Oncotherapy type heparanase MAP vaccine

A technology of heparinase and peptide vaccine, applied in anti-tumor drugs, antibody medical components, peptide/protein components, etc., can solve the problems of short half-life, inability to induce, small molecular weight, weak antigenicity, etc., and achieve major commercial industrialization value , the effect of increasing molecular mass and easy synthesis

Inactive Publication Date: 2011-07-27
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the 9-peptide vaccine has its fatal weaknesses: small molecular weight, weak antigenicity, and it is very easy to be degraded in the body, so the half-life is too short to induce effective CTL responses and achieve the purpose of inducing the body to produce anti-tumor immune effects

Method used

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  • Oncotherapy type heparanase MAP vaccine
  • Oncotherapy type heparanase MAP vaccine
  • Oncotherapy type heparanase MAP vaccine

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0049] Example 1 Obtaining of the polypeptide epitope MAP vaccine of the present invention

[0050] (1) Synthesis of MAP4 polypeptide

[0051] The peptide synthesis was carried out on the ABI431A peptide synthesizer produced by PE Company in the United States. The brief description is as follows: the standard Fmoc protocol was adopted, and arginine was coupled twice, and extended from the carboxy-terminus to the amino-terminus according to the peptide sequence. After the peptide is synthesized, it is cleaved with the corresponding cleavage enzyme, and the crude peptide products of various protections are removed at the same time. Store at -20°C for later use.

[0052] (2) Purification and molecular weight analysis of heparanase MAP epitope

[0053] Purify and analyze the polypeptide MAP epitope obtained by the above implementation examples by RP-HPLC: dissolve the synthesized MAP4 epitope vaccine with DMSO at a concentration of 20 mg / ml, filter through a 0.22 μm fiber membra...

example 2

[0056] Example 2 Anti-tumor effect research of heparanase MAP4 vaccine of the present invention

[0057] Using standard 4h 51 Cr release method

[0058] ① Preparation of effector cells: DC cells were isolated from PBMC cells of HLA-A2 positive healthy blood donors, and 30 μmol / L heparanase antigen peptide was added to prepare effector cells routinely.

[0059] ② Preparation of target cells: KATO-Ⅲ gastric cancer cells (Hpa + , HLA-A2 + ), U2OS osteosarcoma cells (Hpa + , HLA-A2 + ), SW480 colon cancer cells (Hpa + , HLA-A2 + ) was prepared for routine culture and cryopreservation in our laboratory.

[0060] ③ Detection of CTL activity: according to different effector / target cell ratios, use 51 CTL activity detected by Cr release method

[0061] The specific killing efficiency of effector cells was calculated according to the following formula:

[0062]

[0063] Killing effect of effector cells on target cells Figure 25 .

example 3

[0064] Example 3 Study on Heparanase Specificity of Anti-tumor Heparanase MAP4 Vaccine of the Present Invention

[0065] Using standard 4h 51 Cr release method

[0066] ① Effector cell preparation: Same as above

[0067] ② Target cell preparation: MCF-7 breast cancer cells (Hpa - , HLA-A2 + ), MCF-7 breast cancer cells transfected with heparanase gene (MCF-7 / Hpa) were prepared by this research institute. HepG2 liver cancer cells (Hpa + ,HLA-A2.1 ?? ), HepG2 liver cancer cells transfected with HLA-A2.1 gene (HepG2 / HLA-A2.1) (Hpa + , HLA-A2 + ).

[0068] ③ use 51 The Cr release method detects the killing effect of effector cells on heparanase-negative target cells, and then transfects the heparanase-negative target cells with the full-length gene sequence of heparanase, and then detects the killing effect of effector cells on target cells. use 51 The killing effect of effector cells on HLA-A2.1 negative and heparanase positive target cells was detected by Cr release m...

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Abstract

The invention relates to epi-position MAP vaccine which can induce effective antitumor immunity effect in the tumor associated antigen heparanase protein sequence. The vaccine overcomes the defects of single-peptide vaccine that the molecular weight is small and the antigenicity is weak, can induce strong heparanase specific cell toxicity T lymphocyte, and generates killing effect on positive tumor cells of heparanase of different tissues and organs, so that the multi-peptide vaccine can be prepared and used for curing middle or late-period tumors.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, including obtaining heparanase multi-antigen peptide molecules combined with human HLA-A2.1 molecules, as well as DNA and RNA encoding these polypeptides, because these polypeptides combine with human HLA-A2.1 After that, it can induce specific CTL response, effectively kill heparanase-positive tumor cells, and achieve the purpose of treating advanced tumors. Background technique [0002] With the development of immunology, we realized that the recognition of target antigen by CD8+ T lymphocytes is not the full-length sequence of the antigen, but a polypeptide consisting of 8-11 amino acids in the antigen, which is recognized by CD8+ T lymphocytes The amino acid sequence of is called CTL epitope. [0003] Heparanase is a tumor metastasis-related gene successfully cloned by four different laboratories in 1999. The β-D-glucosidase in the expression product can specifically recogniz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K38/47A61P35/00
Inventor 杨仕明汤旭东
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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