapy, chemotherapy and GSH (1.8g/qd, ivgtt), extracting peripheral blood before and after treatment, separating peripheral blood mononuclear cells (same as example 1), and detecting PD-1 in the peripheral blood by using flow cytometry⁺CD8⁺The apoptosis rate of T cells; and detecting and comparing PD-1 of two groups of patients before and after chemotherapy⁺/PD-1^-CD8⁺Levels of IFN-. gamma.and TNF-. alpha.secreted by T cells. Animal experiments: selecting HLA-A2/MAGE-A3 co-positive EC109 as target cell, establishing tumor-bearing mouse model, randomly dividing into five groups of 5 nude mice each, (EC109 group, EC109+ T cell treatment group + GSH, EC109+ T cell treatment group +)^DPPA-1 and EC109+ T cell treatment group + GSH +^DPPA-1 (dose of GSH 240mg/kg/day,^Dthe dose of PPA-1 was 10 mg/kg). Observing and recording the tumor formation time, measuring the major diameter and the minor diameter of the tumor body every 2 days, calculating the tumor volume, and drawing a tumor growth curve. The formula for calculating the tumor volume is the major axisSquare of minor axis/2. On day 9 of tumor formation, 3X 10 was administered through the fundus vein of mice⁶T cell reinfusion. According to the grouping situation, GSH is given and^DPPA-1 is injected into abdominal cavity. After sacrifice, tumor tissue was stripped off, 0.2g of tumor tissue was weighed from each group of mice given a T cell reinfusion, tumor tissue was ground to prepare single cell suspension, and each group of CD8 was detected by flow cytometry⁺Absolute numbers of T cells and expression of internal factors.

2. Results of the experiment

As shown in fig. 4: GSH significantly reduced the post-chemotherapy CD8 in patients compared to controls⁺Apoptosis ratio of T cells (n 10).

FIGS. 5 to 6: GSH enhances CD8 in patients compared to controls⁺Function of T cells, GSH elevation of CD8⁺The ability of T cells to release IFN-. gamma.and TNF-. alpha.s.

FIG. 7: glutathione combination^DPPA-1 synergistically enhanced MAGE-A3 specific CD8⁺Tumor-inhibiting effect of T cells, a: blank control, b: t cell infusion group, c: t cell infusion +^DPPA-1 group, d: t cell infusion + GSH group, e: t cell infusion +^DGroup PPA-1+ GSH; t cell infusion +^DThe PPA-1+ GSH group significantly slowed tumor growth.

FIG. 8: infiltration of T cells in tumor tissue of tumor-bearing mice, T cell infusion +^DPPA-1+ GSH group CD8⁺T cell infiltration was significantly higher than in the other groups.

FIG. 9: functional condition of T cells in tumor tissue of tumor-bearing mice, T cell infusion +^DPPA-1+ GSH group CD8⁺T cell function was significantly higher than in the other groups.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (8)

1. Use of GSH in combination with a PD-1/PD-L1 blocker for the preparation of a medicament for promoting CD8⁺Use in a product for T cell proliferation, wherein the PD-1/PD-L1 blocking agent is^DppA-1, said promotion comprising in vivo promotion and in vitro promotion.
2. 2. An in vitro enhancement of CD8 using GSH as claimed in claim 1 in combination with a PD-1/PD-L1 blocker⁺Method for T cell proliferation, characterized in that CD8 cultured in vitro is administered⁺T cell GSH and^DppA-1, wherein the concentration of GSH is 40-45 μ M,^Dthe concentration of PPA-1 was 18-20. mu.g/mL.
3. 3. Use of GSH according to claim 1 in combination with a PD-1/PD-L1 blocker for the preparation of a medicament for inhibiting CD8⁺Use in a product of T cell apoptosis, wherein the PD-1/PD-L1 blocker is^DppA-1, said inhibition comprising in vivo inhibition and in vitro inhibition.
4. 4. An in vitro inhibition of CD8 using GSH as claimed in claim 1 in combination with a PD-1/PD-L1 blocker⁺Method for apoptosis of T cells, characterized in that CD8 cultured in vitro is administered⁺T cell GSH and^DppA-1, wherein the concentration of GSH is 40-45 μ M,^Dthe concentration of PPA-1 was 18-20. mu.g/mL.
5. 5. Use of GSH according to claim 1 in combination with a PD-1/PD-L1 blocker for the preparation of a medicament for promoting CD8⁺Use in a product with T cell capacity, wherein the PD-1/PD-L1 blocker is^DppA-1, said function referring to the ability to secrete IFN- γ, said promotion including in vivo promotion and in vitro promotion.
6. 6. An in vitro enhancement of CD8 using GSH as claimed in claim 1 in combination with a PD-1/PD-L1 blocker⁺T cell secretionMethod for IFN-gamma capacity, characterized in that in vitro cultured CD8 is administered⁺T cell GSH and^DppA-1, wherein the concentration of GSH is 40-45 μ M,^Dthe concentration of PPA-1 was 18-20. mu.g/mL.
7. 7. Use of GSH according to claim 1 in combination with a PD-1/PD-L1 blocker for the preparation of a medicament for the treatment of CD8⁺Use of a product for a disease associated with T cell dysfunction, wherein said dysfunction is CD8⁺T cells have reduced ability to proliferate and secrete IFN-gamma and increased apoptosis.
8. 8. Use of GSH according to claim 7 in combination with a PD-1/PD-L1 blocker for the preparation of a medicament for the treatment of CD8⁺Use in a product for a disease associated with T cell dysfunction, wherein the disease includes, but is not limited to, esophageal cancer.

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