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. 2021 Jan-Dec;13(1):1914359.
doi: 10.1080/19420862.2021.1914359.

Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1

Weimin Chen  1 , Madhu Pandey  1 , Hong Sun  1 , Andrea Rolong  1 , Mingyan Cao  2 , Dengfeng Liu  2 , Jihong Wang  2 , Lingmin Zeng  3 , Alan Hunter  4 , Shihua Lin  1
Affiliations

Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1

Weimin Chen et al. MAbs. 2021 Jan-Dec.

Abstract

T-cell-mediated immunotherapy has generated much excitement after the success of therapeutic biologics targeting immune checkpoint molecules. Bispecific antibodies (BsAbs) that recognize two antigen targets are a fast-growing class of biologics offering promising clinical benefits for cancer immunotherapy. Due to the complexity of the molecule structure and the potential mechanism of action (MOA) that involves more than one signaling pathway, it is critical to develop appropriate bioassays for measuring potency and characterizing the biological properties of BsAbs. Here, we present a dual target, cell-based reporter bioassay for a BsAb that binds human CTLA-4 and PD-1 and targets two subsequent signaling pathways that negatively regulate T-cell activation. This reporter bioassay is capable of measuring the potency of both antigen target arms in one assay, which would not be achievable using two single target bioassays. This dual target reporter bioassay demonstrates good performance characteristics suitable for lot release, stability testing, critical quality attribute assessment, and biological properties characterization of the CTLA-4/PD-1 BsAb. Furthermore, this assay can capture the synergistic effect of anti-CTLA-4 and anti-PD-1 activity of the BsAb. Compared to single target assays, this dual target bioassay could better reflect the potential MOA of BsAbs and could be used for evaluation of other bispecific biologics, as well as antibody combination therapies.

Keywords: bioassay; bispecific antibody; mechanism of action; potency; qualification.

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Figures

Figure 1.
Figure 1.
Anti-CTLA-4 activity and anti-PD-1 activity of CTLA-4/PD-1 bispecific antibody
Figure 2.
Figure 2.
Design and development of dual target reporter bioassay for CTLA-4 and PD-1 blockage
Figure 3.
Figure 3.
Effect of cell number and assay incubation time on dual target reporter bioassay
Figure 4.
Figure 4.
Factorial design of experiments for assay condition optimization
Figure 5.
Figure 5.
Linearity, specificity, and stability indicating potential of CTLA-4/PD-1 dual target reporter bioassay
Figure 6.
Figure 6.
Comparison of monospecific antibodies, intact BsAb and modified BsAb in CTLA-4/PD-1 dual target reporter bioassay
Figure 7.
Figure 7.
LC-MS analysis and comparison of PD-1/CTLA4 BsAb and mis-pair samples in CTLA-4/PD-1 dual target reporter bioassay
See this image and copyright information in PMC

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