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Dominant sequence of delta 1 chain complementary determining region (CDR) 3 in gamma delta T lymphocytes, and T cell receptor (TCR) transfected cells and application thereof

A technology of complementarity-determining domains and lymphocytes, which is applied in the field of preparation of drugs for the treatment of malignant tumors, and can solve problems such as inability to represent

Active Publication Date: 2012-07-04
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the recognition characteristics of another type of epithelial-derived Vδ1T cells and tumors, the sequence characteristics of the CDR3 region, the recognized tumor antigens, and the molecular mechanism of tumor antigen recognition have not been reported yet.
Vδ1 and Vδ2 T cells are very different in sequence, structure, distribution, recognition of antigen ligands, functions, etc. Therefore, the CDR3 sequence of γδ2 TCR structure cannot represent other CDR3 sequences of other types of TCR and the recognition mechanism of tumors

Method used

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  • Dominant sequence of delta 1 chain complementary determining region (CDR) 3 in gamma delta T lymphocytes, and T cell receptor (TCR) transfected cells and application thereof
  • Dominant sequence of delta 1 chain complementary determining region (CDR) 3 in gamma delta T lymphocytes, and T cell receptor (TCR) transfected cells and application thereof
  • Dominant sequence of delta 1 chain complementary determining region (CDR) 3 in gamma delta T lymphocytes, and T cell receptor (TCR) transfected cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Obtaining the dominant sequence GTM of the complementarity-determining domain 3 (CDR3δ1) of the δ1 chain in tumor-infiltrating γδT lymphocytes (tumor-infiltrating γδT cells, γδTIL) derived from human gastric cancer tissue

[0076] 1. Culture and identification of γδTIL cells in epithelial-derived tumors

[0077] 1. Culture of γδ TIL cells

[0078] A 24-well plate was coated with anti-pan-TCRγδ mAb (purchased from Beckman Coulter), 5 μg / well, and incubated at 37° C. for 2 h. Solid tumor specimens (epithelial origin) such as gastric cancer and kidney cancer collected by aseptic surgery were soaked in RPMI-1640 medium (containing penicillin 200 μg / mL, streptomycin 100 μg / mL, gentamicin 100 μg / mL) for 2 time, 10 min / time; remove necrotic tissue, surrounding blood vessels, and normal tissue; cut the washed tissue into small pieces, wash twice with RPMI-1640 medium (containing penicillin 200 μg / mL, streptomycin 100 μg / mL), 10 min / time times, and then resuspended ...

Embodiment 2

[0113] Example 2. Antitumor function and phenotype analysis of γδ1TIL

[0114] 1. The killing function of γδTIL on tumor cells

[0115] Cytotox 96 non-radioactive cytotoxicity assay reagents kit (Promega, USA) was used to perform the killing test according to the instructions of the kit. W2-γδTIL (derived from gastric cancer tissue) and S2-γδTIL (derived from kidney cancer tissue) were used as effector cells to detect their effects on different tumor cells-gastric adenocarcinoma cell BGC-823 (derived from human gastric cancer tissue) and renal cell carcinoma cell G401 (derived from human kidney cancer) and colon cancer cell HT-29 (derived from human colon cancer) killing efficiency. Target cells 1×10 4 Add to 96-well plate per well, so that the effect-to-target ratios are 2.5:1, 5:1, and 10:1, respectively. The result is as Figure 4 As shown (Figure A is the tumor cell killing rate of W2-γδTIL derived from gastric cancer, and Figure B is the tumor cell killing rate of S2-...

Embodiment 3

[0119] Example 3. Predominant Sequence of Complementarity Determining Domain 3 of δ1 Chain in Human Gastric Cancer Tissue-Derived Tumor-Infiltrating γδT Lymphocytes - Binding of CDR3δ1(GTM) Peptide to Tumor Cells, Tumor Tissue, and Vδ1 Ligand Protein MICA

[0120] 1. Synthesis and biotinylation of GTM peptide

[0121] The dominant sequence-CDR3δ1(GTM) peptide of the complementarity-determining domain 3 of the δ1 chain in human gastric cancer tissue-derived tumor-infiltrating γδT lymphocytes shown in SEQ ID NO: 1 in the sequence table was synthesized by chemical synthesis, and a peptide was directly labeled at the end of the synthesis. Biotin molecule, compared to peptide CH3 (WPHNWWPHFKVK) as a control.

[0122] 2. Detection of binding properties of GTM peptides to different tumor cells by flow cytometry

[0123] BGC-823, G401, lung adenocarcinoma cells GLC-82 (derived from human lung adenocarcinoma), HT-29, and human ovarian cancer cells SKOV3 (derived from human ovarian can...

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Abstract

The invention discloses a dominant sequence GTM of a delta 1 chain complementary determining region (CDR) 3 in human gastric cancer tissue-derived tumor-infiltrating gamma delta T lymphocytes, a T cell receptor (TCR) containing the sequence, and TCR transfected cells. The amino acid residue sequence of the GTM is shown as SEQ ID NO.1 in a sequence table. Experiments prove that GTM peptide can be specifically combined with tumor cells, tumor tissue and V delta 1 T cell ligand MICA protein; and a J.RT3-T3.5 cell platform for expressing the TCR containing the dominant sequence GTM of the delta 1 chain CDR3 in the human gastric cancer tissue-derived tumor-infiltrating gamma delta T lymphocytes on a J.RT3-T3.5 surface is established by a lentivirus expression system, gamma delta 1 tumor-infiltrating lymphocytes (TIL) and a tumor identifying and killing mechanism thereof are simulated in the cell level, the killing activity of the transfected cells TCR gamma 4 delta 1 on the tumor cells is obviously increased, and the killing effect is TCR-dependent. The invention plays an important role in the development of medicines for treating malignant tumors and the adoptive treatment of malignant tumors, and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, polypeptides, coding genes and applications thereof, in particular to GTM, the dominant sequence of complementarity-determining domain 3 of the δ1 chain in human gastric cancer tissue-derived tumor-infiltrating γδT lymphocytes, GTM and its TCR receptor transfected cells and in Application in the preparation of medicines for treating malignant tumors. Background technique [0002] The body's immune system has produced four major antigen recognition mechanisms during the evolution process, which are divided into pattern recognition by macrophages and dendritic cells (DC), specific recognition by αβT cells and B cells, and NK cells targeting the internal self. Abnormally altered recognition and recognition of antigens by γδT cells (Morita CTHH, HHMariuzza RAHH, HHBrenner MBHH. Antigen recognition by human gamma delta T cells: pattern recognition by the adaptive immune system. HHSpringer Semin Immunopath...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K7/06C07K14/725C12N15/12C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A61K38/08A61K38/10A61K38/17A61P35/00A61P35/02
Inventor 何维姜燕崔莲仙
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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