Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.
Clinical trials of chimeric antigen receptor T (CART)–cell therapy in patients with hematologic malignancies, including B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma, B-cell chronic lymphoblastic leukemia, and multiple myeloma (MM) have shown unprecedented therapeutic efficacy (1–7). Most notably, the FDA has approved CD19-directed CART (CART19)-cell therapy for patients with relapsed/refractory B-ALL (8), diffuse large B-cell lymphoma (9, 10), and mantle cell lymphoma (11). Following infusion, CART cells undergo massive in vivo proliferation, which correlates with the development of toxicities after CART-cell therapy and often predicts a stronger response to CART-cell therapy (8, 12, 13). In solid tumors, the application of CART-cell therapy has been largely unsuccessful to date. Multiple mechanisms for the lack of CART-cell activity in solid tumors have been postulated, including poor T-cell expansion, T-cell exhaustion, poor trafficking to tumor sites, and inhibition by the tumor microenvironment (14–19).
To date, there is also a lack of a robust, clinically validated in vivo imaging platform to assess trafficking and expansion of CART cells after infusion. Such a platform would allow (i) real-time, serial monitoring of CART-cell proliferation and trafficking to tumor sites, (ii) the rapid implementation of appropriate strategies to enhance CART-cell activity, and (iii) the potential to predict severe cytokine release syndrome (CRS) associated with massive T-cell expansion after CART-cell administration. Several technologies have been reported for in vivo CART-cell imaging, such as herpes simplex virus-thymidine kinase (HSV-1-TK), Escherichia coli dihydrofolate reductase enzyme (eDHFR), somatostatin receptor 2 (SSTR2), and prostate-specific membrane antigen (PSMA; refs. 20–25). Although these studies demonstrate the ability to label and follow CART-cell trafficking in preclinical studies, clinical experience using any of these reporters has been limited so far (21, 26).
Sodium iodide symporter (NIS) has been widely used in the clinic due to its ability to mediate uptake of a variety of radioisotopes by the thyroid gland. Our group has pioneered the use of NIS in gene therapy and demonstrated applications for oncolytic viral imaging in preclinical studies and clinical trials (27–29). Although incorporation of NIS into CART cells has been reported, no platforms have been developed to track CART-cell expansion or to predict the development of CRS (30, 31). Here, we report NIS as a modality both to track CART-cells by 18F-TFB-PET and to diagnose and potentially predict toxicities after CART-cell administration.
Materials and Methods
Cells lines and primary cells
The CD19+ ALL cell line Nalm6 and CD19− and B-cell maturation antigen (BCMA)− cell line K562 were purchased from ATCC in 2017, and the BCMA+ MM cell line OPM-2 was purchased from DSMZ in 2018. Nalm6 and OPM-2 were used as target cells. K562 served as a negative control. All cell lines were regularly tested for Mycoplasma and phenotype was confirmed by flow cytometry. The number of passages was limited to 10. These cell lines were transduced with a luciferase-ZsGreen lentivirus (Addgene) and sorted with FACS Aria II (BD Biosciences) instrument to 100% purity. Cell lines were maintained in R10 medium as described previously (32). Primary leukemia cells from three different donors were obtained from the Mayo Clinic Biobank for patients with ALL under an Institutional Review Board–approved protocol (IRB 17-008762). All the patients provided signed informed consents. The use of recombinant DNA was approved by the Institutional Biosafety Committee (IBC HIP00000252.16). All cell lines used regularly tested negative for Mycoplasma contamination throughout the whole duration of this study.
Second-generation CD19 and BCMA CAR constructs were synthesized de novo [Integrated DNA Technologies, Inc. (IDT)]. These constructs consisted of a single-chain variable fragment against CD19 (clone FMC63) or BCMA (clone BCMA-02) cloned into a second-generation 4-1BB costimulated CAR in a third-generation lentivirus as described previously (32). The human NIS plasmids were obtained from Imanis Life Science through collaborative research and development and material transfer agreements. Primary cells were cultured in T-cell medium (TCM) made with X-Vivo 15 (Lonza) supplemented with 10% human serum albumin and 1% penicillin-streptomycin-glutamine (Gibco). Lentiviral particles were generated through the transient transfection of plasmids into 293T virus-producing cells (purchased from ATCC), in the presence of Lipofectamine 3000 (Invitrogen), VSV-G, and packaging plasmids (Addgene).
