Application of inhibitor aiming at T cell TIGIT in preparation of antituberculous drugs

Abstract

The invention discloses an application of an inhibitor aiming at T cell TIGIT in preparation of an anti-tuberculosis drug. The inhibitor comprises an Ociperlimab (Ociperlimab), an AGEN1777 (AGEN1777), a Tiragolumab (Tiragolumab) and the like. The TIGIT on the surface of the T cell is blocked, the T cell is promoted to increase the capacity of eliminating intracellular bacteria of macrophages by up-regulating MPEG1, the proliferation of tubercle bacillus is controlled, and evidence is provided for developing antituberculous drugs.

Description

Technical Field

[0001] The present invention relates to an inhibitor, and in particular to application of an inhibitor targeting T cell TIGIT in the preparation of anti-tuberculosis drugs. Background Art

[0002] Tuberculosis is a major global health problem characterized by the persistence of Mycobacterium tuberculosis (Mtb) in the host, usually in the lungs. Over the years, it has been established that tuberculosis is accompanied by a microenvironment in which immunosuppressive cells exist in the lungs. These cells, including regulatory T cells (Tregs) and immunosuppressive cells, have been consistently identified in lungs affected by tuberculosis. These lesions are generally seen as an increase in suppressive immune cells, or an increase in the expression of immune checkpoints or immunosuppressive receptors on the surface of immune cells and a decrease in immune activating receptors. Exploring the mechanism of action of these immunosuppressive receptors or immune checkpoints and efforts to restore immune responses will serve as potential strategies for tuberculosis control. However, the application of anti-PD-1 therapy, which aims to inhibit T cell exhaustion and revitalize the immune system, has produced negative results and exacerbated tuberculosis in humans and animal models. This indicates that the current simple PD-1 blockade is not enough to restore tuberculosis immunity, and the role of more immunosuppressive receptors or immune checkpoints in tuberculosis needs to be explored.

[0003] T cells are a crucial type of cell in the immune system, responsible for cellular immune responses. T cell immunoglobulin with ITIM domain (TIGIT) is a protein expressed on T cells (including CD8 + T cells, CD4 +TIGIT is a type I transmembrane protein on the surface of T cells and Tregs) and natural killer cells (NK cells). The main ligand of TIGIT is CD155 (also known as PVR or NECTIN2), which is expressed on a variety of cell types, including tumor cells, antigen-presenting cells, and endothelial cells. TIGIT inhibits T cell activation and function by binding to CD155 in T cells. DNAM-1 is a co-stimulatory receptor on the surface of T cells, which can enhance T cell activation after binding to CD155. TIGIT and DNAM-1 compete for binding to CD155, thereby blocking the co-stimulatory signal of DNAM-1 and further inhibiting T cell activation. TIGIT is often co-expressed on the surface of T cells with other immune checkpoint molecules such as PD-1. There is a synergistic inhibitory effect between them, which jointly inhibits the anti-tumor activity of T cells. For example, TIGIT and PD-1 can inhibit the activation and effector function of T cells through different mechanisms, resulting in a stronger immunosuppressive effect. However, there are still many gaps in how these molecules specifically affect T cell function, especially in the mechanism of action in different disease contexts. In particular, in the important public health issue of tuberculosis, there has been no research on the expression and function of TIGIT in peripheral blood T cells of tuberculosis-infected patients, which has also hindered the development of therapeutic strategies to improve the immune function of tuberculosis patients by blocking inhibitory immune checkpoints such as TIGIT. Therefore, the existing technology has significant deficiencies in revealing the specific role of TIGIT in tuberculosis and its therapeutic application. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an inhibitor of T cell TIGIT for use in the preparation of anti-tuberculosis drugs, so as to enhance the immune response efficiency of T cells in tuberculosis patients in clearing Mycobacterium tuberculosis (Mtb).

[0005] Technical solution: Use of the inhibitor against T cell TIGIT described in the present invention in the preparation of anti-tuberculosis drugs.

[0006] Preferably, the inhibitor includes Ociperlimab, AGEN1777 or Tiragolumab. Ociperlimab is from BeiGene; AGEN1777 is from Agenus; Tiragolumab is from Roche.

[0007] Preferably, the inhibitor is a TIGIT monoclonal antibody.

