A robust reporter assay for the determination of the bioactivity of IL-4R-targeted therapeutic antibodies

Yu, Xintong; Yu, Chuanfei; Wang, Kaiqin; Liu, Chunyu; Wang, Lan; Wang, Junzhi

doi:10.1016/j.jpba.2021.114033

Cited by 2 publications

(1 citation statement)

References 26 publications

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“…The traditional ADCC assay that uses peripheral blood mononuclear cells or NK cells isolated from human blood as effector cells is not suitable for routine quantitative measurement of ADCC activity due to poor reproducibility and low sensitivity. Our laboratory has established several reporter gene‐based bioactivity determination methods, 10–13 including the ADCC assay for anti‐MERS antibodies, 14 and these methods have obvious advantages compared with traditional methods. For RABV, to date, only a few studies have demonstrated that anti‐RABV antibodies and lymphocytes from human peripheral blood could mediate the lysis of infected cells by ADCC 15,16 …”

Section: Introductionmentioning

confidence: 99%

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti‐rabies virus glycoprotein antibodies

Wang

Cui

et al. 2022

Microbiology and Immunology

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Rabies is a viral disease that is nearly 100% fatal once clinical signs and symptoms develop. Post-exposure prophylaxis can efficiently prevent rabies, and antibody (Ab) induction by vaccination or passive immunization of human rabies immunoglobulin (HRIG) or monoclonal antibodies (mAbs) play an integral role in prevention against rabies. In addition to their capacity to neutralize viruses, antibodies exert their antiviral effects by antibody-dependent cellular cytotoxicity (ADCC), which plays an important role in antiviral immunity and clearance of viral infections. For antibodies against rabies virus (RABV), evaluation of ADCC activity was neglected. Here, we developed a robust cell-based reporter gene assay (RGA) for the determination of the ADCC activity of anti-RABV antibodies using CVS-N2c-293 cells, which stably express the glycoprotein (G) of RABV strain CVS-N2c as target cells, and Jurkat cells, which stably express FcγRⅢa and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT-luc/FcγRⅢa cells). The experimental parameters were carefully optimized, and the established ADCC assay was systematically validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 guideline. We also evaluated the ADCC activity of anti-RABV antibodies, including mAbs, HRIG, and vaccine induced antisera, and found that all test antibodies exhibited ADCC activity with varied strengths. The established RGA provides a novel method for evaluating the ADCC of anti-RABV antibodies.

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Section: Introductionmentioning

confidence: 99%

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti‐rabies virus glycoprotein antibodies

Wang

Cui

et al. 2022

Microbiology and Immunology

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An In Vitro RNA Editing-Based Reporter Assay for Transcriptional Activity of Therapeutic Gene in Gene Therapy Products

Yu,

Zhou,

Wang

et al. 2024

Molecules

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The expression of therapeutic genes is critical for the efficacy of gene therapy products. However, existing methods such as immunological analysis at the protein level or reverse-transcription PCR at the RNA level are unable to accurately quantify the expression activity of the target gene. Herein, an in vitro RNA editing-based reporter assay was developed to detect specific mRNA. The designed sensor RNA could specifically identify the target mRNA, and the reporter gene was activated in a dose-dependent manner because of RNA editing mediated by endogenous adenosine deaminases acting on RNA. Of note, all sensors that targeted different regions, including the gene of interest, tag sequence, and 3′ untranslated region, showed a dose-dependent response pattern. The sensor reporter assay, which was used for quantifying the transcriptional activity of recombinant adeno-associated virus-based gene therapy products, revealed excellent performance in terms of assay specificity, precision (inter-assay relative standard deviation < 15%), accuracy (90–115% recovery), and linearity (R2 > 0.99). The reporter assay could also be employed for other gene therapy vectors, including mRNA and recombinant lentivirus. Thus, a robust and reliable platform was developed for assessing the transcriptional activity of therapeutic genes, thereby offering a powerful tool for the quality control of gene therapy products.

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A robust reporter assay for the determination of the bioactivity of IL-4R-targeted therapeutic antibodies

Cited by 2 publications

References 26 publications

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti‐rabies virus glycoprotein antibodies

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti‐rabies virus glycoprotein antibodies

An In Vitro RNA Editing-Based Reporter Assay for Transcriptional Activity of Therapeutic Gene in Gene Therapy Products

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