Name: dynamic imaging of chimeric antigen receptor t cells with [18f]tetrafluoroborate positron emission tomography/computed tomography
Uploaded: 2022-02-17T13:00:00
Description: Watch this Scientific Journal Video about Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography at JoVE.com

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).&#160;Figures 1, 2, and 4 were created with BioRender.com.

The protocol follows the guidelines of Mayo Clinic&#39;s Institutional Review Board, Institutional Biosafety Committee, and Mayo Clinic&#39;s Institutional Animal Care and Use Committee.
1. NIS+ BCMA-CAR T cell production
NOTE: This protocol follows the guidelines of the Mayo Clinic&#39;s Institutional Review Board (IRB 17-008762) and Institutional Biosafety Committee (IBC Bios00000006.04).
<ol>
	<li>Production of BCMA-CAR, NIS, and luciferase-green flu...

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...

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, tisagenlecleucel (Tisa-Cel)3, and lisocabtagene maraleucel)7, acute lymphoblastic leukemia (Tisa-Cel)5,8, mantle cell lymphoma (brexucabtagene autoleuce)9, and follicular lymphoma (Axi-Cel)10. Most recently, the FDA approved BCMA-directed CAR T cell therapy in patients with multiple myeloma (MM) (idecabtagene vicleucel)11. Moreover, CAR T cell therapy for chronic lymphocytic leukemia (CLL) is in late-stage clinical development and is expected to receive FDA approval within the next three years1.
Despite the unprecedented results of CAR T cell therapy, its widespread use is limited by 1) insufficient in vivo CAR T cell expansion or poor trafficking to tumor sites, which leads to lower rates of durable response12,13&#160;and 2) the development of life-threatening adverse events, including cytokine release syndrome (CRS)14,15. The hallmarks of CRS include not only immune activation resulting in elevated levels of inflammatory cytokines/chemokines but also massive T cell proliferation after CAR T cell infusion15,16. Thus, the development of a validated, clinical-grade strategy to image CAR T cells in vivo would allow 1) CAR T cell tracking in real time in vivo to monitor their trafficking to tumor sites and uncover potential mechanisms of resistance, and 2) monitoring of CAR T cell expansion and potentially predicting their toxicities such as the development of CRS.
Clinical features of mild CRS are high fever, fatigue, headache, rash, diarrhea, arthralgia, myalgia, and malaise. In more severe CRS, patients may develop tachycardia/hypotension, capillary leak, cardiac dysfunction, renal/hepatic failure, and disseminated intravascular coagulation17,18. In general, the degree of elevation of cytokines, including interferon-gamma, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-10, and IL-6, has been shown to correlate with the severity of clinical symptoms17,19. However, the extensive application of &#34;real-time&#34; serum cytokine monitoring to predict CRS is difficult due to the high cost and limited availability. To exploit the beneficial characteristics of CAR T cell therapy, non-invasive imaging of adoptive T cells can be potentially utilized to predict the efficacy, toxicities, and relapse after CAR T cell infusion.
Several researchers have developed strategies to use radionuclide-based imaging with positron emission tomography (PET) or single-photon emission computed tomography (SPECT), which provides high resolution and high sensitivity20,21,22,23,24,25,26,27,28,29,30&#160;for the in vivo visualization and monitoring of CAR T cell trafficking. Among those radionuclide-based imaging strategies, the sodium iodide symporter (NIS) has been developed as a sensitive modality to image cells and viruses using PET scans31,32. NIS+CAR T cell imaging with [18F]TFB-PET is a sensitive, efficient, and convenient technology to assess and diagnose CAR T cell expansion, trafficking, and toxicity30. This protocol describes 1) the development of NIS+CAR T cells through dual transduction with high efficacy and 2) a methodology for imaging NIS+CAR T cells with [18F]TFB-PET&#160;scan. BCMA-CAR T cells for MM are used as a proof-of-concept model to describe NIS as a reporter for CAR T cell imaging. However, these methodologies can be applied to any other CAR T cell therapy.

