T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit a patient's T cells to kill cancer cells. There are increasing numbers of TBDs in clinical trials, demonstrating their widely recognized therapeutic potential. Due to the fact that TDBs engage and activate T cells via an anti-CD3 (aCD3) arm, aCD3 homodimer (aCD3 HD) and high-molecular-weight species (HMWS) are product-related impurities that pose a potential safety risk by triggering off-target T-cell activation through bivalent engagement and dimerization of T-cell receptors (TCRs). To monitor and control the level of unspecific T-cell activation, we developed a sensitive and quantitative T-cell-activation assay, which can detect aCD3 HD in TDB drug product by exploiting its ability to activate T cells in the absence of target cells. This assay provides in-vivo-relevant off-target T-cell-activation readout. Furthermore, we have demonstrated that this assay can serve as a platform assay for detecting T-cell-activating impurities across a broad spectrum of aCD3 bispecific molecules. It therefore has the potential to significantly benefit many T-cell-recruiting bispecific programs.

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit patients' T cells to kill cancer cells. There are many TDBs in clinical trials, demonstrating their widely recognized therapeutic potential. However, their complex, multi-step mechanism of action (MoA), which includes bispecific antigen binding, T-cell activation, and target-cell killing, presents unique challenges for biological characterization and potency assay selection. Here, we describe the development of a single reporter-gene potency assay for a TDB (TDB1) that is MoA reflective and sensitive to binding of both antigens. Our reporter-gene assay measures T-cell activation using Jurkat cells engineered to express luciferase under the control of an NFkB response element. The potencies of select samples were measured both by this assay and by a flow-cytometry-based cell-killing assay using human lymphocytes as effector cells. Correlating the two sets of potency results clearly establishes our reporter-gene assay as MoA reflective. Furthermore, correlating potencies for the same panel of samples against binding data measured by binding assays for each individual arm demonstrates that the reporter-gene potency assay reflects dual-antigen binding and can detect changes in affinity for either arm. This work demonstrates that one reporter-gene assay can be used to measure the potency of TDB1 while capturing key aspects of its MoA, thus serving as a useful case study of selection and justification of reporter-gene potency assays for TDBs. Furthermore, our strategy of correlating reporter-gene potency, target-cell killing, and antigen binding for each individual arm serves as a useful example of a thorough, holistic approach to biological characterization for TDBs that can be applied to other bispecific molecules.

IgG antibodies are important pharmaceutical molecules that successfully treat a variety of human diseases. The neonatal Fc receptor (FcRn) interacts with IgG Fc in the CH2-CH3 domain and plays a key role in IgG antibody homeostasis and affects its pharmacokinetic properties. An in vitro FcRn binding assay could be a highly valuable complementary tool to assess IgG antibody pharmacokinetics in IgG engineering and screening during the early optimization stage. In addition, it could be useful in biological characterization studies for antibody minor variants, process optimization, and comparability study at later stages of antibody development.Here we developed a homogeneous AlphaScreen-based FcRn assay to assess the binding of FcRn to IgG antibody in vitro. The assay is found to be accurate, precise, specific, and simple: donor beads loaded with FcRn and acceptor beads loaded with IgG1 mAb1 are mixed together with sample IgG at various dilutions and incubated for 1 h before acquiring data with a fluorescence reader. This assay can run up to four samples per plate in 2 h, which is time and cost effective compared with other FcRn binding methods such as cell-based fluorescent-activated cell scan and surface plasma resonance. Our data demonstrated that this assay is suitable for assessing the FcRn binding in vitro and provides a platform approach that can be readily applied to various antibodies. (C) 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).