The density gradient technique was used to isolate peripheral blood mononuclear cells (PBMC). T-cell isolation from PBMCs (n = 8) was performed via a negative selection magnetic bead kit (magnetic beads against CD15, CD14, CD 34, CD36, CD56, CD123, CD235a, CD19, and CD16). The purity of T-cell population after the isolation was more than 95%. T cells isolated from normal donors were stimulated using Cell Therapy Systems Dynabeads CD3/CD28 (Life Technologies) at a 1:3 cell:bead ratio. Stimulated T cells were transduced with lentiviral particles at a multiplicity of infection of 3.0, 24 hours after stimulation. NIS+CART cells were generated through dual transduction of both NIS and CAR virus. NIS+ cells were selected by adding 1 μg/mL of puromycin dihydrochloride (Millipore Sigma) to the TCM on days 3, 4, 5, and 6. The expression of NIS and CAR were analyzed by flow cytometry on day 6. CARs were stained with goat anti-mouse IgG. NIS was stained with anti-human ETNL NIS followed by a PE-conjugated anti-rabbit secondary antibody (Supplementary Table S1). ETLN antibody recognizes the cytosolic C-terminus of NIS. Therefore, cells were permeabilized prior to staining. Beads were removed from T cells by using DynaMag-50 (Invitrogen) on day 6. On day 8, CART-cells were harvested and cryopreserved in freezing medium composed of 90% FBS (Millipore Sigma) and 10% DMSO (Millipore Sigma) for planned experiments. CART cells were thawed and rested in TCM 6 to 12 hours prior to the individual experiments.
In vitro125 iodide uptake assay in NIS+CART cells
Untransduced (UTD) T cells or NIS+CART19 cells (300,000 cells) were washed once by Hank's Balanced Salt Solution (HBSS) modified with 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES (STEMCELL Technologies). Cells were then resuspended in HEPES/HBSS or HEPES/HBSS with 1 mmol/L KClO4 and incubated at 37°C, as indicated in the specific experiment. Radioactive substrate solutions were prepared immediately prior to each assay. Na125I in 0.1 mol/L NaOH (PerkinElmer) was diluted in uptake buffer and was added to each tube [500,000 counts per minute (CPM) of 125I in each sample]. Cells were then incubated at 37°C for 60 minutes prior to the assay. After incubation, samples were centrifuged, and the supernatant was aspirated. Cells were washed with cold HEPES/HBSS and centrifuged. Cells were then resuspended in NaOH for quantification on a 2470 Automatic Gamma Counter (PerkinElmer).
Xenograft mouse models
Male and female, 8- to 12-week-old, non-obese diabetic/severe combined immunodeficient bearing a targeted mutation in the IL2 receptor gamma chain gene (NSG) mice were purchased from the Jackson Laboratory (catalog no. 005557) and maintained within the Mayo Clinic Department of Comparative Medicine under an Institutional Animal Care and Use Committee–approved protocol (IACUC A00001767 and A00003102).
Nalm6 xenograft models
Subcutaneous Nalm6 xenografts were established through the subcutaneous injection of 1 × 106 luciferase+ Nalm6 cells in PBS. Tumor burden was assessed by bioluminescence imaging (BLI) using a Xenogen IVIS-200 Spectrum camera (PerkinElmer) to confirm engraftment weekly after Nalm6 inoculation. Once the tumor volume reached 50 mm in diameter, mice were then randomized into groups receiving (i) UTD or (ii) NIS+CART cells (5 × 106 intravenously). Weekly Imaging was performed 10 minutes after the intraperitoneal injection of 10 μL/g d-luciferin (15 mg/mL, Gold Biotechnology). Bioluminescent images were analyzed using Living Image version 4.4 (PerkinElmer). One week after T-cell injection, mice were imaged both with BLI and 18F-TFB-PET/CT as described below. Mice were euthanized after the PET imaging and the experiment was concluded.
OPM-2 xenograft models
The BCMA+ luciferase+ MM OPM-2 cell line was used to establish localized or systemic MM xenografts. For the localized model, mice were implanted with 2.5 × 105 luciferase+ OPM-2 cells subcutaneously in the flank. For the systemic model, 1 × 106 cells were resuspended in PBS and injected via the tail vein. The tumor burden was assessed weekly by BLI using a Xenogen IVIS-200 Spectrum camera to confirm engraftment of OPM-2 cells. In the localized tumor model, engraftment was confirmed 4 weeks after OPM-2 injection. In the systemic model, engraftment was confirmed 3 weeks after OPM-2 inoculation. Mice were then randomized on the basis of tumor burden to receive 5 × 106 cells of (i) UTD, (ii) BCMA-CART cells, or (iii) NIS+BCMA-CART cells. Mice were imaged with BLI and 18F-TFB-PET/CT as described below. Imaging was performed 1 week after T-cell injection in the localized model and 2 weeks after T-cell injection in the systemic model. Mice were euthanized whether they develop paralysis, hunched posture, or abdominal distention. At the conclusion of this experiment, femurs were harvested and bone marrow cells were flushed. Bone marrow cells were stained with APC-Cy7 mouse CD45 (clone 30-F11, catalog no. 103116), brilliant violet (BV) 421 human CD45 (clone HI30, catalog no. 304032), and PE-Cy7 human BCMA (clone 19F2, catalog no. 357507).
