Multiplexing Phenotype and Function for More Biologically Relevant Insights - 13
More Biologically Relevant Insights | CELL CYTOTOXICITY
ADCC Assays: Assays were done in either 96- or 384-well plates. Briefly, target cells were
stained with different concentrations of VL1 encoding dye (Intellicyt, Albuquerque NM)
in order to discriminate the different cell populations. A dilution series of the various antibodies (25,000 - 0.01 ng/ml) were added to the target cells. PBMCs were added at various
E:T ratios (5-40). After overnight incubation, the cells were stained with FL1/BL1 viability
dye (Intellicyt, Cat# 90342) prior to data acquisition on the Intellicyt® iQue Screener PLUS.
Figure 3: Screening of CD20 Antibody Mutants.
VL1 encoded CD20 positive cells were cultured
with increasing amounts of CD20 IgG1, CD20
IgG2 or IgG1 mutants that inhibit either Fc
glycosylation or fucosylation. Published studies
show the glycosylation mutants inhibit ADCC
activity while fucosylation mutants enhances
ADCC. A) Heat maps show target cell death across
the entire screening campaign. The top half
of each plate contained no effector cells while
PBMCs (E:T 40) were added to the wells of the
bottom half of the plates. B) Effector cell death
can occur during ADCC and we observed an antiCD20, dose response loss of monocytes. Profile
maps were created showing wells that had high
ADCC activity with low monocyte death. Line
graph of hits from the profile map rank the wells
containing the highest ADCC activity.
13
| January, 2019
Data Acquisition and Analysis: Data was acquired on the Intellicyt® iQue Screener
PLUS using 7 second sips (~12 μl) per well. Four parameter curve fits, EC50 values,
and heat maps were generated using the integrated ForeCyt software (v6.2). Heat
maps, profile maps and hit ranking from the multi-plate antibody mutation screen
was produced using the Panorama feature in ForeCyt® Software. n
https://www.intellicyt.com/
Multiplexing Phenotype and Function for More Biologically Relevant Insights
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Multiplexing Phenotype and Function for More Biologically Relevant Insights - Contents
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