Assessment Of New Developments In ADCC Assays

The success of biologic therapeutics has begun to reshape today’s pharmaceutical market. The first and most successful of these antibody therapies, Rituximab (Rituxan®; Roche/Genentech), showed worldwide sales in 2009 of $5.6 billion (GEN News Highlights, 2011). This, among others including Trastuzumab (Herceptin®; F Hoffman-La Roche), have shown great promise for treatment of patients with leukemia, lymphomas, breast, and other cancer types due to their specificity and reduced side effects (Zhou, 2007). One of the mechanisms which play a central role in the response to clinical antibody therapy is antibody-dependent cell-mediated cytotoxicity (ADCC) (Wang, 2008). This involves the response of natural killer (NK) cells to bind to specific antibody-coated target cells, such as CD20 and HER2 expressing cells, to promote the death of the target cell.

With many of the existing patents covering these treatments set to expire in the next few years, the development of biologic therapeutics similar to the original drug (biosimilars) has become increasingly important. This is highlighted by the report that Spectrum Pharmaceuticals and Viropro are set to work together to develop a biosimilar to Rituximab (GEN News Highlights, 2011). As a direct result, assays that can assess the ability of a biosimilar to act in a manner similar to the original biologic have also seen increased interest. The current “gold standard” ADCC assay incorporates 51Cr. The procedure involves labeling and incubating target cells with the radioligand, assessment of the labeling procedure, and fi nally performance of the actual assay. Not only is this
time consuming, but involves the use and eventual costly disposal of radioactive material. Newer ADCC assays, however, have recently been developed. They have incorporated luminescent, timeresolved fluorescence (TRF), or homogeneous time-resolved fl uorescence (HTRF) technologies in substitution for the previously used radioligand. These assay chemistries have proven to be easier to use and more amenable to automation, while still delivering accurate results.

As no technology is completely foolproof, it is important to test lead antibodies using a number of different assays. Therefore, an overview of these technologies will be presented here in a toolbox approach. An emphasis will be placed on how each procedure can be automated in 96- or 384-well format to allow for hands-free, higher throughput processing. Experimental data using freshly isolated or cryopreserved NK cells, NK cell lines, or other relevant genetically modifi ed cell lines will be shown to prove the validity of each method.