eBook: Cell and Gene Therapy - 9

Considerable research efforts have been invested
into developing new CAR structures to increase
the scope of targeted cancer types and raise their
antitumor efficacy. One of the bottlenecks in the
process of CAR-T development is evaluating the
biofunction of CAR-T cells. This in vitro process typically
involves a series of labor-intensive co-culture
experiments and immunoassays, where reproducibility
remains a challenge during the validation of
new CAR-T cells due to donor-to-donor variation
and other possible factors. In addition to reproducibility
being an issue, the validation assays themselves
can be problematic.
For example, the use of the radioactive 51
Cr release
assay has a major drawback; data can only be acquired
at a single time point. Moreover, reagent
half-life, protective measures, and waste disposal
are critical factors because of the assay's intrinsic
radioactivity. Nonradioactive assays for CAR-T
functional evaluation are available, although these
require a time-consuming labeling step and suffer
from intra- and inter-assay variability stemming
from inconsistent dye uptake and spontaneous
dye leakage over the course of the assay.
A method of studying CAR-T effector function
that eliminates the concerns of 51
CR release and
dye loading assays is the bioluminescence (BLI)
reporter assay. In BLI reporter assays, target cells
that constitutively express luciferase are co-cultured
with the candidate effector cells and cytotoxicity
is monitored via loss of BLI signal.4
In addition
to their ease of use, the luciferase-expressing
target cells in BLI assays can improve interexperimental
reproducibility.
To provide target cells for immuno-oncology researchers
adopting BLI assays, we generated CAR-T
Target Luciferase Reporter Cells that can be used to
examine the function of CAR-T cells. These reporter
To generate the CAR-T Target Luciferase Reporter
Cells, antibiotic selection and single cell sorting
were performed to isolate stable clones with high
luciferase expression via the introduction of a Lenti-LUC2
luciferase reporter into the parental cell
lines. The target antigen and luciferase were then
verified to have expression stability by comparing
the low-passage and the high-passage reporter
cells. Once stable clones were selected and the expression
of antigen and luciferase was verified, the
reporter cell lines were characterized and authenticated
using tried-and-true methods such as short
tandem repeat (STR) profiling, mycoplasma detection,
and cell growth rate and morphology assays.
The performance of the CAR-T Target Luciferase Reporter
Cells was verified in T cell co-culture experiments.
Commercially available CAR-T cells targeting
CD19, CD20, and HER2 were employed in this
study, with which empty vector-transduced T cells
from the same donor were paired as controls. The
cytotoxicity of the CAR-T cells against target tumor
cells was measured using a luciferase assay, a commercially
available potency assay, and a bright field
and fluorescence live cell imaging assay. Our results
demonstrate that the luciferase reporter syscells
naturally express high levels of clinically relevant
CAR-T target antigens on the cell surface.
ATCC's CAR-T Target Luciferase Reporter Cells
were derived from a variety of highly malignant
liquid and solid cancer types, namely B cell lymphoma,
Burkitt's lymphoma, Non-Hodgkin's B cell
lymphoma, and ductal breast carcinoma. These
novel target cells were generated from parental
tumor cells that have high endogenous expression
of target antigens such as CD19, CD20, and
HER2. Stable luciferase-expressing clones were
engineered to display high signal-to-noise ratios,
aiding in data interpretation.
9
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