Development of a Bioluminescent Cell-Based Assay to ... - Promega

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Fc effector biological activity correlates well with antibody N-glycosylation Relative activity in reporter ADCC Rituximab and Trastuzumab: Good correlation between blended % mixes of fully N-glycosylated and deglycosylated preparations and their relative biological activity in ADCC Reporter Bioassay 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 -0.100 Confidential and Proprietary. Not for Further Disclosure. y = 0.0127x - 0.0314 R² = 0.966 Trastuzumab 0 10 20 30 40 50 60 Percent N-glycosylated sample Relative activity in reporter ADCC 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 y = 0.0125x - 0.0095 R² = 0.9926 Rituximab 0 10 20 30 40 50 60 Percent N-glycosylated sample Small differences in Fc effector activity in ADCC MOA pathway are easily distinguished in the ADCC reporter bioassay 24
Planned ADCC Reporter Bioassay kit configurations Core Kit • Engineered Jurkat Effector cells (NFAT-RE-luc2 / FcgRIIIa); frozen, thaw-and-use • Bio-Glo Luciferase Assay System • RPMI Medium • Low IgG Serum Use with different Abs and target cells Complete Kits (2 types) • Above PLUS: • Target WIL2-S cells; frozen, thaw-and-use or Target Raji cells; frozen thaw-and-use • Control Ab (CD20) Use as assay control, or when WIL2-S or Raji target cells are suitable or target is CD20 Confidential and Proprietary. Not for Further Disclosure. Engineered Jurkat cells Medium Target WIL2-S or Raji Cells + Glo Detection Reagent Serum Control Antibody Core Kit Complete Kits
Page 1 and 2: Development of a Bioluminescent Cel
Page 3 and 4: Signaling pathways activated in eff
Page 5 and 6: Protocol 1. Reference or test antib
Page 7 and 8: FcgRIIIa antibody blocking of ADCC
Page 9 and 10: Bioassay development: Optimizing Ef
Page 11 and 12: Bioassay development: Evaluating cl
Page 13 and 14: Target cells can also be frozen, th
Page 15 and 16: ADCC Reporter Bioassay using frozen
Page 17 and 18: Precision, accuracy and linearity o
Page 19 and 20: ADCC Reporter Bioassay is stability
Page 21 and 22: Additional target cell option: Raji
Page 23: iological activity of therapeutic a
Page 27: For more information Confidential a

Development of a Bioluminescent Cell-Based Assay to ... - Promega

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