TCR for identifying human cytomegalovirus pp65 antigen

A human cytomegalovirus, pp65 technology, applied in the direction of receptors/cell surface antigens/cell surface determinants, animal cells, antiviral agents, etc., can solve serious or even fatal diseases and other problems

Active Publication Date: 2019-10-22
深圳市因诺转化医学研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in individuals with suppressed immune systems, such as AIDS patients or patients who have received organ transplants, reactivation of CMV can lead to severe or even fatal disease (Ulrike Gerdemann, et al. Mol Ther, 2013, 21(11): 2113 -2121)

Method used

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  • TCR for identifying human cytomegalovirus pp65 antigen
  • TCR for identifying human cytomegalovirus pp65 antigen
  • TCR for identifying human cytomegalovirus pp65 antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: HLA-A2-CMV-pp65 495-503 Cloning and sequencing of specific T cells

[0049] Peripheral blood mononuclear cells (PBMC) derived from healthy volunteers with HLA-A2 genotype were stimulated by chemically synthesized short peptide NLVPMVATV (Sangon Bioengineering (Shanghai) Co., Ltd.). After two rounds of peptide stimulation, ELISA was used to detect the reactivity of polyclonal T cells to T2 cells loaded with NLVPMVATV short peptide (target cells) or T2 cells loaded with non-target short peptides (control cells) (the detection index was the cytokine IFN-γ release). Positive polyclonal T cells (target cell OD 450 - control cell OD 450 >1.0) for limiting dilution cloning (1 cell / well, 3 cells / well or 5 cells / well), and after 14 days, ELISA was used to detect the reactivity of T cells and target cells in each well after limiting dilution. Pick positive monoclonal T cells for expansion. After 14 days of amplification, mouse anti-human CD8 antibody (BD Bioscienc...

Embodiment 2

[0072] Example 2: Preparation of HLA-A2-CMV-pp65 495-503 Specific TCR Gene Modified T Cells

[0073] The target TCR sequence cloned above was inserted into the retroviral vector pMSGV1 (addgene) to construct the pMSGV1-A6 TCR vector capable of expressing the TCR. TCR sequence elements such as figure 2 shown. Retrovirus was prepared by transfecting 293GP packaging cells pMSGV1-A6 TCR and pVSV-G plasmid vector, and the virus supernatant was used to transduce T cells. The specific operations were as follows:

[0074] Cell transfection: 293GP cells were seeded into 6-well plates (6x 10 5 / well); on the first day, pMSGV1-A6 TCR and pVSV-G plasmids were co-transfected into 293GP cells (2 μg pMSGV1-A6 TCR and 1.4 μg pVSV-G / well). On the same day, use anti-human CD3 antibody (OKT3) to activate the PBMC of healthy people; on the third day, collect the 293GP culture fluid containing the virus supernatant, and add fresh culture fluid (DMEM culture fluid containing 10% fetal bovine s...

Embodiment 3

[0076] Example 3: HLA-A2-CMV-pp65 495-503 In vitro functional verification of specific TCR gene-modified T cells

[0077] Flow cytometry analysis: target cells are prepared as follows, and will be loaded with target CMV-pp65 495-503 Peptides (natural peptides or peptides containing single amino acid mutations) and non-target peptides EBV-LMP2A 237-245 After the T2 cells (HLA-A2 positive) and TCR-T cells were co-cultured at 37°C for 4 hours, the changes of the cytotoxic function marker CD107a and cytokine IFN-γ of T cells were detected by flow cytometry, as shown in Figure 4 Shown, HLA-A2-CMV-pp65 495-503 Specific TCR gene modified T cells can specifically recognize target cells, release cytokines and exert cytotoxic functions.

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Abstract

The invention relates to the technical field of T cell antigen receptors, in particular to TCR capable of identifying and combining with an HLA-A2-CMV-pp65495-503 antigen complex, a nucleotide sequence encoding the TCR or nucleic acid molecules of a complementary sequence of the nucleotide sequence, a carrier containing the nucleic acid molecules, cells transducing the nucleic acid molecules or carrier, a drug composition containing the TCR, the nucleic acid molecules, the carrier or the cells which are taken as active ingredients and application of the TCR, nucleic acid molecules, carrier, cells and drug composition which are used for preparing drugs for treating tumors or virus infection respectively.

Description

technical field [0001] The present invention relates to the technical field of T cell antigen receptors, in particular to a TCR capable of specifically recognizing and binding to human cytomegalovirus pp65 antigen, and a nucleic acid molecule comprising the nucleotide sequence encoding the TCR or its complementary sequence, and comprising the TCR The carrier of the nucleic acid molecule, and the cell that transduces the nucleic acid molecule or the carrier, and the pharmaceutical composition comprising the TCR, the nucleic acid molecule, the carrier or the cell as an active ingredient, and the TCR, the nucleic acid molecule, the carrier, Cells, uses of pharmaceutical compositions. Background technique [0002] T lymphocytes in the human body are an important part of the human acquired immune system. T cells can specifically recognize and kill cells infected by pathogens or undergoing malignant transformation. The recognition of infected cells or cancer cells by T cells dep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725C12N15/12C12N5/10A61K39/00A61P35/00A61P31/12
CPCC07K14/7051C12N5/0636A61K39/001102A61P35/00A61P31/12C12N2510/00
Inventor 王明军陈磊董莲花
Owner 深圳市因诺转化医学研究院
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