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使用[18F]四氟硼酸盐正电子发射断层扫描/计算机断层扫描对嵌合抗原受体T细胞进行动态成像

Published: February 17, 2022
doi:
10.3791/62334
, , , , , , , , , ,
1T Cell Engineering,Mayo Clinic, 2Division of Hematology,Mayo Clinic, 3Department of Radiology,Mayo Clinic, 4Regenerative Sciences PhD,Mayo Clinic, 5Department of Molecular Medicine,Mayo Clinic, 6Department of Immunology,Mayo Clinic

Summary

该协议描述了非侵入性跟踪T细胞的方法，该T细胞经过基因工程改造，以使用临床可用的平台 在体内 表达嵌合抗原受体。

Abstract

经过基因工程改造以表达嵌合抗原受体（CAR）的T细胞在B细胞恶性肿瘤或多发性骨髓瘤（MM）患者的关键临床试验中显示出前所未有的结果。然而，由于运输和浸润肿瘤部位以及体内缺乏持久性，许多障碍限制了疗效并禁止了CAR T细胞疗法的广泛使用。此外，危及生命的毒性，如细胞因子释放综合征或神经毒性，是主要问题。对CAR T细胞进行高效和灵敏的成像和跟踪，可以评估T细胞的运输，扩增和体内表征，并允许制定策略来克服CAR T细胞治疗的当前局限性。本文描述了在临床前模型中使用[18F]四氟硼酸盐 – 正电子发射断层扫描（[18F]TFB-PET）在CAR T细胞中掺入碘化钠共排（NIS）和CAR T细胞成像的方法。除了用于本研究的方法外，该协议中描述的方法还可以应用于其他CAR构建体和靶基因。

Introduction

嵌合抗原受体 T （CAR T） 细胞疗法是血液系统恶性肿瘤中一种迅速出现且具有潜在治愈意义的方法1，2，3，4，5，6。在CD19指导的CAR T（CART19）或B细胞成熟抗原（BCMA）CAR T细胞治疗后报告了非凡的临床结果2。这导致美国食品和药物管理局（FDA）批准CART19细胞用于侵袭性B细胞淋巴瘤（axicabtagene ciloleucel（Axi-Cel）4，tisagenlecleucel（Tisa-Cel）3和lisocabtagene maraleucel）7，急性淋巴细胞白血病（Tisa-Cel）5，8，套细胞淋巴瘤（brexucabtagene autoleuce）9和滤泡性淋巴瘤（Axi-Cel）10.最近，FDA批准了BCMA指导的CAR T细胞疗法用于多发性骨髓瘤（MM）（idecabtagene vicleucel）患者11。此外，用于慢性淋巴细胞白血病（CLL）的CAR T细胞疗法正处于后期临床开发阶段，预计将在未来三年内获得FDA批准1。

尽管CAR T细胞疗法取得了前所未有的成果，但其广泛使用受到以下因素的限制：1） 体内 CAR T细胞扩增不足或向肿瘤部位的运输不良，导致持久反应率降低12，13和2）危及生命的不良事件的发展，包括细胞因子释放综合征（CRS）14，15.CRS的标志不仅包括导致炎症细胞因子/趋化因子水平升高的免疫激活，还包括CAR T细胞输注后的大量T细胞增殖15，16。因此，开发一种经过验证的临床级策略 来对体内 CAR T细胞进行成像将允许1） 体内 实时CAR T细胞跟踪，以监测它们向肿瘤部位的运输并揭示潜在的耐药机制，以及2）监测CAR T细胞扩增并可能预测其毒性，例如CRS的发展。

轻度CRS的临床特征是高烧、疲劳、头痛、皮疹、腹泻、关节痛、肌痛和不适。在更严重的CRS中，患者可能出现心动过速/低血压，毛细血管渗漏，心功能不全，肾/肝衰竭和弥散性血管内凝血17，18。一般而言，细胞因子（包括干扰素-γ、粒细胞-巨噬细胞集落刺激因子、白细胞介素 （IL）-10 和 IL-6）的升高程度已被证明与临床症状的严重程度相关17，19。然而，由于成本高且可用性有限，广泛应用“实时”血清细胞因子监测来预测CRS是困难的。为了利用CAR T细胞疗法的有益特性，可以潜在地利用过继T细胞的非侵入性成像来预测CAR T细胞输注后的疗效，毒性和复发。

一些研究人员已经开发出使用基于放射性核素的成像与正电子发射断层扫描（PET）或单光子发射计算机断层扫描（SPECT）的策略，其提供高分辨率和高灵敏度20，21，22，23，24，25，26，27，28，29，30CAR T细胞转运的体内可视化和监测。在这些基于放射性核素的成像策略中，碘化钠共排量（NIS）已被开发为使用PET扫描对细胞和病毒进行成像的灵敏方式31，32。使用 [18F]TFB-PET 进行 NIS+CAR T 细胞成像是一种灵敏、高效且方便的技术，可用于评估和诊断 CAR T 细胞扩增、转运和毒性30。该协议描述了1）通过具有高效率的双重转导开发NIS + CAR T细胞和2）用[18F]TFB-PET扫描对NIS + CAR T细胞进行成像的方法。然而，这些方法可以应用于任何其他CAR T细胞疗法。

Protocol

该协议遵循Mayo Clinic机构审查委员会，机构生物安全委员会和Mayo Clinic机构动物护理和使用委员会的指导方针。 1. NIS+ BCMA-CAR-T细胞生产 注意：本议定书遵循梅奥诊所机构审查委员会（IRB 17-008762）和机构生物安全委员会（IBC Bios00000006.04）的指导方针。 生产BCMA-CAR，NIS和荧光素酶 – 绿色荧光蛋白（GFP）编码慢病毒。注：从头合成第?…

Representative Results

图1 表示生成NIS + BCMA-CAR T细胞的步骤。在第0天，分离PBMC，然后通过阴性选择分离T细胞。然后，用抗CD3 / CD28珠刺激T细胞。在第1天，用NIS和BCMA-CAR慢病毒转导T细胞。在第3天、第4天和第5天，计数T细胞并用培养基进料，将浓度调整为1.0×106 / mL。对于NIS转导的T细胞，加入1μg/ mL嘌呤霉素以选择NIS + 细胞。在第6天，通过将细胞放入磁铁中一分钟来去除珠?…

Discussion

本文描述了一种将NIS掺入CAR T细胞并通过[18F]TFB-PET在体内对注入的CAR T细胞进行成像的方法。作为概念验证，NIS + BCMA-CAR T细胞通过双转导产生。我们最近报道说，将NIS掺入CAR T细胞不会损害CAR T细胞在体内的功能和功效，并允许CAR T细胞运输和扩增30。随着CAR T细胞疗法继续从目前的B细胞恶性肿瘤扩展到CLL中的应用，对允许非侵入性体内成像和?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了Mayo Clinic K2R管道（SSK），Mayo Clinic Center for Personalized Medicine（SSK）和Predolin Foundation（RS）的部分支持。图 1、2 和图 4 是使用 BioRender.com 创建的。

Materials

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
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Tags

Keywords: CAR T-cellsPET ImagingNIS ReporterT-cell TraffickingT-cell ExpansionPBMC IsolationT-cell IsolationCell Culture[18F]tetrafluoroborate
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Sakemura, R., Cox, M. J., Bansal, A., Roman, C. M., Hefazi, M., Vernon, C. J., Glynn, D. L., Pandey, M. K., DeGrado, T. R., Siegler, E. L., Kenderian, S. S. Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography. J. Vis. Exp. (180), e62334, doi:10.3791/62334 (2022).
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