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Application of recombinant attenuated listeria in preparation of mesothelin high-expression cancer therapeutic vaccine

A technology for Listeria and cancer treatment, applied in recombinant DNA technology, bacteria, bacterial peptides, etc., can solve problems such as defects and low immunogenicity

Active Publication Date: 2018-10-30
苏州圣苏新药开发有限公司 +1
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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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  • Application of recombinant attenuated listeria in preparation of mesothelin high-expression cancer therapeutic vaccine
  • Application of recombinant attenuated listeria in preparation of mesothelin high-expression cancer therapeutic vaccine
  • Application of recombinant attenuated listeria in preparation of mesothelin high-expression cancer therapeutic vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1. Preparation of vaccine bacteria LiΔactAplcB-Meso

[0102] (1) Synthesis of Mesothelin Gene Fragment

[0103] 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), (the sequence is shown in Sequence 1 of the Sequence Listing).

[0104] (2) Construction of targeting plasmid

[0105] ① Construction of intermediate plasmid pCW203-Meso

[0106] Plasmids pUC57-Meso and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer.

[0107] pUC57-Meso and pCW203 were double-digested with HindⅢ and XhoⅠ, 20 μL of enzyme digestion system: pUC57-Meso or pCW203 2 O Make up the system to 20 μL. Digest the plasmid in a water bath at 37°C for 1 hour, add ...

Embodiment 2

[0144] Embodiment 2: Preparation of vaccine bacteria LmΔactAplcB-Meso

[0145] (1) Synthesis of Mesothelin Gene Fragment

[0146] With embodiment 1 (1).

[0147] (2) Construction of targeting plasmid

[0148] ①The construction of the intermediate plasmid pCW203-Meso is the same as in Example 1(2)①

[0149] ②Construction of targeting plasmid

[0150] Plasmid pCW180 was extracted and eluted with 30 μL Elution Buffer. Mix plasmid pCW180 and intermediate plasmid pCW203-Meso with restriction endonucleases BamHI and XhoI respectively according to the system of (2)① (replace HindIII enzyme with BamHI enzyme), carry out enzyme digestion and dephosphorylation, and gel recovery after electrophoresis The small fragment after pCW203-Meso digestion is the insert fragment (1840bp) and the pCW180 vector backbone (long fragment after digestion, about 8621bp in length). According to the system of (2) ①, mix and connect and transform into E. coli DH5α, spread on LA plate, perform PCR scree...

Embodiment 3

[0167] Example 3: Evaluation of the therapeutic effect of vaccine strains on cancer with high expression of mesothelin

[0168] (1) CT26-MESO tumor cell culture and tumor model establishment

[0169] 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 weeks old BALB / c female mice were injected with 100 μl of cell suspension through the tail vein to establish a mouse lung metastatic tumor model and observe the survival rate.

[0170] (2) Comparing homologous and heterologous priming-boosting immunotherapy on model mice.

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Abstract

The invention relates to application of recombinant attenuated listeria in the preparation of a mesothelin high-expression cancer therapeutic vaccine. By taking attenuated Listeria monocytogenes (Lm for short hereinafter) and attenuated Listeria ivanovii (Li for short hereinafter) as carriers, and special listeria can grow in phagocyte of a host and is a natural T-cell immune activation adjuvant,so that the specific immune response in a tumor microenvironment is effectively improved, the immune tolerance of a body is broken through, the continuous virus infection of the body is eliminated, and good treatment effects on the elimination of an infection focus and the containment of tumor development are achieved. The application has the advantages that the disadvantages that nucleic acid type vaccines and protein polypeptide type vaccines are relatively low in immunogenicity are remedied, the attenuated listeria preserves the properties that an original strain can grow in the cells and finish antigen presentation, and meanwhile, the attenuated listeria has relatively high safety.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation of mesothelin high-expressing cancer therapeutic properties based on recombinant attenuated Listeria [0002] application in vaccines. Background technique [0003] 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. [0004] Mesothelioma is a tumor that arises primarily on the lining of the chest or abdominal cavity and is associated with ex...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P35/00C12N1/21C12N15/74C12R1/01
CPCA61K39/0011A61P35/00C12N15/74C07K14/195Y02A50/30
Inventor 沈海浅
Owner 苏州圣苏新药开发有限公司
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