Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

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 malignancies^1,2,3,4,5,6. 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.......

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.......

Materials

Name	Company	Catalog Number	Comments
22 Gauge needle	Covidien	8881250206
28 gauge insulin syringe	BD	329461
96 well plate	Corning	3595
Anti-human (ETNL) NIS	Imanis	REA009	ETNL antibody binds the cytosolic C-terminus of NIS
Anti-human BCMA, clone 19F2, PE-Cy7	BioLegend	357507	Flow antibody
Anti-human CD45, clone HI30, BV421	BioLegend	304032	Flow antibody
Anti-mouse CD45, clone 30-F11, APC-Cy7	BioLegend	103116	Flow antibody
Anti-rabbit IgG	R&D	F0110	Secondary antibody for NIS staining
BCMA-CAR construct, second generation	IDT, Coralville, IA
BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit	BD	554714
CD3 Monoclonal Antibody (OKT3), PE, eBioscience	Invitrogen	12-0037-42
CTS (Cell Therapy Systems) Dynabeads CD3/CD28	Gibco	40203D
CytoFLEX System  B5-R3-V5	Beckman Coulter	C04652	flow cytometer
Dimethyl sulfoxide	Millipore Sigma	D2650-100ML
Disposable Syringes with Luer-Lok Tips	BD	309646
D-Luciferin, Potassium Salt	Gold Biotechnology	LUCK-1G
D-PBS (Dulbecco's phosphate-buffered saline)	Gibco	14190-144
Dulbecco's Phosphate-Buffered Saline	Gibco	14190-144
Dynabeads MPC-S (Magnetic Particle Concentrator)	Applied Biosystems	A13346
Easy 50 EasySep Magnet	STEMCELL Technologies	18002
EasySep Human T Cell Isolation Kit	STEMCELL Technologies	17951	negative selection magnetic beads; 17951RF includes tips and buffer
Fetal bovine serum	Millipore Sigma	F8067
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Invitrogen	A-21235
Inveon Multiple Modality PET/CT scanner	Siemens Medical Solutions USA, Inc.	10506989 VFT 000 03
Isoflurane liquid	Piramal Critical Care	66794-017-10
IVIS Lumina S5 Imaging System	PerkinElmer	CLS148588
IVIS® Spectrum In Vivo Imaging System	PerkinElmer	124262
Lipofectamine 3000 Transfection Reagent	Invitrogen	L3000075
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation	Invitrogen	L34966
Lymphoprep	STEMCELL Technologies	07851
Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.22 uM, sterile	Thermo Scientific	450-0020
Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.45 uM, sterile	Thermo Scientific	450-0045
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ	Jackson laboratory	05557
OPM-2	DSMZ	CRL-3273	multiple myeloma cell line
pBMN(CMV-copGFP-Luc2-Puro)	Addgene	80389	lentiviral vector encoding luciferase-GFP
Penicillin-Streptomycin-Glutamine (100x), Liquid	Gibco	10378-016
PMOD software	PMOD	PBAS and P3D
Pooled Human AB Serum Plasma Derived	Innovative Research	IPLA-SERAB-H-100ML
Puromycin Dihydrochloride	MP Biomedicals, Inc.	0210055210
RoboSep-S	STEMCELL Technologies	21000	Fully Automated Cell Separator
RPMI (Roswell Park Memorial Institute (RPMI) 1640 Medium)	Gibco	21870-076
SepMate-50 (IVD)	STEMCELL Technologies	85450	density gradient separation tubes
Sodium Azide, 5% (w/v)	Ricca Chemical	7144.8-16
T175 flask	Corning	353112
Terrell (isoflurane, USP)	Piramal Critical Care Inc	66794-019-10
Webcol Alcohol Prep	Covidien	6818
X-VIVO 15 Serum-free Hematopoietic Cell Medium	Lonza	04-418Q