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Tumor antigen peptide and uses thereof in diagnosis and medicines

A technology of tumor antigen peptides and antigens, which is applied in the direction of anti-tumor drugs, anti-animal/human immunoglobulins, drug combinations, etc., and can solve problems such as insufficient activity and inability to fully satisfy the field of cancer immunity

Inactive Publication Date: 2016-03-02
WUXI CHENGBO SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

International Publication No. WO2010 / 047310 Patent Document 2 discloses a PBF-derived tumor antigen peptide that binds to an HLA-A2 antigen. However, its activity as an antigen peptide for cancer diagnosis or treatment is not strong enough to fully satisfy the use in the field of cancer immunity

Method used

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  • Tumor antigen peptide and uses thereof in diagnosis and medicines
  • Tumor antigen peptide and uses thereof in diagnosis and medicines
  • Tumor antigen peptide and uses thereof in diagnosis and medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0173] Synthesis of Example 1 Peptide and Measurement of Its Binding Affinity to HLA-A0201

[0174] The peptide shown as SEQ ID NO: 1 was synthesized by the Fmoc method. The binding affinity of the peptides to HLA-A0201 was measured by the HLA class I stability assay (J Immunol, 2004, 173:1436, which is incorporated herein by reference). In this assay, a peptide derived from influenza matrix protein (RYLRDQQLLGI, SEQ ID NO:3) was used as a positive control and the murine H-2Kb binding peptide VSV8 (RGYVYQGL, SEQ ID NO:4) was used as a negative control. Assays were performed in triplicate. By staining with FITC-labeled anti-HLA-A2 monoclonal antibody BB7.2 (purchased from ATCC) and measuring the fluorescent signal by flow cytometry, and according to the following equation: %MFI increase=[(MFI has a given peptide- MFI without peptide) / (MFI without peptide)] x 100 was used to determine the percentage increase in mean fluorescence intensity (% MFI increase), and to detect the bi...

Embodiment 2

[0175] Example 2 Frequency Analysis of Antigen Peptide-Specific CTL

[0176] Lymphocytes from 5 PBF-positive patients with osteosarcoma were subjected to mixed lymphocyte peptide culture (Limited Dilution / Mixed Lymphocyte Peptide Culture; LD / MLPC) under limiting dilution conditions to induce peptide-specific CTL (CancerSci., 2008, 99:368-375, which is incorporated herein by reference).

[0177] For 2 patients: Patient No. 1 and No. 2, 50 ml of peripheral blood was collected to isolate peripheral blood mononuclear cells (PBMC). PBMCs were incubated in AIM-V medium (Invitrogen) containing 1% human serum and supplemented with 50 μg / ml of the peptide shown in SEQ ID NO: 1 at room temperature for 60 minutes. PBMCs stimulated with peptides at 2 x 10 5 cells / 200 μl / well were plated in U-bottom 96-well microtiter plates and cultured in AIM-V medium containing 10% human serum, 20 U / ml IL-2 and 10 ng / ml IL-7. Seven days after the start of culture, half of the medium in each well was ...

Embodiment 3

[0183] Example 3 Establishment of Antigen Peptide-Specific CTL

[0184] For culturing T cells, B cell lines obtained by transforming B cells from healthy individuals positive for HLA-A0201 with Epstein-Barr virus: NS-EBV-B cells, and cells derived from HLA-A0201 by transforming with Epstein-Barr virus Negative B cell line derived from B cells of patients with osteosarcoma: LCL-S2000 cells (J Orthop Sci., 2003, 8:554-559, which is incorporated herein by reference). The T cells in the wells of patient No. 4 who were positive for tetramers found in Example 2 were plated in a 96-well microplate at a single cell / well. Add 2×10 per well 4 NS-EBV-B cells and 8×10 irradiated NS-EBV-B cells stimulated with the peptide shown in SEQ ID NO:1 4 One irradiated allogeneic PBMC were cultured in 200 μl AIM-V medium containing 10% human serum, 200 U / ml IL-2 and 10 ng / ml IL-7. 14 and 21 days after the start of culture, with 1 × 10 4 NS-EBV-B cells irradiated and stimulated with the peptide s...

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Abstract

The invention discloses a tumor antigen peptide and uses thereof in diagnosis and medicines. The tumor antigen peptide combines with an HLA, is recognized by a cytotoxic T cell, and can be used as an in-vivo or in-vitro CTL induction agent, namely a cancer vaccine. The tumor antigen peptide treats or improves tumors (such as osteosarcoma, kidney cancer and other tumors). The tumor antigen peptide can also be used as a tumor marker for tumors (such as osteosarcoma, kidney cancer and other tumors).

Description

technical field [0001] The present invention relates to tumor antigen peptides. More specifically, the present invention relates to the fragment peptide of tumor antigen protein (papillomavirus binding factor, PBF) and the application of its gene in the field of cancer immunity. Background technique [0002] Cellular immunity, especially cytotoxic T cells (hereinafter referred to as CTLs) play an important role in eradicating tumor cells, virus-infected cells, and the like from a living body. CTLs recognize a complex of antigen peptides (tumor antigen peptides) on tumor cells and MHC (major histocompatibility complex) class I antigens (which are called HLA antigens in humans), and attack and kill tumor cells. [0003] After intracellular synthesis of tumor-specific proteins (ie, tumor antigen proteins), the tumor-specific proteins are degraded intracellularly by proteases to produce tumor antigen peptides. The resulting tumor antigen peptide binds to an MHC class I antigen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N15/12C12N5/0783C07K19/00C07K16/18A61K38/08A61K48/00A61K39/395A61P35/00G01N33/68C12Q1/68
Inventor 李飞
Owner WUXI CHENGBO SCI & TECH DEV
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