K562 xenograft models
The CD19− and BCMA− luciferase+ CML K562 cell line was used to establish localized or systemic CD19−/BCMA− tumor model. For the localized model, mice were implanted with 1 × 106 luciferase+ K562 cells subcutaneously in the flank 2 weeks before T-cell infusion. For the systemic model, 1 × 106 cells were resuspended in PBS and injected via the tail vein, 1 week before T-cell infusion. The tumor burden was assessed weekly by BLI using a Xenogen IVIS-200 Spectrum camera to confirm engraftment of K562 cells as well as visual inspection. For the localized model, mice were treated when the tumor size reached 50 mm in diameter. The mice were randomized according to the tumor burden assessed by IVIS to receive 5 × 106 of (i) UTD, (ii) NIS+CART19, or (iii) NIS+BCMA-CART cells. One week after T-cell infusion, mice were imaged with BLI and 18F-TFB-PET as described below. Mice were euthanized after the imaging with 18F-TFB-PET.
Patient-derived ALL xenografts for CRS
A model for CART-cell toxicity was established using patient-derived xenografts as reported previously (32). Briefly, mice were first intraperitoneally injected with 30 mg/kg busulfan (Selleck Chemicals). The following day, mice received 1–3 × 106 leukemic blasts derived from the peripheral blood of patients with relapsed ALL via tail vein injection. Mice were monitored for human CD19+ cell engraftment for 10 to 13 weeks by peripheral blood sampling via tail vein bleeding. Here, 70 μL peripheral blood was processed with red blood cell lysis using BD FACS Lyse buffer (BD Biosciences). Cells were then stained with APC-Cy7 mouse CD45, BV421 human CD45, and PE human CD19 (clone SJ25C1, catalog no. 340720; Supplementary Table S1). Absolute count of human CD19+ cells was determined using counting beads via flow cytometry. Mouse serum was isolated from the remaining peripheral blood volume to perform cytokine/chemokine assay as described below. When the peripheral blood human CD19+ cell count was ≥10 cells/μL, mice were randomized to receive NIS+CART19 (5 × 106 cells intravenously) or control PBS. Mice were weighed daily and monitored for motor weakness and well-being. The development of CRS was defined by the combination of weight loss, decline motor function, and elevated cytokines. Seven days after treatment, mice underwent 18F-PET/CT imaging as described below. The peripheral blood was collected via tail vein sampling the following day after 18F-PET/CT imaging (day 8) to assess in vivo CART-cell expansion and cytokines. Here, 70 μL mouse peripheral blood was processed with red blood cell lysis using BD FACS Lyse buffer (BD Biosciences) and then used for flow cytometric studies as described below. Mice were euthanized on day 8 to harvest the spleen and liver to confirm the trafficking of CART cells via flow cytometry.
Imaging was performed in the Mayo Clinic Small Animal Imaging Core using an Inveon Multiple Modality PET/CT scanner (Siemens Medical Solutions). 18F-TFB for PET/CT imaging was produced as described previously (33). Forty-five minutes to 1 hour prior to PET imaging, 9.25 MBq of 18F-TFB was delivered to the mice via intravenous injection. CT image acquisition was performed in 5 minutes with 360-degree rotation and 180 projections at 500 μA, 80 keV, and 200 ms exposure. PET Image acquisition began approximately 45 minutes following isotope injection with total acquisition time of 20 minutes.
Co-registered images were rendered and visualized using the PMOD software (PMOD Technologies Ltd.). To calculate standardized uptake value (SUV), the volume of interest (VOI) was determined by the PMOD software. Then, SUV was calculated using the formula as below.
The use and handling of radiotracers were approved by the Institutional Biosafety Committee (IBC HIP00000252).
In vivo sensitivity assay for NIS+CART cells
Male and female, 8- to 12-week-old NSG mice were utilized in this study. NSG mice were purchased from the Jackson Laboratory (catalog no. 005557). NSG mice (naïve mice) were subcutaneously injected in the legs with different doses of NIS+CART19 cells (0.1 × 105–1.25 × 106) or PBS along with matrigel (Corning; 50 μL NIS+CART cells + 50 μL of matrigel), as indicated in the specific experiment. A total of 10 to 15 minutes after the injection of matrigel and NIS+CART-cell mixture, mice were intravenously injected with 9.25 MBq of 18F-TFB, and images were acquired as described above.
In vitro antigen-specific degranulation and cytokine production assays
Intracellular cytokine analysis and T-cell degranulation assays were performed following incubation of CART cells and Nalm6, OPM-2, or K562 cells for 4 hours in the presence of FITC-conjugated CD107a, monensin (catalog no. 554724, BioLegend), human CD49d (clone L25, catalog no. 340976, BD Biosciences), and human CD28 (clone L293, catalog no. 348040, BD Biosciences). After 4 hours, cells were harvested, and intracellular staining was performed after surface staining APC-H7 anti-human CD3 and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit followed by fixation and permeabilization with fixation medium A and B (catalog nos. GAS001S100 and GAS002S100, Life Technologies).
