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A rapid method for identifying cross-reactive activity of high-affinity TCR antigens

A cross-reactive, high-affinity technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., to avoid false positives, simplify the operation process, and demonstrate the effect of improving kurtosis

Active Publication Date: 2022-02-18
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these technologies are all TCR or pMHC for yeast display, and the pMHC or TCR in the other direction exists in the form of tetrameric proteins for interaction screening, and there are no reports on combining with yeast mating technology to study protein interactions

Method used

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  • A rapid method for identifying cross-reactive activity of high-affinity TCR antigens
  • A rapid method for identifying cross-reactive activity of high-affinity TCR antigens
  • A rapid method for identifying cross-reactive activity of high-affinity TCR antigens

Examples

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Embodiment 1

[0067] This example mainly introduces a method for rapidly identifying the cross-reactivity of high-affinity TCR antigens.

[0068] as attached figure 1 As shown, a method for rapidly identifying high-affinity TCR antigen cross-reactivity, comprising the following steps:

[0069] S1. Adjust the TCR and pMHC sequences to constitutive promoters to induce expression, integrate and express the elements into the yeast genome, and display the TCR and pMHC proteins on the surface of yeast with different mating types;

[0070] S2. Yeast mating experiments using different mating-type yeasts integrating TCR and pMHC;

[0071] S3, diploid screening and next-generation sequencing.

[0072] Further, the step of S1 includes:

[0073] Firstly, construct the TCR sequence to be studied into scTCR form, and obtain the peptide mutation library of pMHC;

[0074] Transform the pMHC library to construct the required receiving vector ysynalpha_DEST, and obtain the vector of ysyna-TCR and ysynalp...

Embodiment 2

[0107] This embodiment is carried out on the basis of the above-mentioned embodiment 1, and the similarities with the above-mentioned embodiment 1 will not be repeated.

[0108] This example mainly introduces a specific implementation method of a method for rapidly identifying high-affinity TCR antigen cross-reactivity, including the following steps:

[0109] S1. Adjust the TCR and pMHC sequences to constitutive promoters to induce expression, integrate and express the elements into the yeast genome, and display the TCR and pMHC proteins on the surface of yeast with different mating types;

[0110] S2. Yeast mating experiments using different mating-type yeasts integrating TCR and pMHC;

[0111] S3, diploid screening and next-generation sequencing.

[0112] Further, the step of S1 includes:

[0113] Firstly, construct the TCR sequence to be studied into scTCR form, and obtain the peptide mutation library of pMHC;

[0114] Transform the pMHC library to construct the required...

Embodiment 3

[0149] This example mainly introduces the element design for the integrated expression of TCR and pMHC.

[0150] This embodiment is carried out on the basis of the above-mentioned embodiment 1, and the similarities with the above-mentioned embodiment 1 will not be repeated.

[0151] In the present study, the yeast surface display of TCR and pMHC utilized the α-lectin-target gene surface display system. α-lectin is composed of core subunit Aga1 and binding subunit Aga2, which are connected by disulfide bonds, and the C-terminus of core subunit Aga1 is covalently bound to cell wall glucan. TCR and pMHC are fused to the C-terminal or N-terminal of the Aga2 protein in yeast a-lectin, the expression of the fusion protein is induced by the GAL1 promoter, which is a galactose-inducible system, and the yeast grows in glucose medium When the transcription of the GAL1 promoter is completely inhibited, the Aga2p fusion gene product can be induced to produce the Aga2p fusion gene product...

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Abstract

The present invention relates to a method for rapidly identifying high-affinity TCR antigen cross-reactivity, adjusting TCR and pMHC sequences to constitutive promoters to induce expression, and integrating and expressing the elements into the yeast genome, including the following steps: TCR and pMHC Protein display on the surface of yeast of different mating types; Yeast mating experiments using yeasts of different mating types integrating TCR and pMHC; Diploid screening and next-generation sequencing. The present invention can provide a method for quickly identifying the cross-reactive activity of high-affinity TCR antigens, without the need for pure protein, and only needs to synthesize DNA sequences, using the Golden-gate cloning method, a more complete antigen mutation library can be obtained within one week, which is convenient and quick; The yeast mating system breaks through the limitation of screening at the cell level, and the operation steps are simplified. The antigen sequence interacting with a specific TCR can be obtained in a short time, and the experimental results are reliable.

Description

[0001] technology domain [0002] The invention relates to the field of biotechnology, in particular to a method for quickly identifying the cross-reactivity of high-affinity TCR antigens. Background technique [0003] TCR is a molecule on the surface of T cells that specifically recognizes antigens and mediates immune responses. In the adaptive immune system, TCR specifically recognizes the antigen molecule polypeptide (pMHC) presented by histocompatibility complex molecules, thereby activating T cells and producing immune effects. Tumor cell immunotherapy-TCR-T therapy is a treatment method that uses T cells that specifically bind to the target tumor antigen to kill malignant cells. The high-affinity TCR evolved in vitro can improve the anti-tumor ability of transformed T cells, but it also recognizes non-target antigens due to the problem of antigen cross-reactivity, causing serious side effects to patients. [0004] Currently, one of the methods for studying the cross-re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12N1/19C12N15/12
CPCC12Q1/6869C07K14/7051C07K14/70539
Inventor 蓝勋王莉惠
Owner TSINGHUA UNIV
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