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Novel human leukocyte antigen (HLA)-A2 limiting epitope polypeptide and use thereof

An HLA-A2, epitope peptide technology, applied in the fields of biology and medicine, can solve the problems of structural and functional influence, polypeptide inactivation, amino acid modification and modification methods and combinations, etc.

Inactive Publication Date: 2011-11-23
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

[0009] However, small changes in the amino acids of the polypeptide will have a huge impact on its structure and function, and even cause the polypeptide to lose its original activity (such as binding affinity), and the modification and change of amino acids in the polypeptide sequence and combinations are numerous It is not easy to screen out modified polypeptides with enhanced activity (such as immune activity) from many modifications

Method used

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  • Novel human leukocyte antigen (HLA)-A2 limiting epitope polypeptide and use thereof
  • Novel human leukocyte antigen (HLA)-A2 limiting epitope polypeptide and use thereof
  • Novel human leukocyte antigen (HLA)-A2 limiting epitope polypeptide and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Screening of HLA-A*0201 high-affinity peptides

[0060] In this example, epitope peptides with high affinity to HLA-A*0201 were screened by peptide binding experiments.

[0061] experiment procedure

[0062] First, T2 cells (a type of cells lacking antigen processing ability, purchased from ATCC: CRL-1991) were collected, washed three times with serum-free 1640 medium (purchased from Invitrogen), and the cell concentration was adjusted to 2 × 10 5 A / ml, spread in 24-well plate, 0.5ml / well. Then, add 50 μM candidate polypeptide, 2.5 μg / ml β2 microglobulin (purchased from R&D company) at 37°C, 5% CO 2 Incubate for 18h in total. After incubation, the cells were washed three times with ice PBS (pH 7.2, PBS was a buffer, self-prepared), and FITC-labeled HLA-A2-specific mAb BB7.2 (Sterotec Ltd, Oxford, UK) was added, and the ice bath was 45 After washing with PBS, the mean fluorescence intensity was detected by a flow cytometer (FACSCalibur, Beckton Dickinson...

Embodiment 2

[0073] Example 2. HLA-A2.1 / K b Induction of hPEBP4-derived HLA-A2-restricted polypeptide-specific cytotoxic T lymphocytes in transgenic mice

[0074] Preparation of effector cells

[0075] HLA-A2.1 / K was prepared according to conventional methods (refer to Inaba laboratory method, [Inaba, K. et al., J Exp Med. 1992; 176: 1693-1702]) b Bone marrow-derived dendritic cells (DC) from transgenic mice. Collect DCs cultured (refer to Inaba's laboratory method, ibid.) to day 7, and adjust the cell concentration to 1 × 10 6 cells / ml, add epitope peptides (each epitope peptide in Table 1 of Example 1, final concentration 10 μM / ml) and β2 microglobulin (final concentration 3 μg / ml), 37°C, 5% CO 2 Incubate in the incubator for 3h.

[0076] Collect peptide-sensitized DCs, wash three times with PBS (pH 7.2, PBS as buffer, self-prepared), and adjust the cell concentration to 1×10 6 each / 0.2ml (the amount of immunization is 0.2ml) immunized transgenic mice (HLA-A2.1 / K b Transgenic mic...

Embodiment 3

[0096] Example 3. Culture of human peripheral blood mononuclear cell-derived DCs sensitized with optimized epitope peptide 1F

[0097] Anticoagulated peripheral whole blood of HLA-A2.1+ / hPEBP4+ breast cancer patients was subjected to density gradient centrifugation (room temperature, 400 × g, 30 minutes) in lymphocyte separation medium (Ficoll-Histopaque 1.077, purchased from Sigma), and the interface cells were collected. Put it into a 50ml centrifuge tube, suspend the cells with calcium and magnesium-free PBS (pH7.2)-EDTA (2mM), and then wash the cells once by centrifugation (300×g, 10 minutes), discard the supernatant, and resuspend the cells in calcium and magnesium-free PBS. , and centrifuged again (200×g, 5 minutes) to wash the cells twice to obtain human peripheral blood mononuclear cells (PBMC).

[0098] Suspend PBMCs in complete medium (RPMI1640 with 10% fetal bovine serum) at 1×10 7 Cells / well were plated in 6-well plates at 37°C, 5% CO 2 After 2 hours of incubatio...

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Abstract

The invention provides a novel human leukocyte antigen (HLA)-A2 limiting epitope polypeptide and a use thereof, and specifically relates to an HLA-A2 limiting epitope polypeptide (1F peptide) with a sequence of FLFCQGLEV, and a use of its recombinant protein, its coding nucleotide sequences, related antigen presenting cells and its composition in tumor treatment and prevention utilizing expression of human phosphatidylethanolamine (PE)-bindingprotein 4 (hPEBP4).

Description

technical field [0001] The present invention relates to the fields of biology and medicine, and more particularly to an HLA-A2-restricted epitope polypeptide, as well as the epitope and its related recombinant proteins, coding nucleotide sequences, antigen-presenting cells, and compositions in the expression Use of human phosphatidylethanolamine binding protein (hPEBP4 protein) in tumor therapy and prevention. Background technique [0002] Human phosphatidylethanolamine (PE)-binding protein 4 (hPEBP4), its nucleotide sequence and amino acid can refer to Chinese patent CN02136556.3. It is obtained by primary culture of normal adult bone marrow cells in vitro to obtain bone marrow stromal cells (Bone marrowstromal cell, BMSC) and construct BMSC cDNA library, using the method of large-scale sequencing of cDNA library, a full-length isolated from BMSC cDNA library. The gene, belonging to the phosphatidylethanolamine binding protein (PEBP) family, was registered in the GenBank / E...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K2/00C12N5/0784C12N5/0786C12N5/0781C12N5/071A61K38/08A61K35/14A61K35/12A61P35/00A61K35/15A61K35/17A61K35/33A61K35/44
Inventor 李楠曹雪涛孙伟红
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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