In vitro antigen-specific proliferation assay
For proliferation assays, carboxyfluorescein diacetate succinimidyl ester (CFSE; catalog no. C34554, Life Technologies) labeled CART cells and irradiated Nalm6, OPM-2, or K562 cells were cocultured at 1:1 ratio. All cell lines were lethally irradiated (120 Gy) prior to plating. Cells were cocultured for 5 days, as described in the specific experiments, and then cells were harvested and surface staining with APC-H7 anti-human CD3 and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit was performed (Supplementary Table S1). Phorbol myristate acetate (PMA) and ionomycin (Millipore Sigma) were used as a positive nonspecific stimulant of T cells, at different concentrations as indicated in the specific experiments.
In vitro cytotoxicity assay
Luciferase+ Nalm6, OPM-2, and K562 cells were incubated at the indicated ratios with effector T cells for 24 hours. The killing was calculated by BLI on a Xenogen IVIS-200 Spectrum camera (catalog no. 124262, PerkinElmer) as a measure of residual live cells. Ten minutes prior to imaging, samples were treated with 1 μL d-luciferin (30 μg/mL, Gold Biotechnology) per 100 μL sample volume.
Multiplex analysis of cytokines and chemokines
Cytokine and chemokine profiles from the patient-derived ALL xenograft sera before (day −1) and after (day 8) the treatment of NIS+CART19 cell were interrogated with the HCYTMAG-60K-PX38 Milliplex kit (Millipore Sigma), following the procedure described in the manufacturer's manual. Sera from the mice that did not receive CART cells were used as controls. Sera were diluted 1:2 with human serum matrix (provided within the Milliplex kit) prior to plating. Data were collected using a Luminex instrument (catalog no. 40-012, Millipore Sigma). The xPONENT Software was used to analyze the data (catalog no. MAP0200, Invitrogen).
Multiparametric flow cytometry
Anti-human and anti-mouse antibodies were purchased from BioLegend, eBioscience, or BD Biosciences (Supplementary Table S1). BD FACS lyse buffer (BD Biosciences) was used to lyse red blood cells of mouse (patient-derived ALL xenograft) peripheral blood samples (day −1 and 8 of NIS+CART-cell infusion) prior to staining and flow cytometric analysis. Cells from in vitro culture (in vitro antigen-specific degranulation/cytokine production assays and antigen-specific proliferation assay), mouse peripheral blood, or mouse organs (spleen, liver, and bone marrow) were analyzed by flow cytometry. Prior to staining, cells were washed twice in a flow buffer [PBS supplemented with 1% FBS and 1% sodium azide (Ricca Chemical)], and stained at room temperature. For cell number quantitation, Countbright beads (Invitrogen) were used according to the manufacturer's instructions (Invitrogen). In all analyses, the population of interest was gated on the basis of forward versus side scatter characteristics, followed by singlet gating, and live cells were gated following staining with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen).
Surface expression of the CAR was detected by staining with a goat anti-mouse F(ab')2 antibody. In brief, an aliquot of the CART cells (e.g., 50,000 T cells) was first washed and then resuspended in 50 μL of a flow buffer. Cells were then stained with 1 μL of goat anti-mouse antibody and 0.3 μL of LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for excluding dead cells and incubated in dark for 15 minutes at room temperature. After incubation, cells are washed by adding 150 μL of a flow butter and centrifuged at 650 × g for 3 minutes at 4°C. After surface staining, cells are fixed and permeabilized by adding 100 μL of a fixation medium and incubated for 15 minutes at room temperature. Cells are then washed with 100 μL of a flow buffer. Fixed/permeabilized cells were then resuspended in 50 μL of a permeabilizing buffer. NIS expression was detected by anti-human ETNL (NIS antibody). The NIS antibody recognizes the cytosolic C-terminus of NIS. To stain for NIS, 0.3 ng of anti-human NIS antibody was added along with 50 μL of a flow buffer and incubated for 1 hour at 4°C. Then 100 μL of flow buffer was added and cells were centrifuged at 650 × g for 3 minutes at 4°C.Then 2.5 μL of anti-rabbit secondary antibody along with 50 μL of flow buffer were added and cells were incubated for 30 minutes at 4°C. Cells were then washed and resuspended in 200 μL of a flow buffer and acquired on a flow cytometer. Flow cytometry was performed on a CytoFLEX (Beckman Coulter) three-laser cytometer. Analyses were performed using FlowJo X10.0.7r2 software. See Supplementary Table S1 for specific details of flow antibodies.
All statistics were performed using GraphPad Prism version 8.05 for Windows (GraphPad Software, www.graphpad.com). Statistical tests are described in detail in the representative figure legends.
