Application of combination of PD-1 monoclonal antibody and Tindili in relieving toxic and side effects of PD-1 monoclonal antibody and glutathione

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of a PD-1 monoclonal antibody, namely a combination of Cindili and glutathione in relieving toxic and side effects of the PD-1 monoclonal antibody. According to the application disclosed by the invention, the Tindili monoclonal antibody and the glutathione are combined to intervene the rat lymphocytes, and experimental data shows that compared with non-intervention, the activity of the rat lymphocytes is reduced by singly using the Tindili monoclonal antibody, and the inhibition effect of the Tindili monoclonal antibody on the rat lymphocytes can be slowed down by combining the Tindili monoclonal antibody and the glutathione. Therefore, the invention provides a method for alleviating the toxic and side effects of the PD-1 monoclonal antibody, and the combination of the PD-1 monoclonal antibody and the glutathione can be applied clinically to alleviate the immune-related side effects occurring in the treatment of the PD-1 monoclonal antibody and the Taidili. The invention provides a new method and thought for improving the curative effect of the PD-1 monoclonal antibody, and expands a new way for clinical application of glutathione.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the application of PD-1 monoclonal antibody Sindily combined with glutathione in alleviating its toxic and side effects. Background Art

[0002] In the tumor tissue environment, there are a large number of infiltrating immune cells and main tumor cells coexisting, and the interaction and mutual regulation between them determine the development and changes of tumors. Among them, T lymphocytes are the most important role, which can inhibit or kill tumor cells. Lymphocytes play a vital role in maintaining immune homeostasis, and their functional changes can directly reflect immune homeostasis. Among them, matrix metalloproteinase 9 (MMP-9) is one of the signs of excessive inhibition of lymphocytes. CD8+ T lymphocytes, also known as cytotoxic T lymphocytes (CTLs), are the main effector cells in tumor-infiltrating T cell subsets. They have the ability to directly target and destroy tumor cells by binding to human leukocyte antigen (HLA) class I molecules, and are a more robust biomarker than CD3+. In tumor immunity, CD8+ T cells play an important role in killing tumor cells, and their killing of target cells is mainly through the Fas-FasL pathway and the perforin-granzyme pathway.

[0003] Studies have shown that cancer progression may be caused by the suppression of CD8+ T cells, and this suppression is related to the immune escape of tumor cells caused by the combination of programmed death protein-1 (PD-1) expressed on the CD8+ T cell membrane and programmed death protein-ligand 1 (PD-L1) expressed on the surface of tumor cells. Therefore, blocking this combination is a way to prevent tumor cell immune escape. In current clinical research on tumors, PD-1 blocking therapy has been actively explored for the treatment of tumors.

[0004] PD-1 monoclonal antibody is a fully human monoclonal antibody targeting programmed death receptor 1 (PD-1). It blocks the binding of PD-1 to its ligand PD-L1 in the body, allowing T cells to function normally and then clear tumor cells through autoimmunity. PD-1 monoclonal antibody has developed into a class of anti-tumor immunotherapy drugs.

[0005] Sintilimab is one of the PD-1 monoclonal antibodies. It was approved for marketing by NMPA on December 24, 2018, and is used to treat relapsed or refractory classical Hodgkin's lymphoma after at least two lines of systemic chemotherapy. In clinical applications, it was found that PD-1 monoclonal antibody drugs can cause patients to have varying degrees of immune-related adverse reactions in the lungs, liver, kidneys, gastrointestinal system, endocrine system and skin, and even hyperprogressive disease. Therefore, mitigating the toxic side effects of sintilimab and other PD-1 monoclonal antibodies has become a major clinical problem.

[0006] Glutathione (GSH) is a tripeptide containing γ-amide bonds and sulfhydryl groups, which is composed of glutamic acid, cysteine ​​and glycine. Glutathione has active sulfhydryl groups and is the most important functional group. It can participate in many important biochemical reactions in the body, protect the sulfhydryl groups of important enzyme proteins in the body from oxidation and inactivation, and ensure energy metabolism and cell utilization. At the same time, it can directly reduce free radicals into acidic substances by combining with free radicals in the body through sulfhydryl groups, thereby accelerating the excretion of free radicals and counteracting the damage of free radicals to important organs. As a natural antioxidant, the normal concentration range of glutathione is (1-10mM). Glutathione participates in many cell functions, removes free radicals, protects many substances from oxidation, and helps maintain normal immune system function. Summary of the invention

[0007] The inventors of the present application have shown through experimental studies that the use of PD-1 monoclonal antibody Sindili alone will inhibit lymphocyte viability. In comparison, the PD-1 monoclonal antibody Sindili combined with glutathione can restore lymphocyte cell viability and MMP-9 activity. Glutathione has the function of mitigating the toxic and side effects of PD-1 monoclonal antibody Sindili.

