PET of Adoptively Transferred Chimeric Antigen Receptor T Cells with 89Zr-Oxine
Total Page：16
File Type：pdf, Size：1020Kb
PET of Adoptively Transferred Chimeric Antigen Receptor T Cells with 89Zr-Oxine Michael R. Weist1,2, Renate Starr3, Brenda Aguilar3, Junie Chea1, Joshua K. Miles1, Erasmus Poku1, Ethan Gerdts3, Xin Yang3, Saul J. Priceman3, Stephen J. Forman3, David Colcher1, Christine E. Brown3, and John E. Shively1 1Department of Molecular Immunology, Beckman Research Institute, City of Hope Medical Center, Duarte, California; 2Irell and Manella Graduate School of Biological Sciences, City of Hope Medical Center, Duarte, California; and 3Department of Hematology, City of Hope Medical Center, Duarte, California in CAR T cell therapy has been made over the past several decades, Chimeric antigen receptor (CAR) T cell therapy is a promising clinical as led by the clinical success of CD19-CAR T cells against refrac- approach for reducing tumor progression and prolonging patient tory B-cell malignancies, in which complete responses are achieved survival. However, improvements in both the safety and the potency in most patients (5–8). Despite ongoing progress, challenges remain of CAR T cell therapy demand quantitative imaging techniques to in improving CAR T cell therapy, including poor tumor targeting determine the distribution of cells after adoptive transfer. The purpose (9), normal-tissue toxicity (10), and lack of CAR T cell persistence 89 of this study was to optimize Zr-oxine labeling of CAR T cells and (11). Real-time imaging to determine the dynamics of CAR T cell evaluate PET as a platform for imaging adoptively transferred CAR T trafficking in both patients and animals can help address these cells. Methods: CAR T cells were labeled with 0–1.4 MBq of 89Zr- oxine per 106 cells and assessed for radioactivity retention, viability, issues. Because CAR T cells are manufactured ex vivo, they can and functionality. In vivo trafficking of 89Zr-oxine–labeled CAR T cells readily be labeled for imaging before infusion. PET offers signifi- was evaluated in 2 murine xenograft tumor models: glioblastoma brain cant advantages over other imaging modalities because it is quan- tumors with intracranially delivered IL13Rα2-targeted CAR T cells, and titative, highly sensitive, and subject to minimal tissue attenuation. subcutaneous prostate tumors with intravenously delivered prostate Optimal nuclear imaging is achieved when the radionuclide half-life stem cell antigen (PSCA)–targeted CAR T cells. Results: CAR T cells approximates the biologic action of the imaging target. Of the com- were efficiently labeled (75%) and retained more than 60% of the 89Zr mercially available PET radionuclides, 89Zr has a relatively long over6d.Invitrocytokineproduction, migration, and tumor cytotox- half-life (78.4 h) suitable for imaging CAR T cells over the days icity, as well as in vivo antitumor activity, were not significantly reduced required for trafficking and antitumor responses. when labeled with 70 kBq/106 cells. IL13Rα2-CAR T cells delivered 89Zr-oxine has previously been used for labeling and imaging intraventricularly were detectable by PET for at least 6 d throughout 89 the central nervous system and within intracranial tumors. When murine lymphocytes (12–15) but not human CAR T cells. Zr-oxine intravenously administered, PSCA-CAR T cells also showed tumor has preparation and functional characteristics similar to those of its 111 tropism, with a 9-fold greater tumor-to-muscle ratio than for CAR- SPECT counterpart, In-oxine, which has been used for many types negative T cells. Conclusion: 89Zr-oxine can be used for labeling and of cells (16). 111In-oxine has been used to visualize the trafficking and imaging CAR T cells while maintaining cell viability and function. On the tumor accumulation of g-d-T cells and HER2-primed T cells (17,18). basis of these studies, we conclude that 89Zr-oxine is a clinically trans- In comparison to SPECT, PET has higher resolution (19), making latable platform for real-time assessment of cell therapies. 89Zr-oxine an attractive choice for imaging cells. Another PET-based Key Words: 89Zr-oxine; positron emission tomography; chimeric approach for labeling cells is 18F-FDG, which is inadequate for track- antigen receptor T cells; prostate cancer; glioblastoma ing CAR T cells because of its short half-life (109.7 min) and rela- J Nucl Med 2018; 59:1531–1537 tively poor radioactivity retention (20). On the basis of the existing DOI: 10.2967/jnumed.117.206714 clinical applications of 89Zr (21) and its oxine complex, we selected 89Zr-oxine for labeling CAR T cells and imaging with PET. We present an alternative method for labeling cells with 89Zr- oxine in which 89Zr-oxine is prepared from 89Zr-oxalate and retained within CAR T cells. Before cells were imaged in vivo, we identified The genetic modification of T cells to express chimeric antigen an optimal labeling activity that maintained