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HLA-A2 restriction epi polypeptide originated from post selection cancer gene hRabj and its application

A technology of HLA-A2 and epitope polypeptides, which is applied to the HLA-A2 restricted epitope polypeptides derived from the new candidate oncogene hRabJ and its application field, which can solve the problems of lack of effective prevention and treatment of anti-tumor therapeutic vaccines and other issues

Active Publication Date: 2007-04-18
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there is still a lack of therapeutic vaccines for effective prevention and treatment of anti-tumor, especially synthetic peptide vaccines with high safety. peptide vaccine

Method used

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  • HLA-A2 restriction epi polypeptide originated from post selection cancer gene hRabj and its application
  • HLA-A2 restriction epi polypeptide originated from post selection cancer gene hRabj and its application
  • HLA-A2 restriction epi polypeptide originated from post selection cancer gene hRabj and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1HLA-A * 0201 Screening of high affinity peptides

[0058] In this example, the peptides with HLA-A were screened out through peptide binding experiments.* 0201 high affinity epitope peptide.

[0059] experiment procedure

[0060] First collect T2 cells (a cell lacking antigen processing ability, purchased from ATCC: CRL-1991), wash three times with serum-free 1640, adjust the cell concentration to 2×10 5 cells / ml, spread in 24-well plate, 0.5ml / well. Then mix with 50μM candidate peptide, 2.5μg / ml β2 microglobulin at 37℃, 5% CO 2 A total of 18 hours of incubation in the incubator. The incubated cells were washed three times with ice-cold PBS, FITC-labeled HLA-A2-specific mAb BB7.2 (purchased from Sterotec Ltd, Oxford, UK) was added, ice-bathed for 45 minutes, washed with PBS and detected by flow cytometry mean fluorescence intensity. A positive known peptide CEA HLA-A2 restricted epitope polypeptide CAP-1 (SEQ ID NO: 10, sequence YLSGANLNL) was used as a p...

Embodiment 2

[0068] Embodiment 2 Preparation of adenovirus (pAdRabJ) expressing RabJ

[0069] Using the plasmid containing human RabJ cDNA obtained in Example 1 of Chinese patent 01126826.3 as a template, digested with SalI-XhoI, recombined with plasmid pShuttle-CMV (Stratagene Company) according to conventional methods, and transformed into competent Escherichia coli BL21, The positive clones were picked and identified, purified and sequenced (ABI company, BigDye Terminator kit). The human RabJ-pShuttle-CMV vector with the correct sequence was linearized with PmeI and co-transformed into Escherichia coli BJ5183 (Stratagene) with the pAdeasy adenovirus backbone plasmid (Stratagene). Positive clones were identified by PacI digestion, and the products were analyzed by 0.8% agarose gel electrophoresis. It was confirmed by sequencing that the adenoviral vector containing the coding sequence of human RabJ had been recombinantly produced.

[0070] After the adenoviral vector was linearized wit...

Embodiment 3

[0071] Example 3HLA-A2.1 / K b Induction of hRabJ-derived HLA-A2-restricted polypeptide-specific cytotoxic T lymphocytes in transgenic mice

[0072] Preparation of effector cells:

[0073] Prepare HLA-A2.1 / K by conventional method b Dendritic cells (DC) derived from bone marrow of transgenic mice. Collect the DC cultured to the 7th day, and adjust the cell concentration to 1×10 6 cells / ml, add R41-49 (final concentration 10 μM / ml) and β2 microglobulin (final concentration 3 μg / ml), 37°C, 5% CO 2 Incubate for 3 h in the incubator. Collect the peptide-sensitized DCs, wash them three times with PBS, and adjust the cell concentration to 1×10 6 cells / 0.2ml to immunize mice. Each mouse was injected intraperitoneally with 1×10 6 cells / 0.2ml, a total of three immunizations, with an interval of one week. Seven days after the last immunization, the mouse spleen was removed aseptically, and the red blood cells were dissolved to make a single cell suspension. Splenocyte suspension ...

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Abstract

This invention provides a HLA-A2 restricted epi-position polypeptide come from candidate Oncogene hRabJ, and the use of this epi-position and associated recombination protein, coding nucleotide sequence, antigen presenting cell, combination matter, and specific Immune effector cell aiming directly at this epi-position is used to express hRabJ oncotherapy and prevention.

Description

technical field [0001] The present invention relates to the fields of biology and medicine, and more specifically relates to providing an HLA-A2 restricted epitope polypeptide derived from a candidate oncogene hRabJ, as well as the epitope and its related recombinant protein, coding nucleotide sequence, antigen Application of presenting cells, compositions, and specific immune effector cells targeting the epitope in the treatment and prevention of tumors expressing hRabJ. Background technique [0002] hRabJ (SEQ ID NO: 12) is a novel full-length novel gene discovered during the large-scale sequencing of dendritic cell (dendritic cell, DC) cDNA library, and registered in the GenBank / EMBL database (serial number AF178983 ). hRabJ encodes a protein of 273 amino acids (SEQ ID NO: 13), which is a Rab-like GTP hydrolytic protease (GTPase). RabJ is mainly distributed in tissues such as testis, brain and ovary, can bind and degrade GTP, may be distributed in the nucleus in cells, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N5/00A61K38/17A61K35/12A61P35/00A61K35/15
Inventor 李楠孙伟红陈涛涌曹雪涛
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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