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, 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 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 "real-time" 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 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 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.
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).
2. NIS+ BCMA-CAR T cell imaging with [ 18F]TFB-PET scan
NOTE: This protocol follows the guidelines of Mayo Clinic's Institutional Animal Care and Use Committee (IACUC A00001767-16), IRB, and IBC (Bios00000006.04). OPM-2 is a BCMA+ MM cell line, which is often used as a target cell line for BCMA-CAR T cells39,40.
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 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 × 106/mL.
Figure 2 represents the outline of titrating the lentiviruses. On day 0, resuspend T cells at the concentration of 1.0 × 106/mL in TCM. Then, stimulate T cells with anti-CD3/CD28 beads at a 1:3 cell:beads ratio. Add 100 μL (100,000 cells) of T cells to the colored wells as indicated in Figure 2A. This plate is called a "titer plate." Incubate the titer plate at 37 °C, 5% CO2 for 24 h. On day 1, prepare the dilution plate. Add 100 μL of TCM to the colored wells as indicated in Figure 2B. Then, add 50 μL 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 µL from A6 to B6, and then 50 µL from B6 to C6, repeating until G6. Perform serial dilution for A7 and A8 as well. Then transfer 50 µL 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 μL 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 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 × 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.
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 × 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 µg/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 °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. Please click here to view a larger version of this figure.
Figure 2: Lentivirus titration. (A) On day 0, stimulate T cells with anti-CD3/CD28 beads at a 3:1 beads:cell ratio. Add 100 µL of 1.0 × 106/mL of stimulated T cells to the colored wells as indicated in the cartoon. Then, incubate the titer plate at 37 °C, 5% CO2 for 24 h. (B) Prepare a dilution plate by adding 100 µL of TCM to the colored wells as indicated in the cartoon. Add 50 µL 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 µL from A6 to B6, and then 50 µL from B6 to C6, repeating until G6. Perform serial dilution for A7 and A8 as well. Transfer 50 µL 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. Please click here to view a larger version of this figure.
Figure 3: The generation of NIS+BCMA-CAR T cells and luciferase-GFP positive OPM-2 cells. (A and B) 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) 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 ± SD). (E) 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 µg/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. Please click here to view a larger version of this figure.
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 × 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 × 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 IL2rγnull; luc = luciferase; [18F]TFB-PET/CT = [18F]tetrafluoroborate positron emission tomography/computed tomography. Please click here to view a larger version of this figure.
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 IL2rγnull; [18F]TFB-PET/CT = [18F]tetrafluoroborate positron emission tomography/computed tomography; IVIS = in vivo imaging system; CD = cluster of differentiation; SUV = standardized uptake value. Please click here to view a larger version of this figure.
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. Please click here to download this Video.
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.
The authors have nothing to disclose.
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 | 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 |