[0008] The present invention uses rat spleen lymphocytes and bone marrow lymphocytes as research objects, and adopts the combined intervention of sintilimab and glutathione. It is found that the PD-1 monoclonal antibody sintilimab combined with glutathione can alleviate the inhibitory effect of PD-1 monoclonal antibody sintilimab on the central immune system (bone marrow cells) and peripheral immune system (splenic lymphocytes).

[0009] Therefore, the present invention provides a method for reducing the toxic side effects of PD-1 monoclonal antibody Sintilim. PD-1 monoclonal antibody Sintilim combined with glutathione can be used clinically to alleviate the immune-related side effects occurring during the treatment of PD-1 monoclonal antibody Sintilim.

[0010] In an embodiment, the mitigation of the toxic side effects of the PD-1 monoclonal antibody sintilimab refers to mitigating the inhibitory effect of sintilimab on lymphocytes.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: the results of the present invention show that the combination of PD-1 monoclonal antibody Sindili and glutathione can reduce the inhibitory effect of PD-1 monoclonal antibody Sindili on the peripheral immune system (spleen) and central immune system (bone marrow) of rats. Therefore, the present invention provides a new method and idea for improving the efficacy of PD-1 monoclonal antibody. The present invention combines glutathione with tumor immunotherapy, and puts forward effective suggestions for alleviating the serious immune-related side effects existing in the clinic and ensuring the safety of drug use, which expands new avenues for the clinical application of glutathione. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The graph shows the results of the MTT experiment on rat spleen lymphocytes after 24 hours of drug intervention.

[0013] Figure 2 The figure shows the results of the MTT experiment on rat bone marrow cells after 24 hours of drug intervention.

[0014] Figure 3 Gelatin zymogram of rat splenic lymphocytes is shown.

[0015] Figure 4 The gelatin zymogram of rat bone marrow cells is shown. DETAILED DESCRIPTION

[0016] In order to make the invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0017] Example 1. Extraction and culture of rat spleen lymphocytes and bone marrow cells.

[0018] After rats were killed by cervical dislocation, spleen and bone marrow were obtained from rats. Cells were sieved with a cell sieve under sterile conditions, treated with Tris-NH4Cl red blood cell lysis buffer, and centrifuged. Cells were washed with PBS containing 4% penicillin-streptomycin and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) overnight to remove adherent cells and obtain purer lymphocytes.

[0019] Example 2. PD-1 monoclonal antibody Sindily combined with glutathione can restore the cell viability of rat lymphocytes.

[0020] 2.1 Experimental methods

[0021] Cell viability assay.

[0022] The cell viability experiment was used to detect the effect of PD-1 monoclonal antibody Sindily combined with glutathione on the cell viability of rat spleen lymphocytes and bone marrow cells. Rat spleen lymphocytes (2×10 5 ) and rat bone marrow cells (2×10 5) were inoculated in a 96-well plate and placed in a 37°C, 5% CO2 incubator for stable culture. The drug intervention conditions were set under serum-free conditions: no intervention, low concentration of sintilimab (1 mg / mL), high concentration of sintilimab (10 mg / mL, clinical intravenous injection concentration), low concentration of sintilimab (1 mg / mL) + glutathione (10 mM), high concentration of sintilimab (10 mg / mL) + glutathione (10 mM). After intervention, the spleen lymphocytes and bone marrow cells of rats were cultured in a 37°C, 5% CO2 incubator for 20 h, and the supernatant was collected for gelatin zymogram analysis. The cells were washed three times with PBS and cultured with 5 mg / mL MTT culture medium for another 4 h. After discarding the culture medium, 100 µL of dimethyl sulfoxide was added to each well. The optical density (OD) value was measured at 570 nm on a microplate reader. The effects of various intervention conditions on the cell viability of rat spleen lymphocytes and bone marrow cells were observed and statistically analyzed.

