WO2019001173A1

WO2019001173A1 - Application of scd100 protein in preparing drug for treating chronic hbv infection

Info

Publication number: WO2019001173A1
Application number: PCT/CN2018/087754
Authority: WO
Inventors: 刘嘉; 王璐; 杨尚青; 杨东亮
Original assignee: 华中科技大学同济医学院附属协和医院
Priority date: 2017-06-29
Filing date: 2018-05-22
Publication date: 2019-01-03
Also published as: CN107375902A

Abstract

The present invention provides an application of a sCD100 protein in preparing a drug for treating chronic HBV infection, and further provides a drug for treating chronic HBV infection, with an active component being the sCD100 protein.

Description

Application of sCD100 protein in the preparation of therapeutic drugs for chronic HBV infection

Technical field

The invention relates to the field of immunology and medical technology, in particular to the application of a sCD100 protein for preparing a medicament for treating chronic HBV infection, wherein the sCD100 protein has an anti-viral immune regulation effect in a specific immunotherapy for chronic HBV infection.

Background technique

Hepatitis B virus infection is a global public health problem, and chronic HBV infection is particularly serious in China. At present, clinical antiviral drugs mainly fall into two categories: 1 interferon alpha (such as PEG-IFNα, ie, pegylated interferon), but only induces a sustained antiviral response in about 30% of patients, and the side effects are large. 2 nucleoside (acid) analogues (such as lamivudine, entecavir, etc.), can effectively inhibit viral replication, but no direct immunomodulatory effects, difficult to completely eliminate HBV, long-term or even life-long medication, and the presence of viral resistance Mutation and viremia rebound after stopping the drug ^1-2 . Therefore, it is important to explore more effective CHB immunotherapy strategies.

Cytotoxic T lymphocytes play a key role in clearing HBV-infected hepatocytes ³ . In adaptive cellular immune responses, CD8+ T cell activation depends on dual signaling: 1CD8+ T cell T cell receptor recognition antigen presenting cell surface MHC class I molecule presented antigenic peptide, through the first signal; 2APC and The T cell surface costimulatory molecule interaction provides a costimulatory signal (second signal). Activated CD8+ T cells undergo clonal expansion to differentiate into effector CTLs and memory T cells ⁴ . Studies have shown that viral infection can cause functional differentiation and maturation of APC cells in the liver to induce the production of effector CD8+ T cells, but this process does not depend on the regulation of classical costimulatory molecules (such as CD80/CD86). It suggests that there is a new mechanism of co-stimulatory molecular regulation in intrahepatic T cell immunity ⁵ . At present, it is still unclear how the HBV-specific CD8+ T cells exert their antiviral activity by breaking intrahepatic immune tolerance during acute self-limiting HBV infection.

The literature mentioned above includes: 1. Locarinini, S., Mason, W. S. Cellular and virological mechanisms of HBV drug resistance. J Hepatol 44, 422-431 (2006).

2. Zoulim, F., Locarinini, S. Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 137,1593-1608e1591-1592 (2009).

3. Liu, J., Kosinska, A., Lu, M., Roggendorf, M. New therapeutic vaccination strategies for the treatment of chronic hepatitis B. Virol Sin 29, 10-16 (2014).

4. Schuch, A., Hoh, A., Thimme, R. The role of natural killer cells and CD8 (+) T cells in hepatitis B virus infection. Front Immunol 5, 258 (2014).

5. Thomson, A.W., Knolle, P.A. Antigen-presenting cell function in the tolerogenic liver environment. Nat Rev Immunol 10, 753-766 (2010)

Summary of the invention

In view of this, the present invention provides an immunologically active sCD100 protein for use in a specific immunotherapy for chronic HBV infection, and the sCD100 protein can promote the activation of HBV-specific T cells in the liver to exert an immunotherapeutic effect.

A first aspect of the invention provides the use of the sCD100 protein for the preparation of a medicament for the treatment of chronic HBV infection.

Specifically, the sCD100 protein in the drug has the effect of enhancing the HBV-specific CD8+ T cell immune response in the liver and promoting the clearance of HBV virus.