Development of dual NIS+CART cells
We used two CAR constructs that have been studied extensively in preclinical and clinical studies against B-cell malignancies and MM: CAR19 and anti-BCMA-CAR, respectively (Fig. 1A). We generated NIS+CART19 and NIS+BCMA-CART cells through dual lentiviral transduction (NIS and CAR vectors, see Materials and Methods). On day 6 of expansion, cells were stained for the CAR and NIS and analyzed by flow cytometry. Representative flow plots of NIS+CART cells are shown in Fig. 1B, and the gating strategy is shown in Supplementary Fig. S1A. The summary of NIS+CAR+ transduction efficiency is shown in Supplementary Fig. S1B. No T-cell phenotypic changes were seen after the incorporation of NIS into CART cells (Supplementary Fig. S1C).
Incorporation of NIS into CART cells does not impair their function
To confirm that incorporating NIS into CART cells did not have a negative impact on effector functions, we first investigated the in vitro antigen-specific activity of NIS+CART-cells. Antigen-specific killing of CD19+ Nalm6 cells by NIS+CART19 or CART19 cells was comparable. Similarly, antigen-specific killing of BCMA+ OPM-2 cells by NIS+BCMA-CART or BCMA-CART cells was comparable (Fig. 2A). In a 5-day antigen-specific proliferation assay, we observed a potent and equivalent expansion of both NIS+CART19 and CART19 cells upon stimulation in cocultures with irradiated CD19+ Nalm6 cells (Fig. 2B; Supplementary Fig. S2). NIS+BCMA-CART cells also showed a comparable antigen-specific proliferation to BCMA-CART cells upon stimulation in cocultures with BCMA+ OPM-2 cells (Supplementary Fig. S2 and S3A). Similarly, no differences were observed in antigen-specific degranulation or intracellular cytokines between CART19 and NIS+CART19 cells (Fig. 2C; Supplementary Fig. S4A and S4B) or between BCMA-CART and NIS+BCMA-CART cells (Supplementary Figs. S3B and S4C). To confirm the antigen specificity of NIS+CART cells, the CD19−BCMA− K562 cell line was used as a negative control. NIS+CART19 or NIS+BCMA-CART cells did not exert any effector functions upon stimulation with antigen-negative cell lines in cocultures with the K562 cells (Supplementary Figs. S3D and S5A–S5C). Overall, these results indicate that the incorporation of NIS in CART cells did not inhibit effector functions in vitro.
NIS+CART cells demonstrate specificity for radioiodine and high sensitivity in vivo
Next, we aimed to measure the specificity of dual transduced NIS+CART cells for radioiodine uptake. NIS+CART19 cells or UTD were incubated in the presence of radioactive iodide molecules (125I) with or without the NIS-specific inhibitor potassium perchlorate (KClO4). 125I uptake occurred exclusively in NIS+CART19 cells, and this uptake was appropriately inhibited by KClO4 (Fig. 3A), suggesting that iodide uptake was mediated by NIS+CART cells. We then performed a sensitivity experiment to determine the lowest number of NIS+CART cells that can be detected by 18F-TFB-PET imaging in vivo. Here, NSG mice were subcutaneously injected with different doses of NIS+CART19 cells, and 10 to 15 minutes after, mice were intravenously injected with 18F-TFB. 18F-TFB-PET showed that NIS+CART cells were detectable at numbers as low as 0.4 × 105 cells (Fig. 3B and C). Physiologic uptake of TFB by endogenous NIS was seen in the thyroid/salivary glands, stomach, and bladder, as expected.
PET imaging of NIS+CART cells efficiently detects trafficking to tumor sites
Having demonstrated that NIS+CART cells provided a sensitive method of detection by 18F-TFB-PET and did not have impaired effector functions, we utilized this platform to assess CART-cell trafficking to tumor sites first using a MM xenograft model. Here, NSG mice were engrafted with BCMA+ OPM-2 cells (see systemic model in Materials and Methods; Fig. 4A; Supplementary Fig. S6A). A K562 xenograft model was established as a negative control (Supplementary Fig. S6B). Three weeks after inoculation of OPM-2 cells, tumor burden was assessed by BLI, and mice were then randomized to receive UTD, BCMA-CART, or NIS+BCMA-CART cells via tail vein injection. Mice were then serially imaged with BLI as a measure of disease burden and 18F-TFB-PET to assess CART-cell expansion and trafficking. 18F-TFB-PET suggested trafficking of NIS+BCMA-CART cells to the bone marrow, corresponding to the MM tumor site based on BLI (Fig. 4B; Supplementary Video S1A; Supplementary Fig. S6A). To have a better assessment of NIS+CART-cell trafficking, NIS physiologic uptake was removed from the PET images using PMOD software (Fig. 4B). Supplementary Figure S6A and S6B depicts PET images that include the background NIS signal. Both BCMA-CART and NIS+BCMA-CART cells exhibited similar antitumor activity and resulted in complete remission (Fig. 4C) and prolonged overall survival of the mice (*, P = 0.01, log-rank test; Fig. 4D). 18F-TFB-PET imaging did not detect NIS+BCMA-CART cells in K562 xenografts (Supplementary Fig. S6B). Quantitative assessment of 18F-TFB uptake in the tumor sites revealed significantly higher uptake in OPM-2 xenografts compared with K562 xenografts (Supplementary Fig. S6C). K562 xenografts had a significantly worse survival compared with OPM-2 xenografts (Supplementary Fig. S6D) following treatment with NIS+BCMA-CART cells, indicating antigen-specific activation and antitumor activity of NIS+BCMA-CART cells in vivo. Bone marrow samples were harvested and analyzed by flow cytometry. Analyses confirmed infiltration of OPM-2 cells in the bone marrow, which correlated with BLI findings (Fig. 4E).