cell viability and func- receptors (CARs), which enable these cells to specifically target and tion while maximizing the radioactive signal for imaging. To further 89 kill cancer cells, has become an important immunotherapeutic ap- assess the robustness of this PET imaging platform, we tracked Zr- proach in the treatment of cancer (1–3). CAR T cells combine the oxine–labeled CAR T cells administered via 3 different delivery target specificity of antibodies with the intracellular signaling com- routes: intravenous, intratumoral, and intraventricular. These data ponents of the T cell receptor and costimulatory molecules to re- demonstrate the tumor tropism of adoptively transferred CAR T cells direct T cell–mediated immune responses (4). Significant progress and their distribution based on administration routes. MATERIALS AND METHODS Received Dec. 5, 2017; revision accepted Apr. 17, 2018. For correspondence or reprints contact: John E. Shively, City of Hope Labeling Cells with 89Zr-Oxine Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010. 89Zr-oxalate from 3D Imaging was aliquoted into borosilicate vials. E-mail: [email protected] 111 Published online May 4, 2018. On the basis of the specific activity of aqueous In-oxine prepara- COPYRIGHT © 2018 by the Society of Nuclear Medicine and Molecular Imaging. tions (22), a 50-mL quantity of 6.9 mM 8-hydroxyquinoline (oxine) in IMAGING CAR T CELLS WITH 89ZR-OXINE • Weist et al. 1531 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and sampled at the indicated time points. Radioactivity within the cell was added to each 37 MBq of 89Zr-oxalate, pH was adjusted to 7.4 pellet and supernatant was assayed in a g-counter (Packard Cobra with 1 M HEPES, and the solution was incubated at room temperature Auto-Gamma Counter) to determine retention (% retention 5 cell for 15 min. After 2 washings with Hanks buffered salt solution pellet radioactivity/total radioactivity · 100%). For subcellular frac- (HBSS) (without calcium, magnesium, or phenol red), CAR T cells tionation experiments, 5 · 106 cells were processed per the manufac- (20 · 106/mL) were incubated with 0–1,400 kBq of unpurified 89Zr- turer’s instructions using a subcellular fractionation kit (catalog no. oxine solution per 106 cells for 30 min at 37°C. Past 89Zr- and 111In- 78840; ThermoFisher Scientific). Each fraction was subsequently oxine reports indicate that purification of the oxine complex is not counted for radioactivity. necessary (14,15,22) and that oxine is rapidly lost from labeled cells after washing (23). The radiolabeled cells were washed twice with T Assays for CAR T Cell Function cell medium (IL15 [0.5 ng/mL] and IL2 [50 U/mL]) before in vitro After labeling and incubation under standard culture conditions, the experiments or with HBSS plus 1% human serum albumin before in cytotoxicity, cytokine production, and migration potential of labeled vivo experiments. IL13Ra2-CAR T cells were evaluated. For all experiments, cell via- bility was assessed with 0.1% propidium iodide on a MoxiFlow cas- 89 Labeling Efficiency of Zr-Oxine sette-based flow cytometer (Orflo Technologies) before labeling, after 89 The yield of the Zr-oxine synthesis was determined by instant labeling, and at the indicated time points in culture. For the nonradio- thin-layer chromatography (92% 6 0.1%, n 5 35) and reversed-phase nat nat active Zr-oxine preparation, 10 mLofa17mM ZrCl4 solution in high-performance liquid chromatography (82%) (Supplemental Fig. 1; 1 M oxalic acid was compounded with 260 mL of 6.8 mM oxine in supplemental materials are available at http://jnm.snmjournals.org). 0.1 M HEPES and adjusted to pH 7.4 with 1 M HEPES. Equivalent 89 89 Zr-oxine remained at the origin, whereas unincorporated Zr mi- volumes of natZr-oxine were added at specific activities toxic to CAR grated to the solvent front developed with 0.9% NaCl. A gradient of T cells (1.5 and 12 MBq/106 cells). 0%–100% acetonitrile with 0.1% trifluoroacetic acid on a SunFire C18 The cytotoxicities of 89Zr-oxine–labeled CAR T cells were also ˚ · 89 column (100 A, 3.5 mm, 4.6 150 mm; Waters) separated Zr-oxine monitored using biophotonic luciferase assays as described by Brown 89 89 complexes from unincorporated Zr. Given this high yield, the Zr- et al. (25). A 5 · 104 PBT030-2 quantity of ffLuc-positive cells was oxine solution was immediately used for cell labeling. coincubated with 5–50 · 10489Zr-oxine–labeled IL13Ra2-CAR T Cell Lines cells in 0.2 mL at the specified time points for 7 hours at 5% CO2 ° PBT030-2 is a low-passage, patient-derived IL13Ra2-positive and 37 C. The plates were then supplemented with luciferin to a final tumor line derived from glioblastoma tissue that was obtained in concentration of 0.14 mg/mL, incubated for 10 min in the dark, and accordance with City of Hope Institutional Review Board–approved measured for luminescence (1-s exposure) with the Victor3 plate protocols and cultured as described previously (24).