Hepatocellular carcinoma vaccine targeting secondary lymphoid tissues

An amino acid and nucleotide sequence technology, applied in the direction of cytokines/lymphokines/interferons, drug combinations, cancer antigen components, etc., to achieve the effect of eliminating tumor cells and overcoming HLA restrictions

Active Publication Date: 2019-03-05
NEWISH TECH (BEIJING) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is currently lacking how to actively deliver the GPC3 protein immunogen to CD8α + T cell immunity against autologous embryonal tumor-associated antigens

Method used

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  • Hepatocellular carcinoma vaccine targeting secondary lymphoid tissues
  • Hepatocellular carcinoma vaccine targeting secondary lymphoid tissues
  • Hepatocellular carcinoma vaccine targeting secondary lymphoid tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Embodiment 1: fusion gene and fusion protein

[0127] 1. Construction of Fusion Gene Expression Plasmids

[0128] According to the amino acid sequences of human GPC3 protein (SEQ ID NO: 1) and mouse XCL1 protein (SEQ ID NO: 3), a GPC3-XCL1 fusion protein was constructed. At the same time, in order to ensure the mutual independence of the chemokine and the target protein in terms of spatial structure and exert normal chemotactic function, we designed a linker between GPC3 and XCL1, the corresponding amino acid sequence is: GGGGGSGGGGG (SEQ ID NO: 4 ), the nucleic acid sequence is: GGC GGA GGC GGA GGA TCA GGG GGA GGG GGAGGA (SEQ ID NO: 5). To facilitate the expression and secretion of fusion proteins in mammalian cells and to be targeted to XCR1 in lymph nodes + CD8α + In DC cells, we first replaced the signal peptide located at the amino terminal protein of the fusion protein with the mammalian signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 6), and removed the signal ...

Embodiment 2

[0161] Example 2: Expression and localization of fusion genes in mice

[0162] 1. Plasmid immunization and material collection:

[0163] In vitro experiments, we found that whether the chemokine XCL1 is located at the amino-terminal or carboxyl-terminal of the fusion protein, it can be normally expressed in cells and secreted to the outside of the cell. Chemotactic XCR1 + CD8α + DC cells, therefore, in a further experiment, we selected a fusion gene expression plasmid with XCL1 located at the amino terminal of the fusion protein to immunize mice. According to the previous work in the laboratory, 24 hours before the mouse (C57BL / 6, 6 weeks old), inject 5 mg / ml bupivacaine hydrochloride into the quadriceps muscle of the mouse, 50 μl / mouse, to make the muscle mild Degree of injury, promote muscle cell regeneration to increase the amount of plasmid DNA absorbed by cells. Immunization was carried out on the second day. During the immunization, the mice were first anesthetized wi...

Embodiment 3

[0170] Example 3: The effect of fusion gene on the occurrence of primary liver cancer and its effect on the function of normal liver cells

[0171] 1. Construction of HBV / DEN mouse primary liver cancer model

[0172] In order to further test the intervention effect of the fusion gene expression plasmid on the primary liver cancer in mice, we constructed the HBV transgenic mouse primary liver cancer induced by the chemical carcinogen DEN (diethylnitrosamine, referred to as DEN) Liver cancer model ( Figure 7 A). HBV transgenic male mice (C57BL / 6J-TgN (AlblHBV) 44Bri, established by Chisari in 1985. Purchased from the Experimental Animal Center of Peking University Health Science Center) were injected with DEN solution (25 mg / kg body weight) intraperitoneally at 2 weeks after birth to induce HBV / DEN mouse liver cancer model (abbreviation: HBV / DEN mouse liver cancer model). Mice were sacrificed at the age of 22 weeks, and there were a large number of tumors in the liver and th...

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Abstract

The invention provides a hepatocellular carcinoma vaccine targeting secondary lymphoid tissues. The vaccine comprises fusion protein comprising (a) phosphatidylinositol proteoglycan-3 (GPC3) and (b) lymphocyte chemotactic factor (XCL1) polypeptide of a specifically bonded chemokine receptor 1 (XCR1), nucleic acid encoding the fusion protein and vectors of the nucleic acid, wherein (a) is connectedwith (b) through a linker, and (a) lacks amino acid residues with a cell membrane anchoring effect. The vaccine comprises the fusion protein, the nucleic acid and/or the vectors, and application of the fusion protein, the nucleic acid and/or the expression vectors in prevention and treatment of liver cancers is provided. The vaccine has the effect of intervention of generation and development ofliver cancers, and can be applied to intervention of high-risk population of liver cancer, and for example, the vaccine can be applied to intervention of the generation progress of hepatitis B-relatedpatients to liver cancers.

Description

technical field [0001] The invention relates to the field of cancer prevention and treatment, in particular to liver cancer vaccine and the prevention and treatment of liver cancer. Background technique [0002] Hepatocellular carcinoma (HCC), referred to as liver cancer, is one of the common malignant tumors in my country. Chronic persistent hepatitis B virus (HBV) infection is the main cause of liver cancer in my country (Ming, L. et al., Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China.Hepatology, 2002, 36 (5) : 1214-1220). At present, the main means of liver cancer treatment clinically is surgical resection. Many patients are diagnosed at an advanced stage, and due to the background of chronic HBV infection and liver failure, they cannot be operated on. Due to the lack of effective treatment methods, the 5-year survival rate of liver cancer patients is Very low (Omata M, et al. Asian Pacific Association for the S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K39/00A61P35/00
CPCA61K39/0011C07K14/4725C07K14/521C07K2319/00
Inventor 曲春枫赵宏陈坤蔡建强丁惠国
Owner NEWISH TECH (BEIJING) CO LTD
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