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.

SSK is an inventor on patents in CAR immunotherapy licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis) and Mettaforge (through Mayo Clinic). RS, MJC, and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen. SSK receives research funding from Kite, Gilead, Juno, Celgene, Novartis, Humanigen, MorphoSys, Tolero, Sunesis, Leahlabs, and Lentigen. Figures were created with BioRender.com.

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 malignancies to applications in CLL, there will be a greater need for tools that allow non-invasive in vivo imaging and monitoring of infused adoptive T cells. Dynamic imaging of T cells will enable the validation of adoptive T cell trafficking and potentially allow earlier detection of efficacy and toxicity.
NIS has been investigated and validated as a sensitive modality to image cells and viruses in clinical trials32,42. Physiological accumulation of tracers for NIS is mainly seen in the thyroid/salivary glands, stomach, and bladder, which are not common organs affected by liquid tumors44. Especially in MM, malignant plasma cells are often distributed in the bone marrow or bones, and an extramedullary plasmacytoma in lesions, where the physiological accumulation of tracers for NIS occurs, is a rare phenomenon44,45. Furthermore, NIS is non-immunogenic and therefore suitable for longitudinal imaging studies46. NIS is an intrinsic membrane protein that transports iodide into the cytosol and contains 13 putative transmembrane segments with an extracellular amino terminus site and cytosolic carboxy terminus47.
NIS can be visualized with gamma- or positron-emitting radioisotopes such as technetium-99m (99mTc) pertechnetate, iodide-123 (123I), 131I, 124I, and [18F]TFB43. Recently, [18F]TFB has emerged as a promising iodide analog for NIS-based PET imaging, as it has similar biochemical properties and is radiosynthesized48. One advantage of TFB is that it does not undergo organification in thyroid cells and therefore has a comparatively mild uptake in normal thyroid tissue48. Another advantage of TFB is its short half-life of 109.8 min, while the half-lives of other tracers range from 12 h to 8 days, which could present safety issues for clinical applications49. The main limitation of NIS-based CAR T cell imaging is that tracers, including TFB, do not penetrate the blood-brain barrier (BBB), making it difficult to assess neurotoxicity after CAR T cell treatment50,51,52,53.
Neurotoxicity is associated with the infiltration of T cells and the activation of myeloid cells in the central nervous system. However, in most cases of neurotoxicity after CAR T cell therapy, the integrity of the BBB is disrupted50,54. Therefore, it is unclear whether the tracer is unable to cross the BBB in this compromised setting. Further studies need to be carried out to validate whether neurotoxicity after CAR T cell therapy can be imaged with [18F]TFB-PET. Although the short half-life of [18F]TFB is safe for patients and staff, it makes its procurement difficult for the hospital. Therefore, institutes must be equipped with cyclotrons or have access to a regional facility. The methodology described in the protocol here can potentially be applied to a variety of other CAR T cells via dual transduction to visualize and assess CAR T cells in vivo using [18F]TFB-PET scan.

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.

