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Method for rapidly determining biological activity of IL-2 protein drug and anti-CD25 antibody drug

A biological activity, IL-2 technology, applied in the field of rapid determination of the biological activity of IL-2 protein drugs and anti-CD25 antibody drugs, can solve the problem of not reflecting downstream signaling pathways, increasing economic burden, and inability to assess the applicability of stable indicators and other problems to achieve stable and reliable test results, avoid the possibility of cell contamination, and shorten the experimental period.

Active Publication Date: 2021-04-30
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods used to detect the biological activity of basiliximab, daclizumab and related biosimilars include competitive ELISA and flow cytometry. When the biological activity of the anti-CD25 monoclonal antibody is detected by flow cytometry, the downstream signaling pathway cannot be reflected, and thus the applicability of the stable indicator for the release test cannot be evaluated; when the biological activity of the anti-CD25 monoclonal antibody is detected by flow cytometry, relevant equipment and a large amount of consumables are required. Businesses increase the financial burden

Method used

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  • Method for rapidly determining biological activity of IL-2 protein drug and anti-CD25 antibody drug
  • Method for rapidly determining biological activity of IL-2 protein drug and anti-CD25 antibody drug
  • Method for rapidly determining biological activity of IL-2 protein drug and anti-CD25 antibody drug

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 Construction and screening of monoclonal cell lines stably expressing STAT5-luciferase 1. Experimental materials

[0073] C8166 cells were derived from ATCC; pLV-STAT5-PGK-Zencin was purchased from Jiman Bioengineering Co., Ltd.; IL-2 was purchased from biosystemsACRO; Basilix (anti-CD25 monoclonal antibody drug) was reserved for the monoclonal antibody laboratory (Novartis) ; Bright-Glo luciferase kit was purchased from Promega.

[0074] 2. Lentiviral transduction

[0075] The C8166 cells were transduced by the lentiviral packaging method, and 50 μL of the constructed lentiviral plasmid was added to a 96-well plate, and 50 μL of a density of 1×10 6 C8166 cells per mL, add 50 μL of 10% FBS+1640 medium and 50 μL of the density of 1×10 to another well 6 C8166 cells / mL were used as blank control, centrifuged at 1000 rpm for 30 minutes, taken out and incubated in an incubator for 30 minutes. Add 100 μL of lentiviral plasmid and cell mixture to a 24-well plat...

Embodiment 2

[0082] Comparison of different effector cells in embodiment 2

[0083] 1. Experimental materials

[0084] C8166 cells were derived from ATCC; pLV-STAT5-PGK-Zencin was purchased from Jiman Bioengineering Co., Ltd.; IL-2 was purchased from biosystems ACRO; ); Bright-Glo luciferase kit was purchased from Promega.

[0085] 2. Lentiviral transduction

[0086] HDLM-2 and Mo7e cells were transduced by the lentiviral packaging method, and 50 μL of the constructed lentiviral plasmids were added to the 96-well plate, and 50 μL of the density of 1×10 was added to the same well. 6 HDLM-2 and Mo7e cells / mL were added to the other two wells with 50 μL of 10% FBS+1640 medium and 50 μL of the density of 1×10 6 Each / mL HDLM-2 and Mo7e cells were used as blank control, centrifuged at 1000rpm for 30 minutes, taken out and incubated in the incubator for 30 minutes. Add 100 μL of lentiviral plasmid and cell mixture to a 24-well plate, add 2 mL of 1640 fresh medium containing 10% FBS, and incub...

Embodiment 3

[0093] Verification of embodiment 3 detection method

[0094] 1. Methodological comparison with traditional IL-2 protein drug bioassay methods

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Abstract

The invention discloses a method for rapidly determining the biological activity of an IL-2 protein drug and an anti-CD25 antibody drug. The method comprises the following steps: constructing C8166 effector cells transfected by lentivirus (pLV-STAT5-Luc-PGK-Zencin), screening to obtain monoclonal cell strains for stably expressing STAT5 reporter genes, and stimulating and activating the expression of the reporter genes by using the IL-2 protein drug, and blocking a signal path stimulated and activated by IL2- by using the anti-CD25 antibody drug, and fitting a four-parameter curve according to the measured signal value to measure the biological activity of the IL-2 protein drug and the anti-CD25 antibody drug. The detection method can evaluate the related indication of the drug to the downstream of the cell signal, does not need any human primary tissue-derived cells or other components, and has the advantages of stable color development, short experimental period, simple operation and low cost.

Description

technical field [0001] The invention belongs to the field of biological activity detection of biological medicines, and in particular relates to a method for rapidly measuring the biological activities of IL-2 protein medicines and anti-CD25 antibody medicines. Background technique [0002] As an important biotechnology product, therapeutic monoclonal antibody (referred to as monoclonal antibody) has the advantages of high specificity, strong targeting, and definite curative effect. Antibodies to antigenic epitopes have achieved remarkable curative effects in the treatment of tumors, autoimmune diseases, infectious diseases and transplant rejection. In 2018, the sales of monoclonal antibody drugs accounted for 55.3% of the global biopharmaceutical market. In addition, among the top 20 drugs with global sales in 2019, monoclonal antibody drugs accounted for 9 of the 13 biopharmaceuticals. important parts of. [0003] In the face of the rapid development of antibody drugs, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/66C12N5/10C12N15/65C12N15/867G01N21/76
CPCC12N5/0694C12N15/65C12N15/86C12N2510/00C12N2740/15043C12Q1/66G01N21/763
Inventor 王兰段茂芹于传飞张峰刘春雨付志浩王文波郭莎郭潇黄璟徐苗王军志
Owner NAT INST FOR FOOD & DRUG CONTROL
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