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RGA method for detecting biological activity of anti-CTLA-4 monoclonal antibody and application of RGA method

A CTLA-4, monoclonal antibody technology, applied in the field of biomedicine, can solve the problem of sensitivity to changes in the structure of anti-CTLA-4 monoclonal antibody drugs, and achieve the effects of short experimental period, simple operation, and high precision

Active Publication Date: 2021-06-22
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is still a lack of cell-based biological activity assay methods to detect the biological activity of anti-CTLA-4 monoclonal antibodies. The present invention has developed and verified a reporter gene assay (RGA), by constructing two cell lines to determine The biological activities of the CTLA-4 monoclonal antibody are the target cells stably expressing the anti-CD3 single-chain antibody fragment (scFv), called Raji-CD3scFv cell line, and the stable expression of the CTLA-4 gene and the expression driven by the NFAT response element The effector cells of the luciferase reporter gene, called Jurkat-CTLA-4-NFAT-luc cell line, the method showed good specificity, accuracy, precision and robustness, meanwhile, the method against CTLA-4 The structural changes of monoclonal antibody drugs are sensitive, not only for batch quality release and stability testing, but also for the development and characterization of new anti-CTLA-4 monoclonal antibodies

Method used

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  • RGA method for detecting biological activity of anti-CTLA-4 monoclonal antibody and application of RGA method
  • RGA method for detecting biological activity of anti-CTLA-4 monoclonal antibody and application of RGA method
  • RGA method for detecting biological activity of anti-CTLA-4 monoclonal antibody and application of RGA method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Construction of Raji-CD3scFv cell line and Jurkat-CTLA-4-NFAT-luc cell line

[0061] 1. Construction of Raji cell line stably expressing CD3scFv

[0062] Using NEON TM Raji cells were transfected with CD3scFv plasmid using an electroporation transfection system (Invitrogen). 48h after transfection, Hygromycin B was added for pressure selection. After the cell density and viability were restored, five 96-well plates were plated at a rate of 0.5 cells / well. When the confluence of the cell clones in the wells reaches over 30%, a cell line stably expressing CD3scFv is obtained by flow cytometric screening, and is named Raji-CD3scFv cells.

[0063] 2. Construction of Jurkat cell line stably expressing CTLA-4 gene and luciferase reporter gene expressed by NFAT response element

[0064] (1) Jurkat cells were stably transfected with luciferase reporter gene expressed by NFAT response element

[0065] Using NEON TM The electroporation transfection system (Invitro...

Embodiment 2

[0072] Example 2 Establishment of the RGA method for detecting the biological activity of anti-CTLA-4 monoclonal antibody

[0073] 1. Experimental method

[0074] (1) Sample preparation: Anti-CTLA-4 monoclonal antibody was diluted to 200 μg / mL with diluent, and then serially diluted according to the dilution ratio of 1:4, and 10 concentration gradients were set up, with 2-3 replicate wells for each concentration, 50 μL / Wells were added to a 96-well white plate.

[0075] (2) Preparation of Jurkat-CTLA-4-NFAT-luc effector cell suspension: collect effector cells in good growth state, centrifuge to discard supernatant, resuspend cells with diluent and count, adjust cell density to 6×10 6 pc / mL, spare.

[0076] (3) Preparation of Raji-CD3scFv target cell suspension: collect the target cells in good growth state, centrifuge to discard the supernatant, resuspend the cells with diluent and count them, and adjust the cell density to 6×10 5 pc / mL, spare.

[0077] (4) Cell plating: ...

Embodiment 3R

[0084] The optimization of embodiment 3RGA detection method

[0085] 1. Optimization of antibody dose-effect range

[0086] Use 200 μg / mL as the starting point for dilution of anti-CTLA-4 monoclonal antibody to conduct serial dilutions. The dilution ratios of the serial dilutions are set to 1:3, 1:4 and 1:5 respectively, and 10 concentration points are set. According to the measured chemiluminescence A four-parameter curve fitted to the values ​​determined the range of action of anti-CTLA-4 mAbs.

[0087] Experimental method: Carry out experiment respectively according to the method described in embodiment 2, compare the dose-response curve of different dilution schemes at last, according to whether four parameter curves comprise upper and lower platform and points distributed on upper and lower platform and linear part Whether it is uniform to select the optimal dilution ratio.

[0088] Experimental results: see the experimental results Figure 4 , the results show that wh...

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Abstract

The invention discloses an RGA method for detecting the biological activity of an anti-CTLA-4 monoclonal antibody and application of the RGA method. The method comprises the following steps: constructing target cells capable of stably expressing CD3scFv and effector cells capable of stably expressing CTLA-4 genes and reporter genes, mixing the target cells and the effector cells according to a certain proportion, adding an anti-CTLA-4 monoclonal antibody drug sample and a reference substance to activate a signal channel, and fitting a four-parameter curve according to the measured reporter gene signal value to determine the biological activity of the antibody. The quantitative detection method which is accurate, sensitive, rapid, high in specificity and high in durability is established for biological activity detection of the anti-CTLA-4 monoclonal antibody, and the method has important significance in quality control and clinical application of the anti-CTLA-4 monoclonal antibody.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a method for detecting biological activity, in particular to an RGA method for detecting the biological activity of an anti-CTLA-4 monoclonal antibody and its application. Background technique [0002] Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), also known as CD152, is located in band 3, region 3, long arm of chromosome 2 (2q33), and encodes 233 amino acids. A member of the globulin-related receptor family. CTLA-4 is expressed on the surface of activated T cells. Its high expression in T cells can significantly inhibit the activity of T cells, thereby weakening the ability of T cells to kill cancer cells. It is an important negative regulator of T cell immune response. At the same time, CTLA-4 4 is also the first immune checkpoint discovered as a therapeutic potential in tumor immunotherapy. The activation of T cells depends on two signals at the same time: one is an...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577C12Q1/66C12N5/10C12N15/13C12N15/12C12N15/65
CPCG01N33/6872G01N33/577C12Q1/66C12N15/65C07K16/2809C07K14/70521G01N2333/70521
Inventor 王兰刘春雨于传飞杨雅岚崔永霏段茂芹俞小娟徐苗王军志
Owner NAT INST FOR FOOD & DRUG CONTROL
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