In an embodiment of the present invention, the effect of sCD100 protein was verified by using a chronic replication mouse model of HBV and in vitro administration of sCD100 and HBV-specific polypeptides to treat PBMCs of CHB patients. Specifically, male C57/BL mice aged 6-8 weeks were used to establish a chronic replication mouse model of HBV by injection of plasmid pAAV/HBV 1.2 via tail vein high pressure water, and peripheral blood samples were collected periodically to detect serum HBsAg and HBeAg; In the chronic HBV replication model, sCD100 protein was administered in vivo to detect changes in serum HBsAg and HBeAg levels, and it was found that sCD100 protein can lead to accelerated viral clearance. PBMCs from CHB patients were treated with sCD100 and HBV-specific peptides in vitro, and the HBV-specific CD8+ T cell immune responses in some CHB patients were enhanced by flow cytometry.

A second aspect of the invention provides a medicament for the treatment of chronic HBV infection, the active ingredient of which comprises the sCD100 protein.

Specifically, the pharmaceutical composition for treating chronic HBV infection may further comprise a pharmaceutically acceptable pharmaceutically acceptable excipient or a pharmaceutically acceptable excipient.

The administration method of the medicament for treating chronic HBV infection may be diversified, including but not limited to oral administration, intravenous administration, etc., correspondingly, the dosage form of the medicament may also be diverse, such as a capsule. Such as dosage forms suitable for oral administration, and injection forms such as injections.

The invention has the beneficial effects that the present invention provides a novel immunostimulating factor for promoting activation of HBV-specific CD8+ T cells in the liver, thereby achieving an immunotherapy strategy for promoting HBV virus clearance, and exogenous in the process of chronic HBV infection. sCD100 treatment can promote viral clearance, and sCD100 protein plays an important role in antiviral immune regulation. This finding is of great significance in clinical application.

DRAWINGS

Figure 1 is a schematic diagram showing surface antigen clearance of chronic HBV replication mice after administration of exogenous sCD100 protein;

Figure 2 is a schematic diagram showing the e antigen clearance of chronic HBV replication mice after administration of exogenous sCD100 protein;

Figure 3 is a schematic diagram showing the enhancement of CD8+ T cell immune response in patients with CHB after administration of exogenous sCD100.

Detailed ways

The application of the sCD100 protein provided by the present invention in the preparation of a medicament for treating chronic HBV infection will be further described below in conjunction with specific examples. The embodiments described below are illustrative only and are not to be construed as limiting the invention.

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were commercially available unless otherwise stated.

Example 1

In this example, a mouse HBV chronic replication model was constructed, and the antiviral immune regulation of sCD100 protein in the specific immunotherapy of chronic HBV infection was verified by the model. The specific steps include:

10 male C57BL/6 mice in 6-8 weeks, the plasmid pAAV/HBV1.2 prepared by plasmid company was rapidly pushed through the tail vein of the mouse in 6-8 g by high pressure tail vein injection in 5-8 seconds. Inject into the body, construct a chronic replication model of HBV, and divide the experimental mice into two groups of 5 each. The experimental group was injected with sCD100 protein through the common tail vein on the 7th and 14th day, each time 50 mice were injected; the control group was injected with the corresponding dose of physiological saline. Mouse eyelid peripheral blood was collected on the 4th, 7th, 11th, 14th, 18th, 21st, 25th and 28th day after treatment time, and the supernatant was collected by centrifugation at 10000g for 10 minutes. The serum was diluted 10-fold according to the commercial HBsAg ELISA kit procedure. The HBsAgOD value is shown in Fig. 1. It can be seen from Fig. 1 that administration of exogenous sCD100 protein can prematurely clear surface antigen of chronic HBV-replicating mice.

Example 2

In this embodiment, the mouse HBV chronic replication model of Example 1 was used, and the model was used to verify the antiviral immune regulation of sCD100 protein in the specific immunotherapy of chronic HBV infection. The specific steps include:

The experimental mice were treated as in Example 1. The peripheral blood of the mouse eyelids was collected at the treatment time, and the supernatant was collected by centrifugation at 10000 g for 10 minutes. The HBeAgOD value was measured by 10-fold dilution of the serum according to the commercial HBeAg ELISA kit procedure. As shown in Fig. 2, it can be seen from Fig. 2 that administration of exogenous sCD100 protein can pre-clear e antigen in chronic HBV-replicating mice.