Next, we intended to further demonstrate the efficiency of this technology to assess NIS+CART-cell trafficking to tumor sites using subcutaneous Nalm6 and OPM-2 xenograft models. K562 xenografts were used as negative controls. After engraftment was confirmed by BLI, Nalm6 xenografts were randomized to receive either CART19 or NIS+CART19 cells (5 × 106 intravenously), whereas OPM-2 xenografts were randomized to receive BCMA-CART or NIS+BCMA-CART cells (5 × 106 intravenously). Mice underwent serial BLI to determine disease burden and serial 18F-TFB-PET imaging to track CART-cell trafficking. The administration of 18F-TFB was performed through tail vein injection 45 minutes prior to imaging. To visualize the NIS+CART-cell trafficking clearly in PET images, background signal from thyroid, salivary glands, stomach, and bladder were removed using PMOD software. In both models, 18F-TFB-PET demonstrated trafficking of NIS+CART19 and NIS+BCMA-CART cells to the subcutaneous tumor sites (Fig. 5A and C; Supplementary Video S2) 7 days after CART-cell injection, whereas NIS+CART cells or NIS+BCMA-CART cells did not traffic to K562 tumors (Supplementary Fig. S7A and S7B). Quantitative analysis of 18F-TFB uptake in the tumor sites revealed that Nalm6 xenografts showed significantly higher NIS+CART19 cell accumulation compared with K562 xenografts (Fig. 5B). Similar to Nalm6 xenografts, OPM-2 xenografts revealed higher 18F-TFB uptake of NIS+BCMA-CART cell compared with K562 xenografts (Fig. 5D).
18F-TFB-PET detects massive NIS+CART-cell expansion in a CRS xenograft model
Finally, we explored whether 18F-TFB-PET imaging could detect CART-cell expansion in vivo and whether this correlated with the development of CRS. We used a patient-derived xenograft model for CART-cell toxicity (see Materials and Methods and experimental schema in Fig. 6A). Once mice developed high leukemic burden (defined as human CD19+ cells ≥10 cell/μL, Fig 6B), they were randomized to receive either NIS+CART19 cells (5 × 106 cells intravenously) or PBS. Within 5 to 7 days after NIS+CART19 cell treatment, the majority of treated mice developed muscle weakness, hunched bodies, and weight loss, as expected in this model (Fig. 6C; ref. 32). However, two mice treated with NIS+CART19 cells did not develop any weakness or symptoms and were therefore used as internal controls. 18F-TFB-PET imaging was performed on day 6 post-treatment. In mice treated with vehicle control, 18F-TFB-PET imaging only demonstrated physiologic 18F-TFB uptake in the thyroid and stomach, as expected (Supplementary Fig. S8A). In mice that developed CRS symptoms after NIS+CART19 cell treatment, 18F-TFB-PET imaging revealed extensive 18F-TFB uptake in the bone marrow, spleen, liver, and lungs (“high 18F-TFB uptake,” Fig 6D), whereas in NIS+CART19 cell-treated mice that did not develop CRS symptoms, 18F-TFB-PET imaging detected limited 18F-TFB uptake in the spleen (“low 18F-TFB uptake,” Fig. 6E). Quantitative analysis of 18F-TFB uptake was significantly higher in mice that developed CRS compared with mice that did not develop CRS (Fig. 6F). Multiplex analysis of serum cytokines and chemokines 6 days after CART-cell injection demonstrated a significant elevation of GMCSF, TNFα, IFNγ, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and MIP-1β in mice that developed CRS symptoms, correlating with the high 18F-TFB uptake, but not in mice that did not develop CRS symptoms (Fig. 6G). Flow cytometric analysis of peripheral blood samples 6 days following treatment with NIS+CART19 cells demonstrated a significant expansion of T cells in the mice that developed CRS, but not in the mice with no CRS symptoms (Fig. 6H; Supplementary Fig. S8B). This experiment suggested a correlation between18F-TFB uptake with the development of CRS symptoms, T-cell expansion, and cytokine elevation in this CART-cell model.
