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Reverse hepatitis B chronic virus infection Tim-3 antibody and alpha-galcer combination and application

A chronic infection and antibody technology, applied in the direction of antibodies, drug combinations, antiviral agents, etc., can solve problems such as unclear effects and mechanisms, and achieve the effect of overcoming HBVDNA viral load and reducing HBsAg and liver tissue pgRNA levels

Active Publication Date: 2017-09-05
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunotherapy targeting Tim-3 has shown good therapeutic effects in a variety of tumors, but the role and mechanism of targeting Tim-3 in reversing chronic HBV infection is still unclear

Method used

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  • Reverse hepatitis B chronic virus infection Tim-3 antibody and alpha-galcer combination and application
  • Reverse hepatitis B chronic virus infection Tim-3 antibody and alpha-galcer combination and application
  • Reverse hepatitis B chronic virus infection Tim-3 antibody and alpha-galcer combination and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Flow cytometric detection of Tim-3 expression on the surface of HBV-Tg mouse liver iNKT cells

[0043] 1. Sacrifice the mouse, disinfect the skin, open the abdominal cavity, remove the liver, and keep it moist with PBS.

[0044] 2. Cut the liver into pieces in a mortar, grind it, filter it with a 200-mesh copper mesh, and collect the cell filtrate in a 15ml or 50ml centrifuge tube.

[0045] 3. Centrifuge at 400 rpm for 1 min, and collect the supernatant.

[0046] 4. Centrifuge at 1500 rpm for 8 minutes, and discard the supernatant.

[0047] 5. Resuspend the cell pellet in 5ml of erythrocyte lysate, mix well, incubate at 4°C for 5min, then centrifuge at 1500rpm for 8min, discard the supernatant. During the configuration of 40% percoll solution.

[0048] 6. Add 8ml of 40% percoll solution after centrifugation, resuspend and mix the cells, and centrifuge for 25min with a horizontal rotor at 2500rpm.

[0049] 7. After the centrifugation is completed, the solut...

Embodiment 2

[0055] Example 2 α-Galcer pretreatment, flow cytometry detection of secretion of IFN-γ and CD107a in iNKT cells

[0056] 1. Select 6-8 week-old HBV-Tg mice, inject 2 μg of α-Galcer solution into the tail vein, and the total injection volume is 200 μl. After the injection, observe the state of the mice without abnormalities.

[0057] 2. After 24 hours, the mice were sacrificed, the hepatic mononuclear cells were separated, and the separated cells were washed once with 1640 medium.

[0058] 3. Resuspend the cells with 1640+10% FBS medium, plant in 24-well plate, the plate density is 1*10 6 / hole.

[0059] 4. Adding breamectin (BFA, 1 μg / ml) to block protein secretion, and at the same time adding anti-CD107a (clone number 1D4B, Biolegend) to detect CD107a.

[0060] After 5.2 hours, collect the cells, wash the cells twice with 2ml PBS at 1500rpm, centrifuge for 6min, discard the supernatant, and resuspend the cells in 100μl PBS.

[0061] 6. Add 10 μl of CD1d tetramer or its con...

Embodiment 3

[0066] Example 3 α-Galcer pretreatment, flow cytometry detection of CD8+ T cells and NK cells IFN-γ and CD107a expression

[0067] 1. The methods of mouse treatment, isolation of hepatic mononuclear cells, culture and stimulation of the cells are consistent with the description of IFN-γ in Example 2.

[0068] 2. After washing the cells, resuspend the cells in 100ul PBS, add anti-CD3e (clone number 145-2C11, eBioscience), anti-CD8a (clone number 53-6.7, eBioscience), anti-NK1.1 (clone number PK136, eBioscience ) and anti-CD49b (clone number DX5, BD Pharmingen), incubated at 4°C for 30min.

[0069] 3. Add 2ml of PBS to wash the cells, centrifuge at 1500rpm for 8min, and then label or detect IFN-γ or CD107a as described in 2.

[0070] Among them: CD3+CD8+ cells are defined as CD8 T cells, and CD3-NK1.1 / Dx5+ cells are NK cells.

[0071] The result is as figure 2As shown, to verify the effect of anti-Tim-3 antibody combined with α-Galcer on iNKT cell function, the present inven...

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Abstract

The invention discloses a reverse hepatitis B chronic virus infection Tim-3 antibody and alpha-galcer combination and an application. Research teams find that CD8+T, NK, iNKT and mononuclear macrophage surfaces Tim-3 of HBV (hepatitis B virus) infected patients are increased at different levels, Tim-3 high expression and inherent immunity and adaptive immunity tolerance of the HBV patients are closely related, except for alpha-Galcer, Tim-3 plays a key role in hindering an HBV removal process, the iNKT is activated by the alpha-Galcer, immune cell checking points Tim-3 are intervened, HBV liver immune tolerance can be reversed, and HBV regression is realized. A Tim-3 antibody is combined with the alpha-Galcer in application, HBV can be effectively removed, and HBV load and serum HBsAg and liver tissue pgRNA levels are remarkably reduced.

Description

technical field [0001] The present invention relates to the field of hepatitis B biological treatment research, in particular to a combination of anti-Tim-3 antibody and α-galcer for reversing the chronic infection of hepatitis B virus and α-galcer combined with anti-Tim-3 antibody immunotherapy in eliminating hepatitis B Role in hepatitis virus. Background technique [0002] Hepatitis B virus (HBV) is a hepatotropic DNA virus. HBV infection can induce liver inflammation and cause liver cell damage. Persistent HBV infection can induce liver fibrosis and even hepatocellular carcinoma (hepatocellular carcinoma, HCC), which is an important risk factor that endangers human health. [0003] HBV chronic infection is related to HBV-induced liver immune tolerance. Liver innate immune cells and liver adaptive immune cells in patients with chronic infection are in a state of low response or even no response to HBV, which makes HBV immune escape and continue to expand in liver cells....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/576A61K39/395A61P1/16A61P31/20A61K31/7032
CPCA61K31/7032A61K39/395G01N33/5761A61K2300/00
Inventor 马春红许勇杜宪红武专昌梁晓红高立芬王泽华谭思雨王体潇
Owner SHANDONG UNIV
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