Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work. FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5-20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a relevant characteristic to measure for a number of therapeutic monoclonal antibodies (mAbs) under development. ADCC is a mechanism by which antibody-opsonized, infected, or cancerous cells are destroyed by FcγRIII (CD16)-expressing effector cells. Here we describe three methods that can be used to quantify the ADCC activity of mAbs by measuring distinct aspects of the ADCC mechanism.

Antibody dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MoA) for many monoclonal antibody (mAb) therapeutics. As such, quantitative measurement of ADCC activity is key to drug development. Traditional cell lysis based ADCC assays using PBMCs or NK cell lines can be challenging to develop and implement for routine testing. To provide an alternative to the cell lysis based ADCC assay, a non-cell based measure of ADCC activity was developed to determine the ADCC activity of an anti-CD20 mAb, which measures the ability of the mAb to bind to CD20 antigen and FcγRIIIa, simultaneously. The bridging of CD20 and FcγRIIIa is an essential interaction for the initiation of ADCC activity. This ADCC bridging method is simple, offers the ease of use of a standard ELISA, and shows reproducible dose–response curves in the concentration range of 50–1000 ng/mL. With interassay variability of 7–10% and recovery of 89–115%, the assay demonstrates acceptable precision and accuracy. The assay is able to detect degradative changes in anti-CD20 mAb samples subjected to light and acid exposure, suggesting that it is suitable for use as a stability-indicating method. The assay is also sensitive to mAb fucose levels. A linear relationship between the ADCC bridging assay and the cell lysis ADCC assay was demonstrated, strongly suggesting that the ADCC bridging assay can be used as a surrogate measure of ADCC.