Frozen human T cells collected from a single health donor were activated with CD3/28 beads (ThermoFisher), expanded in vitro, thawed and electroporated with mRNA transcripts encoding c-Met BBZ CAR, mesothelin BBZ CAR and CD19 BBZ CAR as described [11 (link)]. We have previously demonstrated that the cytotoxic activity of mRNA c-Met-CAR T cells was specific against c-Met expressing cell lines such as M30, a human tumor cell line derived from mesothelioma [17 (link)] and cytotoxic activity was not observed against NCI-H522, a lung cancer cell line which lacks c-Met expression [16 (link)]. To confirm the cytolytic activity on c-Met expressing breast cancer cell lines, BT20 (ATCC HTB-19) and TB129 [14 (link)], tumor cells were loaded with 51Cr and incubated with mRNA electroporated CAR T cells (effector cells or E) at various E:T ratios in V-bottom plates for 4 hours. 51Cr released as a result of cell lysis was quantified as described [18 (link)]. All in vitro cell killing assays were performed in duplicates and repeated in at least three independent experiments.
Protocol detail
Cytolytic Activity of CAR T Cells
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Assays
Breast cancer cell lines
C met
Cell
Cell line
Cytotoxic t cells
Donor
Frozen
Human
Lung cancer
Mesothelin
Mesothelioma
Mrna
T cells
Tumor
Tumor cell line
Corresponding Organization : University of Pennsylvania
Other organizations : Moffitt Cancer Center, Fred Hutch Cancer Center, The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Effector to target (E:T) ratio
- Cytolytic activity (measured by 51Cr release)
- Cell lines used (BT20 and TB129 breast cancer cell lines)
- Cell lines loaded with 51Cr
- Incubation time (4 hours)
- Plate format (V-bottom plates)
- Assay performed in duplicates
- Assay repeated in at least three independent experiments
- Positive control: c-Met expressing cell lines (M30 mesothelioma cell line)
- Negative control: c-Met non-expressing cell line (NCI-H522 lung cancer cell line)
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