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New strategy enhancing tumor antigen immunogenicity and application thereof in immunotherapy of lung cancer

A technology for tumor-associated antigens and lung cancer, applied in gene therapy, anti-tumor drugs, antibody medical components, etc. Tumor effect, effect of obvious effect

Inactive Publication Date: 2015-07-08
REDLAND BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The failure of these trials was largely due to the low immunogenicity of tumor antigens and the inability of the vaccine to induce a sufficiently strong immune response

Method used

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  • New strategy enhancing tumor antigen immunogenicity and application thereof in immunotherapy of lung cancer
  • New strategy enhancing tumor antigen immunogenicity and application thereof in immunotherapy of lung cancer
  • New strategy enhancing tumor antigen immunogenicity and application thereof in immunotherapy of lung cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction and Identification of Recombinant Adenovirus Carrying Tumor Fusion Antigen

[0032] Taking the preparation of recombinant adenovirus by inserting the fusion protein coding sequence of lung cancer-associated antigens (PRAME, Muc-1, MAGE-A3) and LC3b protein into adenovirus as an example, the strategy for enhancing the immunogenicity of tumor antigens of the present invention will be further described.

[0033] The LC3b gene sequence was obtained from the NCBI database, as shown in SEQ ID NO.1, and the encoded amino acid sequence was shown in SEQ ID NO.2, and the LC3b gene was obtained by total gene synthesis. The pVAX vector carrying the full-length ORF cDNA sequence of human PRAME is preserved by our laboratory. The full-length ORF cDNA of PRAME is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4.

[0034] The full-length ORF cDNA of the target gene PRAME was fused with the LC3b gene according to conventional mo...

Embodiment 2

[0039] Example 2. Recombinant adenovirus carrying tumor fusion antigen can significantly enhance the immunogenicity of tumor antigen in mouse model

[0040] Further study whether the above-mentioned gene fused with LC3b can play a role in the body and effectively improve the response of T lymphocytes.

[0041] 1. Experimental Materials

[0042] 1.1 Flow cytometry antibody

[0043]Monoclonal antibodies used, anti-mouse anti-CD3-PerCP, anti-CD4-FITC and anti-CD8-APC (BD Biosciences) anti-IFN-γ-PE, anti-TNF-α-PE-cy7, anti-IL- 2-APC-cy7 (BD Pharmingen) was purchased from BD Company.

[0044] 1.2 Tumor antigen peptides

[0045] Tumor peptides were synthesized by our laboratory, and most peptides consisted of 15 amino acids, with 11 amino acid overlaps between each pair. The purity was >80%. It was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 0.4 mg / ml / peptide, and stored at -70°C after aliquoting.

[0046] 1.3 Other reagents

[0047] ELISPOT plate ...

Embodiment 3

[0093] Example 3. Recombinant adenovirus carrying tumor fusion antigen has a good effect on killing tumor cells in a mouse tumor model

[0094] 3.1 Tumor cells and mouse tumor models

[0095]Mouse Lewis lung cancer cells (LLC) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cultivate and expand according to conventional methods, harvest 80-90% confluent Lewis lung cancer (LLC) cells in the logarithmic growth phase, wash twice with calf serum-free culture medium and make a concentration of 5×10 6 / mL of cell suspension, inject LLC cells 5×10 subcutaneously in the right upper armpit of C57BL / 6 5 (0.1mL) per mouse to establish a mouse lung cancer model. After the injection, continue to feed, and observe the eating, spirit, activity status and tumor growth of the mice every day.

[0096] 3.2 Anti-tumor experiment of recombinant adenovirus in mice

[0097] 6-8-year-old female C57BL / 6 mice were randomly divided into 4 groups with 10 mice in each ...

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Abstract

The invention discloses a new strategy enhancing tumor antigen immunogenicity and an application thereof in immunotherapy of lung cancer. In the invention, by means of fusion between a tumor specific antigen and an autophagy gene with high-efficient infection of body APC cells with an adenovirus vector, the synergistic effect of the both can enhance immune response level of human body to a tumor target antigen, thereby achieving a significant antitumor effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a new strategy for enhancing the immunogenicity of tumor antigens and its application in lung cancer immunotherapy. Background technique [0002] Lung cancer is one of the most common malignant tumors in the world, and its mortality rate ranks first among the causes of death from tumor-related diseases in the world. In most cases, patients present with unresectable disease or an incurable disease state. Patients with locally advanced or good general condition of non-small cell lung cancer can receive concurrent chemoradiotherapy, with or without surgery, so that an average of 8 months of disease progression-free survival can be obtained, but the 5-year overall survival rate is still less than 15% . [0003] Therefore, new and more effective lung cancer treatment modalities are urgently needed. In recent years, among the new methods of tumor treatment that people are con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K48/00A61K39/00A61P35/00
Inventor 孙彩军
Owner REDLAND BIOTECH CO LTD
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