Development of reporter gene assays to determine the bioactivity of biopharmaceuticals

Wang, Lan; Yu, Chuanfei; Wang, Junzhi

doi:10.1016/j.biotechadv.2019.107466

Cited by 22 publications

(15 citation statements)

References 97 publications

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“…To determine the biological activities of biopharmaceuticals, biochemical assays, cell-based and animal-based bioassays are often applied. Bioactivity determination methods for biotherapeutics should be developed according to the following principles: MOA-based, QC suitable (small variability, simple operation and transferability) and compliance with good manufacturing practices validation [ 26 ]. Animal-based bioassays are rarely used in the QC due to their high variability, high cost, labor intensiveness and the global trend of the application of 3R principles (replacement, reduction and refinement) in the use of animals.…”

Section: Discussionmentioning

confidence: 99%

“…However, as the binding only involves the initial step of a biological event and is often far away from the end point of a biological response, it cannot completely represent the MOA of biopharmaceuticals. Many cell-based biological assays have been developed and widely used for biological activity determination [ 26 ].…”

Section: Discussionmentioning

confidence: 99%

“…6 and Table 2 ). The FDA has approved some biopharmaceuticals whose bioactivity determinations, as one of critical quality attributes, are dependent on reporter-gene assays [ 52–54 ], indicating their wide acceptance both in industry and regulatory authority [ 26 ].…”

Section: Discussionmentioning

confidence: 99%

“…Ag–Ab association-based potency assays, including Biacore [ 22 ], ELISA [ 23 , 24 ] and flow cytometry (FCM) [ 25 ], were generally and successfully applied for the binding potency analysis. Cell-based function assays were also required along with the product development [ 26 ]. Although cytotoxicity assays, using peripheral blood mononuclear cells (PBMC) or T cells or cytokine-induced killer (CIK) cells as the effector cells [ 27–31 ], are directly measuring the biological activities of a bsAb in killing the tumor cells, these cell-based cytotoxicity assays are hardly validated for evaluation of biological activity of bsAb products for the quality control (QC) due to the unavailability and variability of the effector cells.…”

Section: Introductionmentioning

confidence: 99%

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Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

Xiong

Luo

Zhou

et al. 2021

Antibody Therapeutics

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Background A T cell-redirecting bispecific antibody consisting of a tumor-binding unit and a T cell-binding unit is a large group of antibody-based biologics against death-causing cancer diseases. The anti-CD38 × anti-CD3 bispecific antibody (Y150) is potential for treating multiple myeloma (MM). When developing a cell-based reporter gene bioassay to assess the activities of Y150, it was found that the expression of CD38 on the human T lymphocyte cells (Jurkat) caused the nonspecific activation which interfered with the specific T cells activation of mediated by the Y150 and CD38(+) tumor cells. Methods Here we first knocked-out the CD38 expression on Jurkat T cell line by CRISPR-Cas9 technology, then developed a stable monoclonal CD38(−) Jurkat T cell line with an NFAT-RE driving luciferase expressing system. Further based on the CD38(−) Jurkat cell, we developed a reporter gene method to assess the bioactivity of the anti-CD38 × anti-CD3 bsAb. Results Knocking out CD38 expression abolished the nonspecific self-activation of the Jurkat cells. The selected stable monoclonal CD38(−) Jurkat T cell line assured the robustness of the report genes assay for the anti-CD38 × anti-CD3 bsAb. The relative potencies of the Y150 measured by the developed reporter gene assay were correlated with those by the flow-cytometry-based cell cytotoxicity assay and by the ELISA-based binding assay. Conclusions The developed reporter gene assay was Mechanism of Action (MOA)-reflective for the bioactivity of anti-CD38 × anti-CD3 antibody, and suitable for the quality control for the bsAb product.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

Xiong

Luo

Zhou

et al. 2021

Antibody Therapeutics

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“…The luciferase (and hence the promoter activity) can then be quantified by the measurement of the luminescence produced when the enzyme substrate is added. In this way, the transcriptional activity of the gene of interest (i.e., its expression) can be measured in response to the effects of different modulators of the relevant signaling pathways [8]. The luciferase reaction can also be used in combination with constitutively active promoters, to investigate cytotoxicity or transfection efficiency [9,10].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids

Kenda

Vegelj

Herlah

et al. 2021

IJMS

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Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

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A reporter gene assay for measuring the bioactivity of anti‐LAG‐3 therapeutic antibodies

Wang

et al. 2020

Luminescence

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Although enormous success has been achieved with anti‐PD‐1/PD‐L1 and anti‐CTLA‐4 monoclonal antibodies (mAbs), their unsatisfactory response rate in cancer patients has been driving the research and development of novel immune checkpoint inhibitors (ICIs). Anti‐LAG‐3 mAbs, as one of the most promising candidates, are now being tested for various human cancers at different stages of clinical trials. Here, we describe the development and validation of a reporter gene assay (RGA) to measure the bioactivity of anti‐LAG‐3 mAbs. We established the bioassay based on parental Raji cells and a Jurkat cell line stably transfected with human LAG‐3 gene and luciferase reporter elements controlled by nuclear factor of activated T cell (NFAT) from the IL‐2 promoter. After optimization of key parameters, the established RGA showed excellent precision, specificity, accuracy, and stability. The mechanism of action (MOA) relatedness and the excellent assay performance make the RGA suitable for the characterization, lot release, and stability test of anti‐LAG‐3 mAbs.

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Development of reporter gene assays to determine the bioactivity of biopharmaceuticals

Cited by 22 publications

References 97 publications

Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids

A reporter gene assay for measuring the bioactivity of anti‐LAG‐3 therapeutic antibodies

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