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			<td>22 Gauge needle</td>
			<td>Covidien</td>
			<td>8881250206</td>
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			<td>28 gauge insulin syringe</td>
			<td>BD</td>
			<td>329461</td>
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			<td>96 well plate</td>
			<td>Corning</td>
			<td>3595</td>
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		<tr>
			<td>Anti-human (ETNL) NIS</td>
			<td>Imanis</td>
			<td>REA009</td>
			<td>ETNL antibody binds the cytosolic C-terminus of NIS</td>
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		<tr>
			<td>Anti-human BCMA, clone 19F2, PE-Cy7</td>
			<td>BioLegend</td>
			<td>357507</td>
			<td>Flow antibody</td>
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			<td>Anti-human CD45, clone HI30, BV421</td>
			<td>BioLegend</td>
			<td>304032</td>
			<td>Flow antibody</td>
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		<tr>
			<td>Anti-mouse CD45, clone 30-F11, APC-Cy7</td>
			<td>BioLegend</td>
			<td>103116</td>
			<td>Flow antibody</td>
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		<tr>
			<td>Anti-rabbit IgG</td>
			<td>R&#38;D</td>
			<td>F0110</td>
			<td>Secondary antibody for NIS staining</td>
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		<tr>
			<td>BCMA-CAR construct, second generation</td>
			<td>IDT, Coralville, IA</td>
			<td></td>
			<td></td>
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			<td>BD&#160;Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td>BD</td>
			<td>554714</td>
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			<td>CD3 Monoclonal Antibody (OKT3), PE, eBioscience</td>
			<td>Invitrogen</td>
			<td>12-0037-42</td>
			<td></td>
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		<tr>
			<td>CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td>
			<td>Gibco</td>
			<td>40203D</td>
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			<td>CytoFLEX System&#160; B5-R3-V5</td>
			<td>Beckman Coulter</td>
			<td>C04652</td>
			<td>flow cytometer</td>
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			<td>Dimethyl sulfoxide</td>
			<td>Millipore Sigma</td>
			<td>D2650-100ML</td>
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			<td>Disposable Syringes with Luer-Lok Tips</td>
			<td>BD</td>
			<td>309646</td>
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			<td>D-Luciferin, Potassium Salt</td>
			<td>Gold Biotechnology</td>
			<td>LUCK-1G</td>
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			<td>D-PBS (Dulbecco&#39;s phosphate-buffered saline)</td>
			<td>Gibco</td>
			<td>14190-144</td>
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			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
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			<td>Dynabeads MPC-S (Magnetic Particle Concentrator)</td>
			<td>Applied Biosystems</td>
			<td>A13346</td>
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			<td>Easy 50 EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td>18002</td>
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		<tr>
			<td>EasySep Human T Cell Isolation Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17951</td>
			<td>negative selection magnetic beads; 17951RF includes tips and buffer</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Millipore Sigma</td>
			<td>F8067</td>
			<td></td>
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		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A-21235</td>
			<td></td>
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		<tr>
			<td>Inveon Multiple Modality PET/CT scanner</td>
			<td>Siemens Medical Solutions USA, Inc.</td>
			<td>10506989 VFT 000 03</td>
			<td></td>
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			<td>Isoflurane liquid</td>
			<td>Piramal Critical Care</td>
			<td>66794-017-10</td>
			<td></td>
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			<td>IVIS Lumina S5 Imaging System</td>
			<td>PerkinElmer</td>
			<td>CLS148588</td>
			<td></td>
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			<td>IVIS&#174; Spectrum In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>&#160;124262</td>
			<td></td>
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			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>L3000075</td>
			<td></td>
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		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen</td>
			<td>L34966</td>
			<td></td>
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		<tr>
			<td>Lymphoprep</td>
			<td>STEMCELL Technologies</td>
			<td>07851</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.22 uM, sterile</td>
			<td>Thermo Scientific</td>
			<td>450-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.45 uM, sterile</td>
			<td>Thermo Scientific</td>
			<td>450-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid&#160;Il2rgtm1Wjl/SzJ</td>
			<td>Jackson laboratory</td>
			<td>05557</td>
			<td></td>
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			<td>OPM-2</td>
			<td>DSMZ</td>
			<td>CRL-3273</td>
			<td>multiple myeloma cell line</td>
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			<td>pBMN(CMV-copGFP-Luc2-Puro)</td>
			<td>Addgene</td>
			<td>80389</td>
			<td>lentiviral vector encoding luciferase-GFP</td>
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			<td>Penicillin-Streptomycin-Glutamine (100x), Liquid</td>
			<td>Gibco</td>
			<td>10378-016</td>
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			<td>PMOD software</td>
			<td>PMOD</td>
			<td>PBAS and P3D</td>
			<td></td>
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			<td>Pooled Human AB Serum Plasma Derived</td>
			<td>Innovative Research</td>
			<td>IPLA-SERAB-H-100ML</td>
			<td></td>
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			<td>Puromycin Dihydrochloride</td>
			<td>MP Biomedicals, Inc.</td>
			<td>0210055210</td>
			<td></td>
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			<td>RoboSep-S</td>
			<td>STEMCELL Technologies</td>
			<td>21000</td>
			<td>Fully Automated Cell Separator</td>
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		<tr>
			<td>RPMI (Roswell Park Memorial Institute (RPMI) 1640 Medium)</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td></td>
		</tr>
		<tr>
			<td>SepMate-50 (IVD)</td>
			<td>STEMCELL Technologies</td>
			<td>85450</td>
			<td>density gradient separation tubes</td>
		</tr>
		<tr>
			<td>Sodium Azide, 5% (w/v)</td>
			<td>Ricca Chemical</td>
			<td>7144.8-16</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 flask</td>
			<td>Corning</td>
			<td>353112</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrell (isoflurane, USP)</td>
			<td>Piramal Critical Care Inc</td>
			<td>66794-019-10</td>
			<td></td>
		</tr>
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			<td>Webcol Alcohol Prep</td>
			<td>Covidien</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
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dynamic imaging of chimeric antigen receptor t cells with [18f]tetrafluoroborate positron emission tomography/computed tomography

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.

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 &#215; 106/mL. For NIS-transduced T cells, add 1 &#956;g/mL of puromycin to select NIS+ cells. On day 6, remove t...

Watch this Scientific Journal Video about Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography at JoVE.com

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

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.

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

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

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