Agilent - Breakthroughs in Immunotherapy- 2019 - 37

Breakthroughs in Immunotherapy

is generated from T cells alone, which can be
subtracted as background (Figure 2, black line).
Daudi cell growth is undisturbed by the addition of
T cells and anti-CD19 antibody as a control (Figure
2, green line). However, a rapid decrease in Cell
Index is observed with the addition of the CD19
BiTE in the presence of T cells, indicating that the
Daudi target cells are being killed (Figure 2, orange
line). This data demonstrates the capability of the
CD19 BiTE to enhance T-cell-mediated cytotoxicity
of B cancer cells.

Boosting cytokine and cytolytic
protein secretion
To further study the effect of CD19-BiTE on
T-cell activation and function, cytokine and cytolytic protein secretion was measured. Cells were
cultured as described in Figure 2, and supernatant
was taken at 7, 24, and 48 hours after the addition
of T cells to measure 13 human proteins known to
affect T-cell function with a bead-based multiplex
assay on the NovoCyte flow cytometer (Figure 3).
Consistent with our findings from RTCA, an
increased secretion of CTL-associated proteins
was observed. This data demonstrates that the
presence of CD19-BiTE significantly enhances the
production of cytokines and effector molecules
that mediate and sustain target cell destruction.
37

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Figure 3. CD19-BiTE enhances
the cytotoxic activity of T cells
in a bead-based multiplex
immunoassay. Daudi target
cells were seeded at 50,000
cells/well in a 96-well E-Plate
coated with anti-CD40 antibody.
Eighteen hours post Daudi
cell seeding, enriched human
effector T cells from primary
PBMCs were added at a ratio
of T:Daudi cells of 10:1. At
the same time, CD19-BiTE
(0.33 µg/mL) or an anti-CD19
antibody (0.33 µg/mL) was
added. Supernatant was
collected at 7, 24, and 48 hours
after the addition of T cells
for the bead-based multiplex
immunoassay. The relative
fold change between protein
expression of Daudi + T + CD19BiTE to Daudi + T + αCD19 was
determined for cytokines (A)
and cytolytic proteins (B).

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Agilent - Breakthroughs in Immunotherapy- 2019

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