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In This Article

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

Summary

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

Abstract

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Introduction

Chimeric antigen receptor T (CAR T) cell therapy is a rapidly emerging and potentially curative approach in hematological malignancies1,2,3,4,5,6. Extraordinary clinical outcomes were reported after CD19-directed CAR T (CART19) or B cell maturation antigen (BCMA) CAR T cell therapy2. This led to the US Food and Drug Administration (FDA) approval of CART19 cells for aggressive B-cell lymphoma (axicabtagene ciloleucel (Axi-Cel)4

Protocol

The protocol follows the guidelines of Mayo Clinic's Institutional Review Board, Institutional Biosafety Committee, and Mayo Clinic's Institutional Animal Care and Use Committee.

1. NIS+ BCMA-CAR T cell production

NOTE: This protocol follows the guidelines of the Mayo Clinic's Institutional Review Board (IRB 17-008762) and Institutional Biosafety Committee (IBC Bios00000006.04).

  1. Production of BCMA-CAR, NIS, and luciferase-green flu.......

Representative Results

Figure 1 represents the steps of generating NIS+BCMA-CAR T cells. On day 0, isolate PBMCs and then isolate T cells by negative selection. Then, stimulate T cells with anti-CD3/CD28 beads. On day 1, transduce T cells with both NIS and BCMA-CAR lentiviruses. On days 3, 4, and 5, count T cells and feed with media to adjust the concentration to be 1.0 × 106/mL. For NIS-transduced T cells, add 1 μg/mL of puromycin to select NIS+ cells. On day 6, remove t.......

Discussion

This paper describes a methodology for incorporating NIS into CAR T cells and imaging infused CAR T cells in vivo through [18F]TFB-PET. As proof of concept, NIS+BCMA-CAR T cells were generated via dual transduction. We have recently reported that incorporating NIS into CAR T cells does not impair CAR T cell functions and efficacy in vivo and allows CAR T cell trafficking and expansion30. As CAR T cell therapies continue to expand beyond the current B cell ma.......

Acknowledgements

This work was partly supported through the Mayo Clinic K2R pipeline (SSK), the Mayo Clinic Center for Individualized Medicine (SSK), and the Predolin Foundation (RS). Figures 1, 2, and 4 were created with BioRender.com.

....

Materials

NameCompanyCatalog NumberComments
22 Gauge needleCovidien8881250206
28 gauge insulin syringeBD329461
96 well plateCorning3595
Anti-human (ETNL) NISImanisREA009ETNL antibody binds the cytosolic C-terminus of NIS
Anti-human BCMA, clone 19F2, PE-Cy7BioLegend357507Flow antibody
Anti-human CD45, clone HI30, BV421BioLegend304032Flow antibody
Anti-mouse CD45, clone 30-F11, APC-Cy7BioLegend103116Flow antibody
Anti-rabbit IgGR&DF0110Secondary antibody for NIS staining
BCMA-CAR construct, second generationIDT, Coralville, IA
BD Cytofix/Cytoperm Fixation/Permeabilization Solution KitBD554714
CD3 Monoclonal Antibody (OKT3), PE, eBioscienceInvitrogen12-0037-42
CTS (Cell Therapy Systems) Dynabeads CD3/CD28Gibco40203D
CytoFLEX System  B5-R3-V5Beckman CoulterC04652flow cytometer
Dimethyl sulfoxideMillipore SigmaD2650-100ML
Disposable Syringes with Luer-Lok TipsBD309646
D-Luciferin, Potassium SaltGold BiotechnologyLUCK-1G
D-PBS (Dulbecco's phosphate-buffered saline)Gibco14190-144
Dulbecco's Phosphate-Buffered SalineGibco14190-144
Dynabeads MPC-S (Magnetic Particle Concentrator)Applied BiosystemsA13346
Easy 50 EasySep MagnetSTEMCELL Technologies18002
EasySep Human T Cell Isolation KitSTEMCELL Technologies17951negative selection magnetic beads; 17951RF includes tips and buffer
Fetal bovine serumMillipore SigmaF8067
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647InvitrogenA-21235
Inveon Multiple Modality PET/CT scannerSiemens Medical Solutions USA, Inc.10506989 VFT 000 03
Isoflurane liquidPiramal Critical Care66794-017-10
IVIS Lumina S5 Imaging SystemPerkinElmerCLS148588
IVIS® Spectrum In Vivo Imaging SystemPerkinElmer 124262
Lipofectamine 3000 Transfection ReagentInvitrogenL3000075
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitationInvitrogenL34966
LymphoprepSTEMCELL Technologies07851
Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.22 uM, sterileThermo Scientific450-0020
Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.45 uM, sterileThermo Scientific450-0045
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJJackson laboratory05557
OPM-2DSMZCRL-3273multiple myeloma cell line
pBMN(CMV-copGFP-Luc2-Puro)Addgene80389lentiviral vector encoding luciferase-GFP
Penicillin-Streptomycin-Glutamine (100x), LiquidGibco10378-016
PMOD softwarePMODPBAS and P3D
Pooled Human AB Serum Plasma DerivedInnovative ResearchIPLA-SERAB-H-100ML
Puromycin DihydrochlorideMP Biomedicals, Inc.0210055210
RoboSep-SSTEMCELL Technologies21000Fully Automated Cell Separator
RPMI (Roswell Park Memorial Institute (RPMI) 1640 Medium)Gibco21870-076
SepMate-50 (IVD)STEMCELL Technologies85450density gradient separation tubes
Sodium Azide, 5% (w/v)Ricca Chemical7144.8-16
T175 flaskCorning353112
Terrell (isoflurane, USP)Piramal Critical Care Inc66794-019-10
Webcol Alcohol PrepCovidien6818
X-VIVO 15 Serum-free Hematopoietic Cell MediumLonza04-418Q

References

  1. Porter, D. L., et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Science Translational Medicine. 7 (303), (2015).
  2. Raje, N., et al.

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