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* These authors contributed equally
A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.
Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.
CAR-T cell therapy has shown great promise in hematological malignancies, evident from the 6 FDA-approved CAR-T products since 2017, as reported by the National Cancer Institute1. There are numerous CAR-T cells in clinical trials for targeting solid tumors. Engineering novel CAR targets and optimizing the CAR construct is vital to the efficacy of a CAR-T cell. Choosing the ideal CAR construct for each application is essential for accurate targeting of tumor associated antigens (TAA) while avoiding low levels of TAA expression in normal tissues2.
A CAR construct is primarily made of five compar....
NOTE: All cell culture work is done in a biosafety cabinet with a lab coat, gloves, and following standard aseptic techniques.
1. Generating CAR expressing Jurkats (CAR-J)
A range of E:T ratio between 1:8 and 8:1 for CAR-J1 was evaluated at 72 h which targeted EGFR on TNBC MDA-MB-231 cells. Jurkat cells were transduced with CAR lentivirus with polybrene to generate CAR-J cells as described in step 2. Cytotoxicity of CAR-J1 significantly increased with higher E:T ratio with no difference in killing at 1:8 ratio (Figure 1). More than 50% killing was observed at 4:1 E:T over 72 h. This E:T was used for subsequent experiments with duration reduced to 48 h for rapi.......
Here we have proposed a rapid method to efficiently evaluate the target-specific cytolytic activity induced by CAR expression in Jurkat cells. All CAR constructs have the same scFv but different hinge and transmembrane domains which have been shown to affect CAR-T cells potency13. Further evaluation of non-specific killing by these CAR-J was done by culturing them with antigen knock out (KO) cells. This demonstrates that the killing is tumor antigen specific and not due to basal activation by the .......
MDA-MB-231 were a kind gift from Dr. Shane Stecklein. The authors acknowledge funding from the University of Kansas Cancer Center to conduct this research.
....Name | Company | Catalog Number | Comments |
15 mL Conical Tube (Sterile) | Midwest Scientific | #C15B | Any similar will work |
50 mL Conical Tube (Sterile) | Thermo Scientific | 339652 | Any similar will work |
Black/Clear 96 well plate | Falcon | 353219 | Celligo has a list of compatible plates |
Celigo 4 Channel Imaging Cytomenter | Nexcelcom Bioscience | 200-BFFL-5C | Any similar will work |
Celigo Software | Nexcelcom Bioscience | Version 5.3.0.0 | Any similar will work |
Cell Culture Incubator | Thermo Scientific | HeraCell 160i | Any similar will work |
Cell Culture Treated Flasks (T75, various sizes, Sterile) | TPP | 90076 | Any similar will work |
CFSE | Tonbo | 13-0850-U500 | Any similar will work |
Cytek Muse Cell Analyzer | Cytek | 0500-3115 | Any similar will work |
DMEM | Gibco | 11995-040 | Any similar will work |
FBS | Gemini bio-products | 900-108 | Any similar will work |
Flow Cytometer | Cytek, BD, etc | Aurora, LSR II, etc | Any similar will work |
FlowJo Sortware | Becton Dickinson & Company | Version 10.7.1 | Any similar will work |
Fluorobrite DMEM | Gibco | A18967-01 | Any similar will work |
GraphPad Software | GraphPad | Version 9.3.1 (471) | Any similar will work |
Multichanel Pipette | Thermo Scientific | Finnpipette F2 | Any similar will work |
PBS | Gibco | 10010-031 | Any similar will work |
PenStrep | Gibco | 15070-063 | Any similar will work |
Pipette tips (Sterile, filtered, 1 mL, Various sizes) | Pr1ma | PR-1250RK-FL, etc | Any similar will work |
Pipettors | Thermo Scientific | Finnpipette F2 | Any similar will work |
Propidium Iodide | Invitrogen | P1304MP | Any similar will work |
RPMI | Corning | 10-041-cv | Any similar will work |
Serological Pipette Aid | Drummond Scientific | 4-000-105 | Any similar will work |
Serological Pipettes (Sterile, various sizes) | Pr1ma | PR-SERO-25, etc | Any similar will work |
Sodium Pyruvate | Corning | 25-000-CI | Any similar will work |
Sterile Reservoirs | Midwest Scientific | RESE-2000 | Any similar will work |
Table top centrifuge | Eppendorf | 5810R | Any similar will work |
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