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Mesothelin high-expression cancer treatment vaccine based on recombinant attenuated listeria monocytogenes

A technology for Listeria monocytogenes and cancer treatment, applied in the biological field, can solve problems such as defects and low immunogenicity

Inactive Publication Date: 2018-10-26
南京颂悦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key to its success is to induce a sufficient immune response, but the current nucleic acid and protein peptide vaccines have low immunogenicity and obvious defects

Method used

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  • Mesothelin high-expression cancer treatment vaccine based on recombinant attenuated listeria monocytogenes
  • Mesothelin high-expression cancer treatment vaccine based on recombinant attenuated listeria monocytogenes
  • Mesothelin high-expression cancer treatment vaccine based on recombinant attenuated listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1. Preparation of vaccine bacteria LmΔactAplcB-Meso

[0068] (1) Synthesis of Mesothelin Gene Fragment

[0069] Query the mesothelin protein gene from NCBI, then optimize it according to the codons of Listeria monocytogenes, add a HindⅢ enzyme cleavage site upstream, add an XhoI enzyme cleavage site downstream, and finally obtain the DNA sequence directly by synthesis (provided by the company The cloning plasmid pUC57-Meso was obtained), and the sequence is shown in Sequence 1 of the Sequence Listing.

[0070] (2) Construction of targeting plasmid

[0071] ① Construction of intermediate plasmid pCW203-Meso

[0072] Plasmids pUC57-Meso and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer.

[0073] pUC57-Meso and pCW203 were double-digested with HindⅢ and XhoⅠ, 20 μL of enzyme digestion system: pUC57-Meso or pCW2032 O Make up the system to 20 μL. Digest the plasmid in a water bath at 37°C for 1 hour, ...

Embodiment 2

[0110] Example 2: Evaluation of the therapeutic effect of vaccine strains on tumors with high expression of mesothelin

[0111] (1) CT26-MESO tumor cell culture and tumor model establishment

[0112]Use RPMI1640 Medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in 5% CO 2 Cultivate CT26-MESO cells in an incubator until the density reaches about 80%, rinse the cells with Hank’s solution for 2 to 3 times, digest with trypsin and resuspend with PBS for counting, and prepare the cells to a concentration of 5×10 6 / ml of cell suspension, 6-8 week-old BALB / c female mice were injected with 100 μl of cell suspension into the tail vein to establish a mouse tumor model and observe the survival rate.

[0113] (2) Immunotherapy of model mice

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Abstract

The invention relates to a mesothelin high-expression cancer treatment vaccine based on recombinant attenuated listeria monocytogenes. The attenuated listeria monocytogenes (Lm) is used as a carier, the characteristics that the Lm can specifically grow in a host phagocyte and is a natural T cell immune activated reagent are utilized, specific immune response in a tumor microenvironment can be effectively improved, the immune tolerance of a body can be broken, persistent viral infection of the body can be cleaned, and an excellent treatment effect for cleaning the focus and inhibiting tumor development can be achieved. The mesothelin high-expression cancer treatment vaccine has the advantages of overcoming the defect of relatively low immunogenicity of nucleotide and protein polypeptide vaccines, the attenuated listeria remained with original strains can grow in cells, so that higher safety is achieved while the characteristic of antigen presentation can be completed.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a cancer therapeutic vaccine with high mesothelin expression based on recombinant attenuated Listeria monocytogenes. Background technique [0002] Studies have found that mesothelin is a cell surface glycoprotein whose expression is usually limited to the mesothelium (peritoneum, pericardium, and pleura), and found that in mesothelioma, pancreatic cancer, lung cancer, and ovarian cancer, mesothelin protein is significantly High expression. (MesothelinA New Target for Immunotherapy, Raffit Hassan, Tapan Bera, and Ira Pastan doi: 10.1158 / 1078-0432. CCR-03-0801 Clin Cancer Res June 15, 2004 10; 3937) This feature makes mesothelin an ideal target for recombinant therapy. Tool antigens of Listeria monocytogenes in tumor therapy. [0003] Mesothelioma is a tumor that arises primarily on the lining of the chest or abdominal cavity and is associated with exposure to asbestos fibers in the wor...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P35/00C12N1/21C12N15/74
CPCA61K39/0011A61K2039/522A61K2039/523A61K2039/53A61P35/00C07K14/195C12N15/74
Inventor 沈海浅
Owner 南京颂悦生物科技有限公司
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