<table><tbody><tr><td>22 Gauge needle</td><td>Covidien</td><td>8881250206</td><td></td></tr><tr><td>28 gauge insulin syringe</td><td>BD</td><td>329461</td><td></td></tr><tr><td>96 well plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Anti-human (ETNL) NIS</td><td>Imanis</td><td>REA009</td><td>ETNL antibody binds the cytosolic C-terminus of NIS</td></tr><tr><td>Anti-human BCMA, clone 19F2, PE-Cy7</td><td>BioLegend</td><td>357507</td><td>Flow antibody</td></tr><tr><td>Anti-human CD45, clone HI30, BV421</td><td>BioLegend</td><td>304032</td><td>Flow antibody</td></tr><tr><td>Anti-mouse CD45, clone 30-F11, APC-Cy7</td><td>BioLegend</td><td>103116</td><td>Flow antibody</td></tr><tr><td>Anti-rabbit IgG</td><td>R&amp;amp;D</td><td>F0110</td><td>Secondary antibody for NIS staining</td></tr><tr><td>BCMA-CAR construct, second generation</td><td>IDT, Coralville, IA</td><td></td><td></td></tr><tr><td>BD&amp;nbsp;Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td><td>BD</td><td>554714</td><td></td></tr><tr><td>CD3 Monoclonal Antibody (OKT3), PE, eBioscience</td><td>Invitrogen</td><td>12-0037-42</td><td></td></tr><tr><td>CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td><td>Gibco</td><td>40203D</td><td></td></tr><tr><td>CytoFLEX System&amp;nbsp; B5-R3-V5</td><td>Beckman Coulter</td><td>C04652</td><td>flow cytometer</td></tr><tr><td>Dimethyl sulfoxide</td><td>Millipore Sigma</td><td>D2650-100ML</td><td></td></tr><tr><td>Disposable Syringes with Luer-Lok Tips</td><td>BD</td><td>309646</td><td></td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>Gold Biotechnology</td><td>LUCK-1G</td><td></td></tr><tr><td>D-PBS (Dulbecco&#039;s phosphate-buffered saline)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Dynabeads MPC-S (Magnetic Particle Concentrator)</td><td>Applied Biosystems</td><td>A13346</td><td></td></tr><tr><td>Easy 50 EasySep Magnet</td><td>STEMCELL Technologies</td><td>18002</td><td></td></tr><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></tr><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></tr><tr><td>Inveon Multiple Modality PET/CT scanner</td><td>Siemens Medical Solutions USA, Inc.</td><td>10506989 VFT 000 03</td><td></td></tr><tr><td>Isoflurane liquid</td><td>Piramal Critical Care</td><td>66794-017-10</td><td></td></tr><tr><td>IVIS Lumina S5 Imaging System</td><td>PerkinElmer</td><td>CLS148588</td><td></td></tr><tr><td>IVIS&amp;reg; Spectrum In Vivo Imaging System</td><td>PerkinElmer</td><td>&amp;nbsp;124262</td><td></td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>Invitrogen</td><td>L3000075</td><td></td></tr><tr><td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td><td>Invitrogen</td><td>L34966</td><td></td></tr><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&amp;nbsp;Il2rgtm1Wjl/SzJ</td><td>Jackson laboratory</td><td>05557</td><td></td></tr><tr><td>OPM-2</td><td>DSMZ</td><td>CRL-3273</td><td>multiple myeloma cell line</td></tr><tr><td>pBMN(CMV-copGFP-Luc2-Puro)</td><td>Addgene</td><td>80389</td><td>lentiviral vector encoding luciferase-GFP</td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100x), Liquid</td><td>Gibco</td><td>10378-016</td><td></td></tr><tr><td>PMOD software</td><td>PMOD</td><td>PBAS and P3D</td><td></td></tr><tr><td>Pooled Human AB Serum Plasma Derived</td><td>Innovative Research</td><td>IPLA-SERAB-H-100ML</td><td></td></tr><tr><td>Puromycin Dihydrochloride</td><td>MP Biomedicals, Inc.</td><td>0210055210</td><td></td></tr><tr><td>RoboSep-S</td><td>STEMCELL Technologies</td><td>21000</td><td>Fully Automated Cell Separator</td></tr><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><tr><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></tbody></table>

