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

Abstract

The present invention discloses the use of an inhibitor targeting T-cell TIGIT in the preparation of an anti-tuberculosis drug. The inhibitor includes ociperlimab, AGEN1777, tiragolumab, etc. The present invention blocks TIGIT on the surface of T cells, promoting 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.

Description

Technical Field

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

[0002] Tuberculosis (TB) is a major global health problem characterized by the persistence of Mycobacterium tuberculosis (Mtb) within the host, typically in the lungs. For many years, it has been established that TB is accompanied by a microenvironment within the lungs that harbors immunosuppressive cells. These cells, including regulatory T cells (Tregs) and immunosuppressive cells, have been consistently identified in TB-affected lungs. These lesions are generally considered to result from an increase in suppressive immune cells or an increase in immune cell surface expression of immune checkpoints or immunosuppressive receptors, combined with a decrease in activating receptors. Exploring the mechanisms of action of these immunosuppressive receptors or immune checkpoints and efforts to restore immune responses are potential strategies for TB control. However, the use of anti-PD-1 therapies, designed to suppress T cell exhaustion and reinvigorate the immune system, has yielded negative results, exacerbating TB in humans and animal models. This suggests that current PD-1 blockade alone is insufficient to restore TB immunity, and further investigation of the roles of additional immunosuppressive receptors or immune checkpoints in TB is needed.

[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 present on the surface of T cells (T cells and Tregs) and natural killer cells (NK cells). The primary ligand for 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 that enhances T cell activation upon binding to CD155. TIGIT and DNAM-1 compete for binding to CD155, thereby blocking DNAM-1's co-stimulatory signal 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. They interact synergistically, jointly suppressing the anti-tumor activity of T cells. For example, TIGIT and PD-1 can inhibit T cell activation and effector function through different mechanisms, resulting in a stronger immunosuppressive effect. However, there are still many gaps in the understanding of how these molecules specifically affect T cell function, especially their mechanisms of action in different disease contexts. In particular, in the important public health issue of tuberculosis, the expression and function of TIGIT in peripheral blood T cells of tuberculosis-infected patients have not been studied to date. This 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, existing technologies have significant deficiencies in revealing the specific role of TIGIT in tuberculosis and its therapeutic applications. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an inhibitor targeting T cell TIGIT for use in the preparation of anti-tuberculosis drugs, thereby enhancing the immune response efficiency of T cells in tuberculosis patients to eliminate 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; and 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 eliminate 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 existing technology, 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 in vitro Mtb (H37Rv△leuD) infection of healthy human 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 3 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; (A) is a schematic diagram of the experimental process, in which PMA-differentiated CFSE-labeled THP-1 cells were infected with Mtb(H37RvΔleuD)-mScarlet, and these macrophages were co-cultured with Jurkat cells pretreated with IgG or αTIGIT for 24 hours, and dead cells were excluded by FVS-700 staining. PE-Texas Red was used to assess the number of intracellular bacteria, and a colony forming unit (CFU) assay was performed to quantify the intracellular bacterial load; (B) Flow cytometric analysis of the inhibitory efficiency of TIGIT neutralizing antibody in inhibiting TIGIT expression in Jurkat cells; (C) Statistical graph of the inhibitory efficiency of TIGIT neutralizing antibody in inhibiting TIGIT expression in Jurkat cells; (D) Flow cytometric analysis 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 forming unit analysis of the number of tuberculosis bacteria in THP-1 cells; (G) Statistical graph of the number of tuberculosis bacteria in THP-1 cells by colony forming units;

[0020] Figure 3 Figure 1 shows how inhibition of TIGIT on the surface of T cells affects the release of cytokines and cytotoxic granules in a co-culture environment. (A) In a co-culture system of Mtb-infected THP-1 cells and Jurkat cells, inhibition of TIGIT results in decreased mRNA expression of GZMA and GZMB, while increased mRNA expression of MPEG1. (B) TIGIT inhibition affects MPEG1 protein expression in a co-culture environment. Western blot analysis of MPEG1 protein expression 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 primary human 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 will be further described below with reference to 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 a TIGIT monoclonal antibody (MAB7898, Monoclonal Mouse IgG2B Clone #741182, R&D System). The tuberculosis disease model is a blood-type disseminated pulmonary tuberculosis, and the disease model cells include monocytes, macrophages and T lymphocytes.