[0008] Further preferably, the TIGIT monoclonal antibody is: MAB7898, Monoclonal Mouse IgG2B Clone #741182, R&D System.

[0009] Preferably, the tuberculosis includes primary pulmonary tuberculosis, hematogenously disseminated pulmonary tuberculosis, secondary pulmonary tuberculosis, tuberculous pleurisy or bronchial tuberculosis.

[0010] Preferably, the tuberculosis is hematogenously disseminated pulmonary tuberculosis.

[0011] Preferably, the cells of the tuberculosis disease model include monocytes, macrophages or T lymphocytes.

[0012] Preferably, the drug is a drug that targets and blocks TIGIT.

[0013] Preferably, the drug is a drug that enhances T cells to clear intracellular tuberculosis bacteria.

[0014] The anti-tuberculosis drug of the present invention includes an inhibitor targeting T cell TIGIT.

[0015] Preferably, the dosage form of the anti-tuberculosis drug is a suspension or an injection.

[0016] Preferably, the anti-tuberculosis drug further comprises a pharmaceutically acceptable carrier, and the carrier comprises a liposome, a cell carrier or a nanoparticle.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention blocks TIGIT on the surface of T cells, promotes T cells to enhance the ability of T cells to clear intracellular bacteria in macrophages by upregulating MPEG1, thereby achieving control of tuberculosis proliferation and providing evidence for the development of anti-tuberculosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Peripheral blood T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi and TIGIT lo Expression flow cytometry and statistical results, and peripheral blood T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56+ ) hi and TIGIT lo Expression flow cytometry and statistical results; (A) T cell subsets in peripheral blood of tuberculosis patients and healthy controls (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi and TIGIT lo Expression flow cytometry; (B) T cell subsets in peripheral blood of tuberculosis patients and healthy controls (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi Expression statistics; (C) is the peripheral blood T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) lo Expression statistics; (D) is the T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi and TIGIT lo Expression flow cytometry; (E) T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi Statistical graph of expression; (F) T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56+ ) lo Statistical graph of expression; statistical significance was determined using the unpaired Mann-Whitney U test, with p < 0.05 indicating significance;

[0019] Figure 2 Figure 1 is a diagram showing the effect of TIGIT inhibition on T cell clearance of intracellular tuberculosis in THP-1 cells; (A) is a schematic diagram of the experimental process, using PMA-differentiated CFSE-labeled THP-1 cells to be infected with Mtb(H37RvΔleuD)-mScarlet, these macrophages were co-cultured with Jurkat cells pretreated with IgG or αTIGIT for 24 hours, FVS-700 staining was used to exclude dead cells, and PE-Texas Red was used to evaluate the number of intracellular bacteria, and a colony forming unit (CFU) experiment was performed to quantify the intracellular bacterial load; (B) Flow cytometric graph of the inhibitory efficiency of inhibiting TIGIT expression in Jurkat cells using TIGIT neutralizing antibodies; (C) Statistical graph of the inhibitory efficiency of inhibiting TIGIT expression in Jurkat cells using TIGIT neutralizing antibodies; (D) Flow cytometric graph of the percentage of Jurkat cells clearing intracellular Mtb in THP-1 macrophages; (E) Statistical graph of the percentage of Jurkat cells clearing intracellular Mtb in THP-1 macrophages by flow cytometry; (F) Colony formation graph of the number of Mycobacterium tuberculosis in THP-1 cells counted by colony forming units; (G) Statistical graph of the number of Mycobacterium tuberculosis in THP-1 cells counted by colony forming units;

[0020] Figure 3 The present invention shows that the inhibition of TIGIT on the surface of T cells affects the release levels of cytokines and cytotoxic particles in a co-culture environment; (A) In a co-culture system of THP-1 cells infected with Mtb and Jurkat cells, inhibition of TIGIT leads to a decrease in the mRNA expression of GZMA and GZMB, while an increase in the mRNA expression of MPEG1; (B) Inhibition of TIGIT affects the expression of MPEG1 protein in a co-culture environment; Western blot analysis of the expression of MPEG1 protein in Jurkat cells (left) and THP-1 cells (right) in a co-culture environment;

[0021] Figure 4 This is a representative flow cytometric graph of the number of tuberculosis bacteria in macrophages co-cultured with human primary T cells of the present invention;

[0022] Figure 5 This is a statistical graph showing the percentage of intracellular tuberculosis bacteria in macrophages co-cultured with human primary T cells of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0024] Example 1

[0025] TIGIT inhibitors affect the bactericidal function of T cells in the preparation of anti-tuberculosis drugs. The specific process is as follows:

[0026] The TIGIT inhibitor used in this example is TIGIT monoclonal antibody (MAB7898, Monoclonal Mouse IgG2B Clone #741182, R&D System). The tuberculosis disease model is blood-type disseminated pulmonary tuberculosis, and the disease model cells include mononuclear macrophages and T lymphocytes.