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 the beads by placing the cells in the magnet for a minute. Then, put the de-beaded cells from the tube into a new flask. Take an aliquot of cells (e.g., 50,000 cells) and stain with antibodies to check the expression of NIS and CAR on the surface of T cells using flow cytometry. On day 8, count the T cells and cryopreserve with freezing media at the concentration of 10 &#215; 106/mL.
Figure 2&#160;represents the outline of titrating the lentiviruses. On day 0, resuspend T cells at the concentration of 1.0 &#215; 106/mL in TCM. Then, stimulate T cells with anti-CD3/CD28 beads at a 1:3 cell:beads ratio. Add 100 &#956;L (100,000 cells) of T cells to the colored wells as indicated in Figure 2A. This plate is called a &#34;titer plate.&#34; Incubate the titer plate at 37 &#176;C, 5% CO2 for 24 h. On day 1, prepare the dilution plate. Add 100 &#956;L of TCM to the colored wells as indicated in Figure 2B. Then, add 50 &#956;L of freshly thawed lentiviruses into the first row (e.g., A6, A7, or A8 as depicted in Figure 2B). Perform serial dilution by transferring 50 &#181;L from A6 to B6, and then 50 &#181;L from B6 to C6, repeating until G6. Perform serial dilution for A7 and A8 as well. Then transfer 50 &#181;L of the diluted virus to the titer plate. Twenty-four hours after transferring the virus from the dilution plate to the titer plate, feed the cells with 100 &#956;L of TCM. On day 3, stain cells with antibodies and analyze the expression of NIS and BCMA on the T cells via flow cytometry.
Figure 3A,B show the representative flow plots of BCMA-CAR T or NIS+BCMA-CAR T cells. T cells are gated on FSC/SSC, followed by singlet and live-cell discrimination. Over 90% of cells are NIS+ cells (Figure 3B). Figure 3C shows the representative flow plot for the composition of NIS+BCMA-CAR T cells. Similar to Figure 3A,B, T cells are gated on FSC/SSC, followed by singlet and live-cell discrimination. Figure 3D shows the T cell expansion curve from days 0 to 8. There are no fold expansion differences between UTD, BCMA-CAR T, or NIS+BCMA-CAR T cells. Figure 3E depicts the GFP expression on OPM-2 cells after the transduction of lentivirus that encodes GFP and luciferase followed by puromycin selection.
Figure 4&#160;is the outline of imaging NIS+BCMA-CAR T cells in vivo with [18F]TFB-PET. Inoculate six to eight-week-old mice with 1.0 &#215; 106 cells of luciferase+ OPM-2 cells via tail vein injection on day -21. Assess the tumor burden by BLI on day -1. On day 0, randomize the mice according to the tumor burden to be treated with NIS+BCMA-CAR T cells through tail vein injection or monitored without any treatment (untreated xenograft). Image the mice with BLI on day 6. Perform [18F]TFB-PET on day 7 to image NIS+BCMA-CAR T cells.
Figure 5A shows the representative BLI 20 days after the inoculation of luciferase+OPM-2 cells into the NSG mice. Figure 5B shows the representative PET imaging a week after administration of NIS+BCMA-CAR T cells. [18F]TFB uptake is observed in the sternum, spines, pelvis, and femurs. In addition, physiological uptake of [18F]TFB is seen in the thyroid and stomach. Figure 5C shows the representative flow plots of femur-derived bone marrow harvested from the untreated xenograft or NIS+BCMA-CAR T cell-treated mice. Bone marrow samples are stained with mouse CD45, human CD45, human CD3, and human BCMA. Cells are gated on FSC/SSC, followed by singlet, live, and human-cell discrimination. Bone marrow samples derived from untreated xenograft show BCMA+ cells whereas NIS+BCMA-CAR T cell treated mouse shows CD3+ cells, which support the [18F]TFB-PET finding.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62334/62334fig01.jpg" /> 
Figure 1: NIS+BCMA-CAR T cell production schema. Normal donor CD3 T cells (isolated from peripheral blood mononuclear cells) using negative bead selection. T cells are plated at 1.0 &#215; 106/mL and expanded in TCM using anti-CD3/CD28 beads added on day 0 of culture and removed on day 6. T cells are dually transduced with lentiviruses encoding NIS or BCMA-CAR on day 1 (MOI=5.0). NIS+BCMA-CAR T cells are treated with 1 &#181;g/mL of puromycin on days 3, 4, and 5. T cells are expanded in culture for 8 days. T cells are cryopreserved in FBS with 10% DMSO for future experiments. T cells are thawed and rested overnight at 37 &#176;C before all experiments. Abbreviations: CD = cluster of differentiation; TCM = T cell expansion medium; BCMA = B cell maturation antigen; CAR = chimeric antigen receptor; NIS = sodium iodide symporter; GFP = green fluorescent protein; PBMCs = peripheral blood mononuclear cells; MOI = multiplicity of infection; FBS = fetal bovine serum; DMSO = dimethylsulfoxide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62334/62334fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62334/62334fig02.jpg" /> 
