Published: February 17th, 2022
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.
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.
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
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).
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.......
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.......
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.....
|22 Gauge needle
|28 gauge insulin syringe
|96 well plate
|Anti-human (ETNL) NIS
|ETNL antibody binds the cytosolic C-terminus of NIS
|Anti-human BCMA, clone 19F2, PE-Cy7
|Anti-human CD45, clone HI30, BV421
|Anti-mouse CD45, clone 30-F11, APC-Cy7
|Secondary antibody for NIS staining
|BCMA-CAR construct, second generation
|IDT, Coralville, IA
|BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit
|CD3 Monoclonal Antibody (OKT3), PE, eBioscience
|CTS (Cell Therapy Systems) Dynabeads CD3/CD28
|CytoFLEX System B5-R3-V5
|Disposable Syringes with Luer-Lok Tips
|D-Luciferin, Potassium Salt
|D-PBS (Dulbecco's phosphate-buffered saline)
|Dulbecco's Phosphate-Buffered Saline
|Dynabeads MPC-S (Magnetic Particle Concentrator)
|Easy 50 EasySep Magnet
|EasySep Human T Cell Isolation Kit
|negative selection magnetic beads; 17951RF includes tips and buffer
|Fetal bovine serum
|Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647
|Inveon Multiple Modality PET/CT scanner
|Siemens Medical Solutions USA, Inc.
|10506989 VFT 000 03
|Piramal Critical Care
|IVIS Lumina S5 Imaging System
|IVIS® Spectrum In Vivo Imaging System
|Lipofectamine 3000 Transfection Reagent
|LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation
|Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.22 uM, sterile
|Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.45 uM, sterile
|multiple myeloma cell line
|lentiviral vector encoding luciferase-GFP
|Penicillin-Streptomycin-Glutamine (100x), Liquid
|PBAS and P3D
|Pooled Human AB Serum Plasma Derived
|MP Biomedicals, Inc.
|Fully Automated Cell Separator
|RPMI (Roswell Park Memorial Institute (RPMI) 1640 Medium)
|density gradient separation tubes
|Sodium Azide, 5% (w/v)
|Terrell (isoflurane, USP)
|Piramal Critical Care Inc
|Webcol Alcohol Prep
|X-VIVO 15 Serum-free Hematopoietic Cell Medium
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