An Orthogonal Approach to ADCC Biosimilar Testing

19th August 2015

Category: Biosimilars

Tags: , ,

By: Debbie Allan PhD, 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 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.

Promega (LINK) have developed a genetically engineered effector cell line that expresses the FcγRIIIa receptor (V158 and F158 variants are available) on the cell surface and the NFAT (nuclear factor of activated T cells) respone element produces firefly luciferase upon activation. The assay format replicates many features of the classical ADCC assay with the antibody binding it’s specific antigen on the surface of the target cell. The effector cell preparation is then added which binds the Fc portion of the antibody. This binding interaction activates the gene transcription through the NFAT signalling pathway of the effector cell resulting in a luminescence readout which is proportional to the amount of antibody bound in the initiating event. In contrast to classical ADCC assays in which background death may be observed, this method produces a substantially lower background. The effector cells are available as continuous cultures allowing for a greater degree of consistency between different assay setups and result in methods with significantly less variation in day to day responses making direct comparison between samples tested on different days easier without fear of data being negatively affected from an individual PBMC donor.

BioOutsource have further developed the standard Reporter ADCC assay to provide methods with further reduced levels of variability in a method termed the Functional ADCC Bridging Assay (FABA). The FABA method replaces the target cell preparation with a recombinant version of the antibody’s target antigen and in doing so, methods that were previously impossible to perform for the lack of target-expressing cell line can be rapidly developed (Diagram A).

Diagram A

Diagram A

The format is also advantageous as it can easily be adapted to assess the impact of varying concentrations of antigen, a surrogate for evaluation of multiple target cell lines, differing in the expression levels of the antigen, that are typically conducted using the standard ADCC assay formats. The FABA platform has successfully be applied to Humira (adalimumab), Enbrel (etanercept), Avastin (bevacizumab) and Remicade (infliximab) (Diagram B).

Diagram B

Diagram B

Together, the standard Promega ADCC and FABA formats provide easy, reliable and reproducable data which is invaluable at an early stage of biosimilar development and during lot release activities where consistency becomes critical, but also increasingly useful as an orthogonal method to support classical ADCC during demonstration of analytical similarity.

 

 

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