An in vitro FcRn- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans.

A cell-based assay employing Madin-Darby canine kidney cells stably expressing human neonatal Fc receptor (FcRn) heavy chain and ß2-microglobulin genes was developed to measure transcytosis of monoclonal antibodies (mAbs) under conditions relevant to the FcRn-mediated immunoglobulin G (IgG) salvage pathway. The FcRn-dependent transcytosis assay is modeled to reflect combined effects of nonspecific interactions between mAbs and cells, cellular uptake via pinocytosis, pH-dependent interactions with FcRn, and dynamics of intracellular trafficking and sorting mechanisms. Evaluation of 53 mAbs, including 30 marketed mAb drugs, revealed a notable correlation between the transcytosis readouts and clearance in humans. FcRn was required to promote efficient transcytosis of mAbs and contributed directly to the observed correlation. Furthermore, the transcytosis assay correctly predicted rank order of clearance of glycosylation and Fv charge variants of Fc-containing proteins. These results strongly support the utility of this assay as a cost-effective and animal-sparing screening tool for evaluation of mAb-based drug candidates during lead selection, optimization, and process development for desired pharmacokinetic properties.

Development of a label-free FcRn-mediated transcytosis assay for in vitro characterization of FcRn interactions with therapeutic antibodies and Fc-fusion proteins.

The neonatal Fc receptor (FcRn) binds to the Fc domain of IgG in a pH-dependent manner, guides the intracellular movement of the bound antibodies and protects them from lysosomal degradation. Proper characterization of Fc-FcRn interactions is fundamental to successful design, development, and production of Fc-containing therapeutic proteins because of the potential impact of such interactions on their in vivo pharmacokinetic behaviors. Here, we describe the development and characterization of a cell-based, label-free FcRn-mediated transcytosis assay that provides a functional readout to reflect the totality of Fc-FcRn interactions, including pH-dependent association and dissociation, as well as the intracellular trafficking of Fc-containing molecules in complex with FcRn. Our study demonstrates that this transcytosis assay can be used to evaluate FcRn binding of therapeutic antibodies and Fc-fusion proteins, including wild-type and engineered Fc variants with varying FcRn binding affinities, as well as oxidized and aggregated antibody samples. These results support the utility of an FcRn-dependent transcytosis assay for evaluation of both Fc-FcRn interactions and the structural integrity of Fc-containing therapeutic proteins pertinent to their pharmacokinetic behavior in vivo.

Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays.

In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., 51Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness.

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice.

The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.