Example 3

Randomly take 4 patients who met the diagnostic criteria of chronic hepatitis B. 3 ml of iliac vein blood was taken into the heparin tube, and repeated reversal to ensure adequate anticoagulation; the lymphocyte separation tube empty tube was centrifuged at 2000 °C for 1 minute at 2000 °C, and the collected subjects were collected. The whole blood is transferred into the lymphocyte separation tube; centrifuged at 800g for 15 minutes at room temperature 20 ° C, the centrifuge acceleration and deceleration is adjusted to 3 steps; the upper PBMC layer is aspirated and transferred to a new 15 ml centrifuge tube; in a new tube Add 5 ml of red blood cell lysate, centrifuge at 300 g for 10 minutes at room temperature, discard the supernatant, and gently scrape the centrifuge tube; resuspend the PBMC by adding 2 ml of PBS buffer. Using 12-well cell culture plate, each well was added the following ingredients: 1ml PBMC (10 ⁶ cells), 1μL aCD28,2.5μL IL-2, exogenous soluble CD100 protein (1μg / ml) was placed in a cell culture incubator Cultivate for 10 days. The cells were removed at 5-7 days of culture, and 500 μL of 1640 complete medium and 1.25 μL of IL-2 were added to each 1 ml of cells. A new 96-well plate was taken and 200 μL of cells were added to the wells. Centrifuge at 1500 rpm for 5 minutes. The cells were resuspended by adding 200 μL of 1640 complete medium to each well. Centrifuge at 1500 rpm for 5 minutes. The BFA solution was diluted to 1:1000 in a new culture plate and incubated at 37 ° C for 2-4 hours. Centrifuge for 5 minutes at 1500 rpm after incubation. The supernatant was discarded, and the cells were resuspended in 200 μL of cold PBS and centrifuged at 1500 rpm for 5 minutes. The supernatant of the centrifuged cells was discarded, and 100 μL of the diluted FVD was added to each well of the cells. Incubate for 15 minutes at 4 ° C in the dark. At 1500 rpm x 5 minutes, the supernatant was discarded. The cells were resuspended in 200 μL PBS + 1% FCS solution. At 1500 rpm x 5 minutes, the supernatant was discarded. Prepare 1:1000 CD4/CD8/CD3 (100 μL per well). Incubate for 15 minutes at 4 ° C in the dark. At 1500 rpm x 5 minutes, the supernatant was discarded. The cells were resuspended in 200 μL PBS + 1% FCS solution. At 1500 rpm x 5 minutes, the supernatant was discarded. The cells were resuspended in 100 μl of Fixation Buffer and incubated for 15 minutes at 4 °C. 1500 rpm × 5 minutes, discard the supernatant. The cells were resuspended using 100 μL of Perm Buffer. At 1500 rpm x 5 minutes, the supernatant was discarded. 1:1000 IFN-γ (100 μL per well) was prepared using Perm Buffer. The cells were resuspended in 100 μl of IFN-γ antibody dilution and incubated at 4 ° C for 20 minutes. At 1500 rpm x 5 minutes, the supernatant was discarded. The cells were resuspended in 200 μL of PBS and transferred into a FACS tube to measure the frequency of expression of IFN-γ by CD8+ T cells using flow cytometry. The results are shown in Figure 3. From the results of Figure 3, administration of exogenous sCD100 treatment enhanced the CD8+ T cell immune response in CHB patients.

The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalents, improvements, etc., which are within the spirit and scope of the present invention, should be included in the protection of the present invention. Within the scope.

Claims

The use of sCD100 protein in the preparation of a medicament for treating chronic HBV infection.
The use of the sCD100 protein according to claim 1 for the preparation of a medicament for treating chronic HBV infection, characterized in that the sCD100 protein in the medicament has the effect of enhancing the HBV-specific CD8+ T cell immune response in the liver and promoting the clearance of HBV virus. .
A medicament for treating chronic HBV infection, characterized in that the active ingredient comprises sCD100 protein.