Application Note


glycosylation, and assayed using either the ADCC Reporter Bioassay or a lytic LDH release ADCC bioassay in which PBMCs were used as effector cells. Target cells were SK-BR-3 in both assay formats. For both assays, biological activity reflects down- stream effects of effector cell FcRIIIa crosslinking by antibody bound to target cells. Biological activity was determined and expressed relative to fully N-glycosy- lated trastuzumab, and plotted against per- cent parent N-glycosylated trastuzumab in the blend.


Figure 2: Correlation of relative activity in ADCC bioassay with percent parent fully N-glycosylated trastuzumab in blended mixtures of parent and deglycosylated antibody


Jurkat cells that express both the FcRIIIa (V158) receptor and nuclear factor of acti- vated T-cells (NFAT) response element upstream of a luciferase reporter gene. The cells are provided in frozen, thaw-and-use format. Cross-linking of mAbs bound to target cells at the Fab region and to the effector cells at the Fc region results in activation of the NFAT pathway via the ADCC mechanism of action, just as occurs in primary NK effector cells in classic ADCC. The ADCC Reporter Bioassay can be performed optimally in a single day (6hr induction) and the readout is simple detection of luminescence from the effec- tor cells by addition of Bio-Glo™ luciferase assay reagent.


The frozen, thaw-and-use formatted cells and optimised protocol result in a bioassay with low variability and high repeatability. There is no need for PBMC/NK cell collec- tion from donors and there are few manip- ulations because the assay is homogeneous. A schematic of the bioassay principle is illustrated in Figure 1.


Results


Data from the ADCC Reporter Bioassay correlates well to that from a classic LDH release ADCC assay, as seen in Figure 2. In this study, trastuzumab was N-deglycosy- lated (PNGase F), blended with the fully N- glycosylated parent preparation to create test samples representing different % N-


Data shown in Figure 3, for which two different target cell types were used, demon- strates that the ADCC Reporter Bioassay generates equivalent results when per- formed in 96-well and 384-well formats.


Conclusions


The ADCC Reporter Bioassay is a suitable alternative to classic and cumbersome PBMC-based ADCC assays. Importantly, the assay reflects ADCC mechanism of action and gives equivalent rank order of biological activity of antibodies as classic ADCC assay. Since it is much easier to per- form, more precise and scalable to 384- well format, it enables expanded use of ADCC bioassay in earlier stages of biolog- ics drug discovery and potentially in QC lot release.


Figure 3: Comparison of 96-well and 384-well format using WIL2-S Target Cells (Panel A) or Raji Target Cells (Panel B). The standard protocol uses a 96-well plate. For 384-well plate experiments, one-fifth of each component was used


Drug Discovery World Spring 2013


References 1 Medicines in Development: Biologics, 2013 Report. PhRMA, 2013. 2 Guideline on similar biological medicinal products containing monoclonal antibodies; European Medicines Agency, Committee for Medicinal Products for Human Use, EMA/CHMP/BMWP/403543/2010, Draft, 2010. 3 Scientific Considerations in Demonstrating Biosimilarity to a Reference Product; US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), Draft Guidance, Feb 2012. 4 Surowy, T et al. Low variability ADCC Bioassay GEN 32(7), 2012.


41


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80