Figure 2: Lentivirus titration. (A)&#160;On day 0, stimulate T cells with anti-CD3/CD28 beads at a 3:1 beads:cell ratio. Add 100 &#181;L of 1.0 &#215; 106/mL of stimulated T cells to the colored wells as indicated in the cartoon. Then, incubate the titer plate at 37 &#176;C, 5% CO2 for 24 h. (B)&#160;Prepare a dilution plate by adding 100 &#181;L of TCM to the colored wells as indicated in the cartoon. Add 50 &#181;L of freshly thawed virus to A6, A7, or A8 (e.g., BCMA-CAR to A6, NIS to A7, and luciferase-GFP to A8). Then, serially dilute the virus by transferring 50 &#181;L from A6 to B6, and then 50 &#181;L from B6 to C6, repeating until G6. Perform serial dilution for A7 and A8 as well. Transfer 50 &#181;L of the diluted virus to the titer plate. (C) On day 3, stain the cells with corresponding antibodies and analyze the titer plate by flow cytometry. Abbreviations: CD = cluster of differentiation; TCM = T cell expansion medium; BCMA = B cell maturation antigen; CAR = chimeric antigen receptor; NIS = sodium iodide symporter; GFP = green fluorescent protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62334/62334fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62334/62334fig03.jpg" /> 
Figure 3: The generation of NIS+BCMA-CAR T cells and luciferase-GFP positive OPM-2 cells. (A and B)&#160;Cells are gated on FSC/SSC, followed by singlet and live-cell discrimination. Representative flow plots of (A) untransduced T and BCMA-CAR T cells and (B) UTD and NIS+BCMA-CAR T are shown. NIS+BCMA-CAR T cells are generated by co-transduction of two viruses on day 1 of T cell expansion, as described in Figure 2. On day 6, cells are stained for CARs and NIS. (C)&#160;The phenotypic analysis of NIS+BCMA-CAR T. The representative flow plot of NIS+BCMA-CAR T cells is shown. (D) Summary of the UTD, BCMA-CAR T, or NIS+BCMA-CAR T cell growth kinetics. Incorporation of BCMA-CAR and/or NIS does not impact T cell expansion (two-way ANOVA, n=3 biological replicates, mean &#177; SD). (E)&#160;Flow cytometric analysis of luciferase-GFP-transduced OPM-2. OPM-2 cells are transduced with lentivirus encoding luciferase-GFP with puromycin resistance. Forty-eight hours after transduction, OPM-2 cells are treated with 2 &#181;g/mL of puromycin. Cells are expanded for two more days, and the expression of GFP is analyzed via flow cytometry. Abbreviations: CD = cluster of differentiation; BCMA = B cell maturation antigen; CAR = chimeric antigen receptor; NIS = sodium iodide symporter; GFP = green fluorescent protein; UTD = untransduced; FSC/SSC = forward scattering/side scattering; ANOVA = analysis of variance; n.s.= not significant; SD = standard deviation; FL-1-A = area of fluorophore 1; FITC-A = area of fluorescein isothiocyanate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62334/62334fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62334/62334fig04.jpg" /> 
Figure 4: Scheme for in vivo trafficking assay in a systemic OPM2 xenograft model. Inject six to eight-week-old NSG mice with 1.0 &#215; 106 of luciferase-positive OPM-2 cells via the tail vein on day -21. On day -1, perform bioluminescent imaging on the mice to confirm the engraftment of OPM-2 cells. On day 0, inject mice with 5.0 &#215; 106 of NIS+BCMA-CAR T cells. Image mice with [18F]TFB-PET/CT on day 7 to assess the trafficking of NIS+BCMA-CAR T cells. Abbreviations: BCMA = B cell maturation antigen; CAR = chimeric antigen receptor; NIS = sodium iodide symporter; BLI = bioluminescent imaging; NSG = immunocompromised NOD-scid IL2r&#947;null; luc = luciferase; [18F]TFB-PET/CT = [18F]tetrafluoroborate positron emission tomography/computed tomography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62334/62334fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62334/62334fig05.jpg" /> 
Figure 5: In vivo trafficking assay in a systemic OPM2 xenograft model. (A and B) BCMA+luciferase+OPM2 cells are intravenously injected into NSG mice. Mice receive NIS+BCMA CAR T cells three weeks after the inoculation of OPM-2 cells. (A) BLI confirms the engraftment of OPM-2 cells. (B) [18F]TFB-PET reveals NIS+BCMA-CAR T cell trafficking to the bone marrow. (C) To confirm that OPM-2 cells engraft in the bone marrow and NIS+BCMA-CAR T cells traffic to the tumor site, mice are euthanized after the imaging, and the bone marrow is harvested. Flow cytometric analysis revealed that OPM-2 cells are engrafted in the bone marrow (left), and NIS+BCMA-CAR T cells are present in the bone marrow (right). Abbreviations: BCMA = B cell maturation antigen; CAR = chimeric antigen receptor; NIS = sodium iodide symporter; BLI = bioluminescent imaging; NSG = immunocompromised NOD-scid IL2r&#947;null; [18F]TFB-PET/CT = [18F]tetrafluoroborate positron emission tomography/computed tomography; IVIS = in vivo imaging system; CD = cluster of differentiation; SUV = standardized uptake value. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62334/62334fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video S1: Three-Dimensional (3D) rendering of PET/CT data showing the in vivo distribution of [18F]TFB in the thyroid, stomach, and bone marrow. Abbreviations: BCMA = B cell maturation antigen; CAR = chimeric antigen receptor; NIS = sodium iodide symporter; PET/CT = positron emission tomography/computed tomography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62334/Supplemental Video S1.mov" target="_blank">Please click here to download this Video.</a>

