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T cell antigen receptor, polymer complex thereof, and preparation method and application of polymer complex

A cell antigen and complex technology, applied in the direction of antibody mimics/scaffolds, animal cells, antibody medical components, etc., can solve the problem of insignificant killing effect of T cell receptors

Pending Publication Date: 2022-01-04
CHINA IMMUNOTECH BEIJING BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this application, the killing effect of T cell receptors on target cells is not obvious

Method used

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  • T cell antigen receptor, polymer complex thereof, and preparation method and application of polymer complex
  • T cell antigen receptor, polymer complex thereof, and preparation method and application of polymer complex
  • T cell antigen receptor, polymer complex thereof, and preparation method and application of polymer complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0169] Example 1: Construction and Effect Detection of CMV Antigen Epitope Tetramer

[0170] 1. Construction of CMV epitope tetramer

[0171] 1) α chain and β2m chain (whose amino acid sequence is shown in SEQ ID NO: 25) of HLA-A*1101 (whose amino acid sequence is shown in SEQ ID NO: 24 and its nucleotide sequence is shown in SEQ ID NO: 25) expressing sequence optimization NO: 26, the nucleotide sequence is shown in SEQ ID NO: 27). Wherein, the structure of the α-chain is that the extracellular region sequence of the corresponding HLA-type α-chain is linked with the Avi-tag sequence, separated by BamHI restriction sites to provide biotinylation sites. The β2m chain has the signal peptide sequence removed and two amino acids (M and A) added in front of the mature peptide sequence. The expression vector is PET28a+, and the expression strain is transetta or BL21. The concentration of IPTG was 0.5mM, and the expression was induced for 4h. Extraction of α-chain and β2m-catenin ...

Embodiment 2

[0187] Embodiment 2: Construction of pHAGE-TCR-RFP carrier

[0188] 1. Obtain β and α gene fragments of CMV pp65 epitope-specific TCR

[0189] 1) Use the HLA-A*1101-ATVQGQNLK-tetramer purchased from MBL or made by our company, and stain the peripheral blood according to the product manual, and perform flow cytometry single cell sorting on the T cells positive for the tetramer staining, cDNA was obtained by reverse transcription ( IV Reverse Transcriptase, Invitrogen). According to the multiplex PCR (Multiplex PCR) principle, the variable region fragment of the TCRβ gene was amplified by two rounds of PCR (KOD-Plus-Neo, TOYOBO).

[0190] The reverse transcription primer is: TRBC1-TCAGGCAGTATCTGGAGTCATTG (SEQ ID NO: 136)

[0191] PCR amplification primers are:

[0192] Upstream primer 1: T cell receptor β variable region--TRBV_F1 (SEQ ID NO: 56 to 95, wherein SEQ ID NO: 60, 59 can also be separately used as the variable region fragment of the β gene of C44 and C45 TRBV_F1)...

Embodiment 3

[0211] Example 3: Detection of membrane expression and affinity of TCR by pMHC tetramer staining

[0212] 1. Construction of endogenous TCR knockout JurkatT cell line

[0213] Based on the sequence characteristics of TCR in Jurkat cells, guide sequences were designed in the constant regions of the α chain and β chain (TRA_oligo1-CACCGTCTCTCAGCTGGTACACGGC (SEQ ID NO: 20), TRA_oligo2-AAACGCCGTGTACCAGCTGAGAGAC (SEQ ID NO: 21), TRB_oligo1-CACCGGGCTCCAAACACAGCGACCTC (SEQ ID NO :22), TRB_oligo2-AAACGAGGTCGCTGTGTTTGAGCCC (SEQ ID NO: 23)).

[0214] The guide sequences of the synthesized α-chain and β-chain were respectively constructed into sgRNA-LentiCRISPR-puro and sgRNA-LentiCRISPR-BSD lentiviral vectors, and co-transfected with packaging plasmids psPAX2, pMD2.G and PEI transfection reagents in a certain ratio to 293T Cells, the cell culture supernatants of 48h and 72h were collected, and the concentrated two viruses simultaneously infected the human JurkatT cell line. 48 hours a...

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PUM

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Abstract

The invention provides a T cell antigen receptor, an immune cell for expressing the T cell antigen receptor (TCR), and a preparation method and application of the T cell antigen receptor. The TCR can be specifically activated by virus antigen peptide presenting cells, the release level of extracellular cytokines IFN gamma and IL2 and the release amount of lactic dehydrogenase are improved, and target cells are remarkably killed.

Description

technical field [0001] The present invention relates to the technical field of T cell antigen receptor capable of recognizing CMV pp65, in particular to various TCRs and their use in treating and / or preventing CMV-related diseases. Background technique [0002] T cell antigen receptor (T cell receptor, TCR) is crucial to the cellular immune function of the immune system, and TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), The combination of antigen-specific TCR and pMHC complex triggers direct physical contact and interaction between T cells and antigen-presenting cells, leading to a series of subsequent cell signal transmission and other physiological responses, so that T cells with different antigen specificities can target cells exert an immune effect. [0003] CN107827959A discloses a method for isolating antigen-specific T cells and cloning antigen-specific TCR genes in the antigen-specific T cells. The...

Claims

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Application Information

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IPC IPC(8): C07K14/725C07K16/08C07K19/00C12N15/12C12N15/13C12N5/10A61K39/00A61P35/00A61P35/02A61P31/22G01N33/68G01N33/569
CPCC07K14/7051C07K14/70539A61P35/00A61P31/22C07K16/088C12N5/0636G01N33/56994C12N2800/22A61P35/02G01N33/6893C12N2510/00C07K2319/00A61K39/4632A61K39/464838A61K2239/38A61K39/4611G01N33/574A61K39/42C07K14/005C12N2710/16134C12N2710/16122G01N2333/045A61K2039/505A61K38/00A61K35/17C07K2317/565C07K2317/622C07K2319/03A61K2039/5158A61K39/4631C12N15/86C12N15/907C12N2740/15043
Inventor 陈花刘光娜雷蕾刘芳贾乐梅黄道盛赵学强林欣
Owner CHINA IMMUNOTECH BEIJING BIOTECH CO LTD
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