[0027] 1. Peripheral blood samples were collected from individuals registered at Nantong Sixth People's Hospital (a specialized TB hospital) who were receiving standardized anti-TB treatment. All participants were excluded from infectious diseases or tumors. Healthy controls (HC) were recruited through a physical examination center, had never been exposed to pulmonary TB patients, had a negative PPD test result, and had no clinical symptoms of TB. The collected peripheral blood samples were stained after removing red blood cells using an erythrocyte lysis buffer and blocking Fc. Cell membrane staining included CD3, CD4, CD8, CD56, and TIGIT.

[0028] 2. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll density gradient centrifugation. Peripheral blood was transferred to a 15 ml centrifuge tube, Ficoll separation buffer was added, and the tube was centrifuged at 500 × g for 30 minutes. The PBMC layer was collected, washed with PBS, and centrifuged at 300 × g for 10 minutes. 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] 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. The cells were then treated with Brefeldin A (10 μg / ml) and Monensin (3 μM) for 4 hours and stained with antibodies against CD3, CD4, CD8, CD56, and TIGIT. Following live / dead cell staining with Fixable Viability Stain 700 (1:1000 dilution, 30 minutes, 4°C), the cells were fixed. TIGIT expression in T cell subsets was assessed 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, multiplicity of infection 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 of PE-Texas Red by the CytoFLEX S instrument. + The number of intracellular bacteria was measured by colony forming units (CFU).

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

[0033] THP-1 cells were lysed with 0.1% Triton X-100, diluted with a gradient of sterile PBS, and evenly plated on 7H10 agar plates (Sigma-Aldrich). The plates were incubated at 37°C for 30 days, and the number of colonies grown on the plates was counted.

[0034] 6. Western Blot Experiment

[0035] Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The lysate was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk and incubated with primary antibodies against MPEG1 (PA1-29037, Thermofisher) and β-actin (60008-1-Ig, Proteintech). The membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody, 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 harvested for RNA isolation using Trizol reagent (Cat. No. B511311, Sangon). Reverse transcription was performed using the HiScript III kit (R312, Vazyme). qPCR analysis was performed on a StepOne / StepOnePlus real-time PCR system using SYBR-Green (Q711, Vazyme). Primers used are listed in the following table:

[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 monocytes were isolated using the Human Monocyte Isolation Kit (#19359, STEMCELL™) according to the manufacturer’s instructions. + 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, with the medium replaced every 48-72 hours to ensure optimal cell growth and activity.

[0043] 10. Statistical methods

[0044] Data normality was assessed using the Shapiro-Wilk test. For two groups of data, between-group comparisons were performed using the Student's t-test or the Mann-Whitney U test (unpaired samples and nonnormal distribution); 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 in vitro Mtb (H37Rv△leuD) infection of healthy human peripheral blood T cell subsets (CD3 + CD4 + , CD3 + CD8 + , CD3 + CD56 + ) hi and TIGIT lo The expression flow cytometry graph and statistical result graph 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 figure shows the effect of TIGIT inhibition on T cell clearance of intracellular tuberculosis bacteria in THP-1 cells, indicating that after TIGIT on the surface of Jurkat cells is inhibited, its ability to clear intracellular tuberculosis bacteria can be significantly enhanced.

[0047] Figure 3This figure shows that the inhibition of TIGIT on the surface of T cells in the present invention affects the release of cytokines and cytotoxic particles in a co-culture environment, which 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 figures are representative flow cytometric graphs of the number of tuberculosis bacteria in macrophages co-cultured with human primary T cells of the present invention and quantitative graphs of the percentage of intracellular bacteria content. The results 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. Use of an inhibitor targeting T cell TIGIT in the preparation of an anti-tuberculosis drug, wherein the inhibitor is the TIGIT monoclonal antibody MAB7898.

2. The application 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.

3. The application according to claim 1, characterized in that The tuberculosis is hematogenously disseminated pulmonary tuberculosis.

4. The application according to claim 1, characterized in that The drug is a drug that targets and blocks TIGIT.

5. The application according to claim 1, characterized in that: The drug is a drug that enhances T cells to eliminate intracellular tuberculosis bacteria.

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