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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3.3K Views.  Mayo Clinic. Er is tot op heden geen klinisch gevalideerd chimere antigeenreceptor T-cel beeldvormingsplatform. De natriumjodidesymporter vertegenwoordigt een gevoelige en klinisch relevante verslaggever om Car T-cellen in beeld te brengen door PET-scan. TLP PET-beeldvorming van NIS CAR T-cel stelt onderzoekers in staat om niet-invasief geïnfundeerde cellen in vivo te volgen en heeft een potentieel om de werkzaamheid en toxiciteit van CAR T-celtherapie te voorspellen.Efficiënte beeldvorming van C...

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

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Immunology and Infection

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Medicine

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioengineering

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Automation of a Positron-emission Tomography PET Radiotracer Synthesis Protocol for Clinical Production

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

Chemistry

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose analogue, 18F-fluordeoxyglucose (FDG-fPET), it is now possible to measure the dynamics of glucose metabolism with temporal resolutions approaching those of functional magnetic resonance imaging (fMRI). This direct measure of glucose uptake has enormous potential for understanding normal and abnormal brain function and probing the effects of metabolic and neurodegenerative diseases. Further, new advances in hybrid MR-PET hardware make it possible to capture fluctuations in glucose and blood oxygenation simultaneously using fMRI and FDG-fPET.
The temporal resolution and signal-to-noise of the FDG-fPET images is critically dependent upon the administration of the radiotracer. This work presents two alternative continuous infusion protocols and compares them to a traditional bolus approach. It presents a method for acquiring blood samples, time-locking PET, MRI, experimental stimulus, and administering the non-traditional tracer delivery. Using a visual stimulus, the protocol results show cortical maps of the glucose-response to external stimuli on an individual level with a temporal resolution of 16 s.

This manuscript describes two radiotracer administration protocols for FDG-PET (constant infusion and bolus plus infusion) and compares them to bolus administration. Temporal resolutions of 16 s are achievable using these protocols.

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose ...

Behavior

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

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

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