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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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)

  1. Purchase Jurkat cells, clone E6-1 from ATCC. Thaw 1 x 106 cells in a T-75 flask and culture them in T-75 flasks. Maintain them in suspension at 0.6 x 106 cells per mL using Roswell Park Memorial Institute (RPMI) media supplemented with 10% FBS in an incubator at 37 °C w.......

Representative Results

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.......

Discussion

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 .......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
15 mL Conical Tube (Sterile)Midwest Scientific#C15BAny similar will work
50 mL Conical Tube (Sterile)Thermo Scientific339652Any similar will work
Black/Clear 96 well plateFalcon353219Celligo has a list of compatible plates
Celigo 4 Channel Imaging CytomenterNexcelcom Bioscience200-BFFL-5CAny similar will work
Celigo SoftwareNexcelcom BioscienceVersion 5.3.0.0Any similar will work
Cell Culture IncubatorThermo ScientificHeraCell 160iAny similar will work
Cell Culture Treated Flasks (T75, various sizes, Sterile)TPP90076Any similar will work
CFSETonbo13-0850-U500Any similar will work
Cytek Muse Cell AnalyzerCytek0500-3115Any similar will work
DMEMGibco11995-040Any similar will work
FBSGemini bio-products900-108Any similar will work
Flow CytometerCytek, BD, etcAurora, LSR II, etcAny similar will work
FlowJo SortwareBecton Dickinson & Company Version 10.7.1Any similar will work
Fluorobrite DMEMGibcoA18967-01Any similar will work
GraphPad SoftwareGraphPadVersion 9.3.1 (471)Any similar will work
Multichanel PipetteThermo ScientificFinnpipette F2Any similar will work
PBSGibco10010-031Any similar will work
PenStrepGibco15070-063Any similar will work
Pipette tips (Sterile, filtered, 1 mL, Various sizes)Pr1maPR-1250RK-FL, etcAny similar will work
Pipettors Thermo ScientificFinnpipette F2Any similar will work
Propidium IodideInvitrogenP1304MPAny similar will work
RPMICorning10-041-cvAny similar will work
Serological Pipette AidDrummond Scientific4-000-105Any similar will work
Serological Pipettes (Sterile, various sizes)Pr1maPR-SERO-25, etcAny similar will work
Sodium PyruvateCorning25-000-CIAny similar will work
Sterile ReservoirsMidwest ScientificRESE-2000Any similar will work
Table top centrifugeEppendorf5810RAny similar will work

References

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CAR T cellsJurkat CellsCytotoxicityFluorescent ImagingHigh throughput ScreeningCAR Construct OptimizationCancer Immunotherapy

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