The Impact of Human Serum on ADCC Assays
3rd July 2018
By: Laura McAleer, Technical Services Scientist,
Antibody-Dependent Cellular Cytotoxicity (ADCC) is a mechanism adopted by the immune system to remove infected or cancerous cells. Antibodies bind a specific antigen on the target cell surface, which in turn binds the FcγRIIIa receptor on effector cells (e.g Natural Killer (NK) Cells) via the Fc region, resulting in the lysis of the target cell. Although in vitro ADCC assays reflect the mode of action, these methods are beset with practical challenges and require significant expertise to design and successfully perform in a routine manner. Complications arise through the variability associated with the use of primary peripheral blood mononuclear cells (PBMC) or NK preparations with both donor-specific and genotype-dependent responses frequently observed. Different ADCC formats can be applied that impart differing levels of sensitivity of the assay with some methodologies resulting in a high background due to nonspecific lysis of target cells.
Whilst these classical ADCC assays are critical in the development of a biosimilar, there is certainly a need for a more reliable, easy to use tool to support the demonstration of analytical similarity. There are numerous considerations for the evaluation of ADCC activity of therapeutic antibodies which is primarily based upon the mechanism of action of the antibody. However, it may be necessary to demonstrate secondary ADCC mechanism of action or even the absence of ADCC activity. At Sartorius Stedim BioOoutsource we offer a highly robust and standardized approach for evaluating ADCC activity or lack thereof in your therapeutic antibody.
Figure 1. An overview of ADCC activity
The presence of human serum in the ADCC assay should, in theory, create a more physiological assay by imitating the whole blood environment. However, the presence of high levels of IgG1 in the serum results in competition for the Fc receptors on the NK cells, thus lowering potential ADCC activity. The complement factors in the serum also contribute to the diminished ADCC response for human IgG1 therapeutic antibodies.
Figure 2. An overview of ADCC activity in the presence of human serum
We performed a study to compare the performance of several anti-TNF-α molecules, including a biosimilar molecule in our total PBMC ADCC assay in the presence and absence of varying concentrations of human serum. It was expected that the observed ADCC activity may be reduced or be eliminated when the methodology was conducted in a relevant biological matrix due to general serum components, such as IgG molecules. The graphs below (Figure 3) demonstrate the ADCC activity of infliximab innovator (blue, reference standard) against the infliximab biosimilar (Red, Test material). As expected the relative potency of the infliximab biosimilar was reduced compared to the innovator, and returned a relative potency of around 42%. This is well described in our Remsima® case study – The Importance of ADCC in Assessing Clinically Relevant Differences. However, in the presence of heat inactivated human serum at concentrations as low as 2.5%, the ADCC activity of both molecules were significantly diminished, and no differentiation in relative potency could be determined.
Figure 3. The effect of varying concentration of human serum on the relative potency of innovator and biosimilar anti-TNF-α monoclonal antibodies
As expected our investigation showed that assessment of test materials under physiologically relevant concentrations of serum, significantly reduced the ADCC activity of anti-TNF-α molecules to the point where no activity was observed.
Therefore, it is not possible to determine the difference in ADCC activity between anti-TNFα molecules in the presence of human serum.
We offer off-the-shelf and custom assays. Contact us to discuss your testing requirements