Characterization of a single reporter-gene potency assay for T-cell-dependent bispecific molecules

Lee, Ho‐Young; Register, Ames C.; Shim, Jeongsup; Contreras, Edward; Wu, Qiang; Jiang, Guoying

doi:10.1080/19420862.2019.1640548

Cited by 9 publications

(11 citation statements)

References 45 publications

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“…Simultaneous ligation of target and effector cells induces T-cell activation, followed by the killing of target cells. Although therapeutically effective, their multiple MoA, including simultaneous target and effector-cell engagement, T-cell activation and target-cell killing, shows challenges for the development and selection of a cell-based potency bioassay [ 38 ]. A single potency assay that measures all key aspects of the MoA is desirable.…”

Section: Discussionmentioning

confidence: 99%

“…FCM-based cell-killing assay with double fluorescent labeling [ 45 , 46 ] or standard chromium release assays [ 47 , 48 ] were quantified for the death of tumor cell more accurately. However, because of their high assay variability and longtime consume of the procedure, which makes them difficult to sustain over a product’s lifetime from development through commercialization, cell-killing assays are not suitable for the QC purpose [ 38 ].…”

Section: Discussionmentioning

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%

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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“…We selected the engineered Jurkat cells rather than NK cells, because Jurkat cells share the same ability to activate the appropriate pathways for cell activation after the crosslinking of FcγRIIIa as that of NK cells 55,56 and Jurkat cells are much more simple to handle than NK cell line. The ADCC reporter assay using Jurkat/NFAT-luc/FcγRIIIa is more suitable for release control, batch-to batch consistency and stability tests for mAbs with ADCC activity where accuracy and precision are important, and engineered Jurkat cells are commonly employed in the surrogate ADCC measurement of mAbs 41,49 and cytotoxicity measurement of CD3 based bispecific antibodies (BiTEs) 57 in industry.…”

Section: Discussionmentioning

confidence: 99%

Development of an antibody-dependent cellular cytotoxicity reporter assay for measuring anti-Middle East Respiratory Syndrome antibody bioactivity

Cao

Wang

et al. 2020

Sci Rep

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Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.

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“…To date, TCRL-Fab candidates had to be converted into the final TCB format to allow functional screening. Even though the characteristics in TCB-killing assays can be anticipated from TCB activation assays, i.e., reporter Jurkat cells that are co-incubated with target cells in the presence of the TCB-candidates in question, 25 these assays still require the labor-intense production and purification of TCB molecules.…”

Section: Introductionmentioning

confidence: 99%

CAR-J cells for antibody discovery and lead optimization of TCR-like immunoglobulins

Jöst

Darowski

Challier

et al. 2020

mAbs

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T-cell bispecific antibodies (TCBs) are a novel class of engineered immunoglobulins that unite monovalent binding to the T-cell receptor (TCR) CD3e chain and bivalent binding to tumor-associated antigens in order to recruit and activate T-cells for tumor cell killing. In vivo, T-cell activation is usually initiated via the interaction of the TCR with the peptide-HLA complex formed by the human leukocyte antigen (HLA) and peptides derived from intracellular proteins. TCR-like antibodies (TCRLs) that recognize pHLA-epitopes extend the target space of TCBs to peptides derived from intracellular proteins, such as those overexpressed during oncogenesis or created via mutations found in cancer. One challenge during lead identification of TCRL-TCBs is to identify TCRLs that specifically, and ideally exclusively, recognize the desired pHLA, but not unrelated pHLAs. In order to identify TCRLs suitable for TCRL-TCBs, large numbers of TCRLs have to be tested in the TCB format. Here, we propose a novel approach using chimeric antigen receptors (CARs) to facilitate the identification of highly selective TCRLs. In this new so-called TCRL-CAR-J approach, TCRL-candidates are transduced as CARs into Jurkat reporter-cells, and subsequently assessed for their specificity profile. This work demonstrates that the CAR-J reporter-cell assay can be applied to predict the profile of TCRL-TCBs without the need to produce each candidate in the final TCB format. It is therefore useful in streamlining the identification of TCRL-TCBs.

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Characterization of a single reporter-gene potency assay for T-cell-dependent bispecific molecules

Cited by 9 publications

References 45 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

Development of an antibody-dependent cellular cytotoxicity reporter assay for measuring anti-Middle East Respiratory Syndrome antibody bioactivity

CAR-J cells for antibody discovery and lead optimization of TCR-like immunoglobulins

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