[0027] 1. Peripheral blood samples were collected from individuals registered at the Nantong Sixth People's Hospital (a specialized hospital for tuberculosis) who were receiving standardized anti-tuberculosis treatment. All participants were excluded from infectious diseases or tumors. The healthy control group (HC) was recruited through the physical examination center, had never been in contact with patients with pulmonary tuberculosis, had a negative PPD test, and had no clinical symptoms of tuberculosis. The collected peripheral blood samples were stained after removing red blood cells and blocking Fc with red blood cell lysis buffer. Cell membrane staining includes CD3, CD4, CD8, CD56, and TIGIT.

[0028] 2. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood using Ficoll density gradient centrifugation. Peripheral blood was transferred into a 15 ml centrifuge tube, and Ficoll separation solution was added and centrifuged at 500 × g for 30 min. The PBMC layer was collected, washed with PBS, and centrifuged at 300 × g for 10 min. PBMCs were resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, IL-2 (10 ng / ml), 2.5 ng / μl anti-CD3 (16-0037-81, ThermoFisher), 5 ng / μl anti-CD28 (16-0281-82, ThermoFisher) and M-CSF (50 ng / ml).

[0029] 3. PBMCs were co-cultured with Mycobacterium tuberculosis (Mtb, H37Rv△leuD or H37Rv△leuD-mScarlet) at a multiplicity of infection (MOI) of 1:1 for 24 hours, and then treated with BrefeldinA (10μg / ml) and Monensin (3μM) for 4 hours, followed by staining with antibodies against CD3, CD4, CD8, CD56 and TIGIT. After live / dead cell staining with Fixable Viability Stain 700 (1:1000 dilution, 30 minutes, 4°C), the cells were fixed. Finally, the expression of TIGIT in T cell subsets was detected by flow cytometry.

[0030] 4. T cell cytotoxicity assay

[0031] First, THP-1 cells were labeled with CFSE (5 μM, 565082, BD), and then induced to differentiate using PMA (10 ng / ml) for 24 hours. After coinfection with Mtb (H37Rv△leuD-mScarlet, MOI = 1) for 4 hours, the adherent cells were treated with Jurkat cells in a co-culture system, and co-cultured for 24 hours using IgG or neutralizing antibody αTIGIT (effector cell to target cell ratio of 4:1). After the co-culture phase, the cells were washed and then incubated with Fixable Viability Stain 700 in a dark room for 30 minutes. After the cells were rinsed to remove Fixable Viability Stain 700, the cells were transferred to a tube for analysis by the CytoFLEX S instrument PE-Texas Red + The number of intracellular bacteria was measured by colony forming units (CFU).

[0032] 5. Colony forming unit (CFU) count

[0033] THP-1 was lysed with 0.1% Triton X-100 to break the cell membrane, diluted with gradient sterile PBS, and evenly spread on 7H10 agar culture plates (Sigma-Aldrich). The culture plates were placed in a 37°C constant temperature incubator for 30 days, and the number of colonies grown on the culture plates was counted.

[0034] 6. Western Blot Experiment

[0035] Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% skim milk, and then incubated with primary antibodies MPEG1 (PA1-29037, Thermofisher), β-actin (60008-1-Ig, proteintech). The membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies, and protein bands were detected using an enhanced chemiluminescence kit. Densitometry was performed using Image-Pro Plus software.