In this study, we report the development and characterization of NIS as a sensitive, specific, and clinically relevant reporter platform to image CART cells by PET scan, to track their trafficking and in vivo expansion, and to potentially predict their toxicities. We show that the incorporation of the NIS transgene into CART cells did not impair their function or antitumor activity in vitro or in vivo. 18F-TFB-PET imaging of NIS+CART cells successfully detected trafficking to tumor sites in xenograft models. 18F-TFB-PET imaging of NIS+CART cells also efficiently detected CART-cell in vivo expansion and correlated with the development of clinical and laboratory CRS markers in models of toxicity after CART-cell administration.
The two major challenges for the wider application of CART-cell therapy in the clinic are limited tumor responses and the development of life-threatening toxicities. Despite the significant overall response rates after CART-cell therapies in hematologic malignancies, rates of durable responses are low, and most patients relapse within the first 1–2 years (7, 12, 34). CART-cell activity in solid tumors is extremely limited, and mechanisms for suboptimal activity are complex, including lack of ideal targets, inadequate T-cell expansion, poor trafficking to tumor sites, and inhibition by the tumor microenvironment (14–19, 35).
Because CART-cell therapy is becoming widely used in the clinic, the associated toxicities, which are dissimilar from conventional cancer therapies, are being increasingly recognized as potentially life-threatening, challenging to predict and treat, and detrimental to the broader adoption of CART-cell therapy beyond select treatment centers. The most commonly observed toxicity of CART-cell therapies is CRS, which often requires close monitoring and treatment in the intensive care unit (36). The pivotal clinical trials reported an incidence of grade ≥3 CRS in 13% to 22% of patients after CART-cell therapy (8, 12, 37). In the clinic, the development of CRS is associated with increased proliferation of T-cells and elevation of inflammatory and effector cytokines (8, 12, 13, 38).
Development of an efficient system to image CART cells in vivo can therefore represent a strategy to monitor both their trafficking to tumor sites and expansion. Several in vivo imaging strategies for adoptive cell therapy using reporter gene systems have been described. Constitutive expression of a transporter represents an attractive strategy because reporter expression is not affected by cell division (39, 40). HSV-1-TK has been widely utilized to image adoptive cells in vivo (41–44). Najjar and colleagues have successfully imaged CART cells in vivo using engineered T cells that express CD19-directed CAR and HSV-1-TK (45). This reporter system was also tested in IL13Rα2-directed CART cells in a clinical trial for patients with glioblastoma (21). Although this platform was able to efficiently detect trafficking of HSV-1-TK+CART cells, there was nonspecific uptake of the tracer, 18F-FHBG (fluoro-3-(hydroxymethyl) butylguanine), in the brain before infusion of CART cells. The high background seen with HSV-1-TK transporter is a major limitation for the accurate assessment of cell trafficking or expansion. Another disadvantage of HSV-1-TK platform is its immunogenicity, which can lead to immune-mediated elimination of infused cells. This has been demonstrated in early clinical trials of adoptive immunotherapy of HSV-1-TK+ T cells (26, 46).
The SSTR2 reporter system has also been investigated as an imaging platform for CART cells. It has been demonstrated to have high sensitivity with the ability to detect as low as 50,000 target cells via PET scan (25). The main challenge of SSTR2 is its expression in endogenous T cells, which can interfere with T-cell activation and potentially negatively impact antitumor efficacy (25). eDHFR has been introduced as another strategy for CART-cell imaging (23). Although eDHFR allows high sensitivity in imaging CART cells in vivo, similar to HSV-1-TK, eDHFR has a significant disadvantage due to its immunogenicity, which could result in patient rejection of eDHFR+ cellular products.
PSMA-targeted PET imaging has also been described as a sensitive reporter to image CART cells that are engineered to express PSMA (20). This system possesses high sensitivity, does not impair CART-cell functions, and efficiently tracks CART-cell trafficking. However, at least an hour is needed for the PSMA+CART cells to fully uptake the 18F-DCFPyL radiotracer.
NIS is an alternative reporter system which has been shown to be a sensitive strategy for PET imaging in viral and cell therapy clinical trials (33, 47–49). There are several advantages for this reporter platform over others. First, the endogenous expression of NIS is restricted to the thyroid, salivary glands, and the stomach at low levels (28); therefore, it does not interfere with imaging of the vast majority of other organs (49). Second, to detect positive cells, imaging of NIS+ cells depends on the ATP-driven cellular Na+/K+ gradient (28, 50), resulting in an enhanced overall sensitivity for this platform (28). Third, unlike HSV-1-TK or eDHFR, NIS is a human protein and therefore does not carry any risk for immunogenicity. Finally and most importantly, the NIS transgene is uniquely characterized by its rapid uptake of radiotracers. The majority of 18F-TFB uptake by NIS+ cells occurs over the first 10 minutes, with some slower uptake occurring up to 30 minutes after the administration of the tracer (33). In the other reporter systems, such as SSTR2 or HSV-1-TK, more than 1 hour is needed for the tracer to be taken up by the targeted cells. 18F-TFB, the most commonly used radiotracer for NIS, has a short half-life of 110 minutes which, when combined with its rapid clearance, provides a lower amount of radiation to the patient. The advantages and disadvantages of different reporter systems used in CART-cell imaging have been reviewed previously (51).