[0023] 2.2 Experimental results

[0024] like Figure 1 As shown, the experimental results showed that compared with no intervention, sintilimab intervention significantly reduced the cell viability of spleen lymphocytes; compared with low concentration of sintilimab, low concentration of sintilimab + glutathione combined to restore the cell viability of spleen lymphocytes, there was a significant statistical difference.

[0025] like Figure 2 As shown, the experimental results showed that compared with no intervention, sintilimab intervention significantly reduced the cell viability of bone marrow cells; compared with high concentration of sintilimab, the combination of high concentration of sintilimab + glutathione restored the cell viability of bone marrow cells, with significant statistical differences.

[0026] Example 3. Sintilimab combined with glutathione can alleviate the activity of MMP-9 in rat lymphocytes.

[0027] 3.1 Experimental methods

[0028] Gelatin zymography.

[0029] The activity of matrix metalloproteinase MMP-2 / 9 was determined by gelatin zymography. The supernatant after intervention was mixed with an equal volume of sample buffer and loaded, 24 µL per well, and the gel was run at 100 V for vertical electrophoresis. The gel was eluted twice with elution buffer for 1 hour each time, and incubated in active incubation buffer overnight. The gel was stained with Coomassie Brilliant Blue R-250, eluted until white bands appeared on the background, photographed with Bio-Rad gel imaging system, and analyzed with Image Lab 6.1.

[0030] 3.2 Experimental results

[0031] like Figure 3 As shown, the experimental results showed that compared with no intervention, low concentration of sintilimab reduced the MMP-9 activity of rat splenic lymphocytes and high concentration callback; compared with sintilimab, the combination of sintilimab + glutathione alleviated the MMP-9 activity of rat splenic lymphocytes.

[0032] like Figure 4 As shown, the experimental results showed that compared with no intervention, sintilimab reduced the activity of MMP-9 in rat bone marrow cells; compared with sintilimab, the combination of sintilimab + glutathione alleviated the activity of MMP-9 in rat bone marrow cells.

[0033] Based on the comprehensive experimental results, compared with no intervention, Sindilimab reduced the viability of rat spleen lymphocytes and bone marrow cells, and the combination of Sindilimab + glutathione can restore the viability of rat spleen lymphocytes and bone marrow cells, with statistically significant differences. Compared with no intervention, Sindilimab reduced the MMP-9 activity of rat spleen lymphocytes and bone marrow cells, and the combination of Sindilimab + glutathione can alleviate the MMP-9 activity of rat spleen lymphocytes and bone marrow cells, with statistically significant differences. This shows that the combination of PD-1 monoclonal antibody Sindilimab and glutathione can slow down the inhibitory effect of PD-1 monoclonal antibody Sindilimab on the peripheral immune system (splenic lymphocytes) and the central immune system (bone marrow cells).

Claims

1. The application of PD-1 monoclonal antibody Sindili combined with glutathione in alleviating its toxic side effects.

2. The use according to claim 1, characterized in that: The PD-1 monoclonal antibody sintilide combined concentration is 10 mg / mL, and the glutathione combined concentration is 10 mM.

3. The use according to claim 1, characterized in that: The said mitigation of its toxic side effects refers to mitigation of the inhibitory effect of the PD-1 monoclonal antibody Sindili on rat lymphocytes.

4. The use according to claim 1, characterized in that: The PD-1 monoclonal antibody Sindili can reduce the cell viability and MMP-9 activity of rat lymphocytes.

5. The use according to claim 1, characterized in that: The PD-1 monoclonal antibody Sindily combined with glutathione can restore the cell viability and MMP-9 activity of rat lymphocytes.

6. The use according to claim 5, characterized in that: The rat lymphocytes are spleen lymphocytes and bone marrow cells.

7. The use according to claims 1-6, characterized in that: The present invention provides a method for alleviating the toxic side effects of PD-1 monoclonal antibody Sintilim. The PD-1 monoclonal antibody Sintilim combined with glutathione can be used clinically to alleviate the immune-related side effects occurring during the treatment of PD-1 monoclonal antibody Sintilim.

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