[0036] 7. Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0037] Differentiated THP-1 cells infected with Mtb (H37Rv△leuD) and co-cultured with Jurkat cells were collected for RNA isolation using Trizol reagent (Cat. No. B511311, Bio-Tech). Reverse transcription was performed using HiScript III kit (R312, Vazyme). qPCR analysis was performed on StepOne / StepOnePlus real-time PCR system using SYBR-Green (Q711, Vazyme). The primers used are as follows:

[0038]

[0039] 8. Isolation and culture of human T cells

[0040] Human T cells were isolated from freshly prepared peripheral blood mononuclear cells (PBMCs) using EasySep TM Human CD3 Positive Selection Kit (#17751, STEMCELL TM ). The isolated T cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. To maintain T cell activation, 2.5 ng / μl anti-CD3 (16-0037-81, ThermoFisher), 5 ng / μl anti-CD28 (16-0281-82, ThermoFisher) and 20 ng / mL recombinant human IL-2 protein were added to the culture medium and cultured for 24 hours.

[0041] 9. Isolation and culture of human monocytes

[0042] Human monocytes were isolated from freshly prepared PBMCs using EasySep TMHuman monocyte isolation kit (#19359, STEMCELLTM) was used to isolate CD14 + CD16 - Monocytes were cultured in complete RPMI 1640 medium (10% FBS and 1% penicillin-streptomycin). Monocytes were differentiated into macrophages by adding M-CSF (50 ng / ml). All primary cells were maintained in a humidified incubator at 37°C and 5% CO2, and the medium was changed every 48-72 hours to ensure optimal growth and activity of the cells.

[0043] 10. Statistical methods

[0044] Data normality was assessed using the Shapiro-Wilk test. For two groups of data, Student's t-test or Mann-Whitney U test (unpaired samples and non-normal distribution) was used for comparison between groups; p < 0.05 was considered significant.

[0045] Figure 1 Peripheral blood T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi and TIGIT lo Expression flow cytometry and statistical results, and peripheral blood T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi and TIGIT lo The expression flow cytometry graphs and statistical results show that the percentage of cells with high expression of TIGIT in peripheral blood T cell subsets of tuberculosis patients at the in vivo level is reduced, while the percentage of cells with high expression of TIGIT in peripheral blood T cell subsets infected with Mtb in vitro is increased.

[0046] Figure 2 This is a diagram showing the effect of TIGIT inhibition on T cell clearance of intracellular tuberculosis bacteria in THP-1 cells according to the present invention, indicating that after TIGIT on the surface of Jurkat cells is inhibited, their ability to clear intracellular tuberculosis bacteria can be significantly enhanced.

[0047] Figure 3This diagram shows that the inhibition of TIGIT on the surface of T cells affects the release of cytokines and cytotoxic particles in a co-culture environment. It shows that in a co-culture environment, after inhibiting TIGIT on the surface of Jurkat cells, the expression of the bactericidal substance MPEG1 in Jurkat cells increases, and the expression of MPEG1 protein in THP-1 co-cultured with Jurkat cells also increases.

[0048] Figures 4-5 The representative flow cytometric graphs of the number of tuberculosis bacteria in macrophages co-cultured with human primary T cells of the present invention and the quantitative graphs of the percentage of intracellular bacteria content show that after TIGIT on the surface of human T cells is inhibited, the ability of T cells to clear intracellular tuberculosis bacteria can be enhanced.

Claims

1. Application of an inhibitor targeting T cell TIGIT in the preparation of anti-tuberculosis drugs.

2. The application according to claim 1, characterized in that: The inhibitors include Ociperlimab, AGEN1777 or Tiragolumab.

3. The application according to claim 1, characterized in that: The inhibitor is a TIGIT monoclonal antibody.

4. The use according to claim 1, characterized in that: The tuberculosis includes primary pulmonary tuberculosis, hematogenously disseminated pulmonary tuberculosis, secondary pulmonary tuberculosis, tuberculous pleurisy or bronchial tuberculosis.

5. The use according to claim 1, characterized in that: The tuberculosis is hematogenously disseminated pulmonary tuberculosis.

6. The use according to claim 1, characterized in that: The drug is a drug that targets and blocks TIGIT.

7. The use according to claim 1, characterized in that: The drug is a drug for enhancing T cells to clear intracellular tuberculosis bacteria.

8. An anti-tuberculosis drug, characterized in that: Including inhibitors targeting T cell TIGIT.

9. The anti-tuberculosis drug according to claim 8, characterized in that: The dosage form of the anti-tuberculosis drug is a suspension or an injection.

10. The anti-tuberculosis drug according to claim 8, characterized in that: Also included are pharmaceutically acceptable carriers, including liposomes, cell carriers or nanoparticles.

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