The incorporation of NIS in CART-cell imaging has been reported in two studies (30, 52). Both studies validated our findings that NIS is a sensitive reporter platform for CART-cell imaging. Emami-Shahri and colleagues report a preclinical study using NIS for real-time trafficking of PSMA-directed CART cells. NIS+CART cells were able to eliminate tumor cells in vivo with a high sensitivity. SPECT/CT was used to visualize CART cells, resulting in lower resolution of assessment of CART-cell trafficking, and no characterization of CART-cell expansion or antitumor activity was performed (30). Volpe and colleagues reported the PET imaging of NIS+CART cells in breast cancer models. In our study, we chose to utilize mouse models of hematologic malignancies to allow us to study both CART-cell trafficking and their in vivo expansion in toxicity models. We demonstrated that the incorporation of NIS in CART19 or BCMA-CART cells did not impair their antitumor activity or other effector functions. We also showed that NIS is a sensitive and specific platform for CART-cell imaging to detect their trafficking to tumor sites and in vivo expansion, correlating with the development of CRS. The main limitation of the NIS-based imaging platform is its background uptake in some organs, such as the thyroid, stomach, and salivary glands due to their endogenous expression of NIS. This will pose a challenge to the visualization of T cells trafficking to these organs but does not represent a major issue in patients with hematologic malignancies or solid tumors.
In summary, the NIS transporter system provides a sensitive, clinically applicable platform for direct visualization of CART cells by 18F-TFB-PET imaging. This platform can be used for dynamic in vivo monitoring of CART-cell expansion and trafficking, as well as for the assessment of CART-associated toxicities.
R. Sakemura reports grants from Henry J. Predolin Foundation during the conduct of the study, as well as a patent for CAR immunotherapy licensed to Humanigen. M.J. Cox reports a patent and royalties in the field of CART-cell therapy, outside of this work, that is licensed to Humanigen. S.A. Parikh reports grants and other support from Pharmacyclics, Janssen, and AstraZeneca; grants from TG Therapeutics, Merck, AbbVie, and Ascentage Pharma; and other support from Genentech, Innate Pharma, Adaptive Biotechnologies, and GlaxoSmithKline outside the submitted work. T.R. DeGrado reports a patent for 18F-tetrafluoroborate chemistry pending. K.-W. Peng reports other support from Imanis Life Sciences outside the submitted work, as well as a patent for NIS reporter gene technology licensed and with royalties paid from Imanis Life Sciences. S.S. Kenderian reports grants from Novartis, Gilead/Kite, Juno/Bristol-Myers Squibb, Humanigen, Lentigen, Leahlabs, Morphosys, Sunesis, and Tolero outside the submitted work, as well as patents and royalties in the field of CART-cell therapy, outside of this work, that are licensed to Novartis, Humanigen, and Mettaforge. No disclosures were reported by the other authors.
R. Sakemura: Conceptualization, data curation, formal analysis, investigation, methodology, writing–original draft, project administration. A. Bansal: Formal analysis, investigation, visualization, methodology. E.L. Siegler: Writing–review and editing. M.J. Hefazi: Investigation, writing–review and editing. N. Yang: Investigation. R.H. Khadka: Investigation, visualization. A.N. Newsom: Investigation. M.J. Hansen: Investigation. M.J. Cox: Investigation, writing–review and editing. C. Manriquez Roman: Investigation. K.J. Schick: Investigation. I. Can: Investigation. E.E. Tapper: Investigation. W.K. Nevala: Investigation. M.M. Adada: Data curation. E.D. Bezerra: Data curation. L.A. Kankeu Fonkoua: Data curation. P. Horvei: Investigation. M.W. Ruff: Investigation. S.A. Parikh: Writing–review and editing. M.K. Pandey: Resources. T.R. DeGrado: Resources, writing–review and editing. L. Suksanpaisan: Resources, software, formal analysis, supervision, investigation, visualization, methodology. N.E. Kay: Writing–review and editing. K.-W. Peng: Supervision, writing–review and editing. S.J. Russell: Supervision, writing–review and editing. S.S. Kenderian: Conceptualization, resources, supervision, funding acquisition, methodology, project administration, writing–review and editing.
This work was supported through grant K12CA090628 (S.S. Kenderian), the Mayo Clinic Center for Individualized Medicine (S.S. Kenderian), the Predolin Foundation (R. Sakemura), the Schulze Foundation (S.S. Kenderian), and the Exact Sciences Corporation (S.S. Kenderian). The authors are grateful to Dianna L. Glynn and Cynthia J. Vernon for their technical assistance during PET imaging.
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