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Fluorescein marked protein tetramer as well as preparation method and application thereof

A fluorescein-labeled, tetramer technology, applied in the field of fluorescein-labeled protein tetramer and its preparation, can solve the problems of poor detection sensitivity of CAR-T target protein, inability to detect whether CAR can be recognized, etc. The effect of improved detection sensitivity

Active Publication Date: 2020-05-05
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For routine detection of CAR expression, protein L protein and anti-Fab antibody can be used, but protein L only recognizes κ light chain type CAR, and anti-Fab antibody only recognizes the Fab structure of CAR, even if the expression of CAR is detected, it cannot Detect whether the CAR on the surface of the T cell membrane can recognize its target antigen
Only using the corresponding target antigen of CAR to detect the expression of transfected CAR is the most effective, but the detection sensitivity of monomeric CAR-T target protein is poor, especially in the PK test of CAR-T therapy

Method used

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  • Fluorescein marked protein tetramer as well as preparation method and application thereof
  • Fluorescein marked protein tetramer as well as preparation method and application thereof
  • Fluorescein marked protein tetramer as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1. Preparation and Application of Immune Checkpoint Protein Tetramer (PDCD1 / PD1 C-Fc-avi-PE Tetramer)

[0039] 1. Preparation of Biotinylated Human PD-1 / PDCD1Avi Tag Protein

[0040] 1.1. Reagents and instruments

[0041] TransT1 competent cells (full gold, CD501-01); TransT1 competent cells (ThermoFisher, C505003); tryptone (Sigma, T7293-1KG), yeast extract (Sigma, Y1625-1KG); agar (Sigma, A7002 -1KG); Max plasma Kit (promega, A2492); Bacterial shaker (Uno, HN211B); Bacterial incubator (Tianjin Tester, DH3600B) Nanodrop 2000; Expi293F cells (Invitrogen, Cat#R790-07); cells Expression medium, prepared by the laboratory; Polyethyleneimine, PEI (Polysciences, Cat#23966). Cell shaker incubator (Uno, HNY-2102)

[0042] 1.2. Solution preparation

[0043] 1.2.1.LB medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1000ml, autoclave, cool to room temperature, add Amp, the final concentration is 100ug / ml, set aside.

[0044] 1.2.2.LB solid me...

Embodiment 2

[0099] Example 2. Preparation and application of CAR-T target protein tetramer (CD19 Fc-Avi-PE tetramer)

[0100] 1. Preparation of Biotinylated Human CD19 FcAvi Tag Protein

[0101] 1.1. Reagents and instruments

[0102] TransT1 competent cells (full gold, CD501-01); TransT1 competent cells (ThermoFisher, C505003); tryptone (Sigma, T7293-1KG), yeast extract (Sigma, Y1625-1KG); agar (Sigma, A7002 -1KG); Max plasma Kit (promega, A2492); Bacterial shaker (Uno, HN211B); Bacterial incubator (Tianjin Tester, DH3600B) Nanodrop 2000; Expi293F cells (Invitrogen, Cat#R790-07); cells Expression medium, prepared by the laboratory; Polyethyleneimine, PEI (Polysciences, Cat#23966). Cell shaker incubator (Uno, HNY-2102).

[0103] 1.2. Solution preparation

[0104] 1.2.1.LB medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1000ml, autoclave, cool to room temperature, add Amp, the final concentration is 100ug / ml, set aside.

[0105]1.2.2.LB solid medium: tryptone ...

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Abstract

The invention provides a fluorescein marked protein tetramer as well as a preparation method and application thereof. The fluorescein marked protein tetramer is formed by combining fluorescein markedstreptavidin and a biotin marked target protein. The biotin marked target protein is in recombinant expression in an HEK293 cell, one Avi Tag is coexpressed at an N-end or a C-end, and furthermore, under the action of a BirA enzyme, a lysine residue group of Avi Tag is connected with biotin. The biotin marked target protein and the fluorescein marked streptavidin are mixed and reacted to obtain the protein tetramer in direct fluorescence marking. The protein tetramer in direct fluorescence marking, which is provided by the invention, can be applied to detection on positive rates of neutralizing antibodies or CAR-T (chimeric antigen recetor-T) cells of target proteins of stream screening immune checkpoints, and as streptavidin and biotin both play a role of cascade amplification, the detection sensitivity of the protein tetramer is greatly improved when being compared with that of fluorescein marked proteins prepared by using other methods.

Description

technical field [0001] The present invention relates to a fluorescein-labeled protein tetramer and its preparation method and application, in particular to a fluorescein-labeled protein tetramer and its preparation method, and its use in screening immune checkpoint proteins Neutralizing antibodies or the application of detecting the positive rate of CAR-T cells. Background technique [0002] In recent years, cancer immunotherapy represented by Immune Checkpoint inhibitors and CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) therapy has become the first-line treatment for many tumors. The surprising effects of PD-1 inhibitors and CAR-T cell therapy in the treatment of multiple cancers have also established a new position for immunotherapy in tumor treatment, especially the emerging CAR-T cell therapy, known as New hope for a cancer cure. [0003] The currently approved immune checkpoint inhibitors are mainly neutralizing antibodies against immune checkpoint proteins, ...

Claims

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Application Information

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IPC IPC(8): C07K14/705G01N33/68G01N33/569C12N15/85G01N33/58
CPCC07K14/70521C12N15/85G01N33/56966G01N33/582G01N33/68G01N2333/70521
Inventor 石晓娟苗景赟闫爽刘丛张晓慧
Owner ACROBIOSYSTEMS INC
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