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Dynamisk billeddannelse af kimære antigenreceptor-T-celler med [18F]tetrafluorborat positronemissionstomografi/computertomografi

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

Denne protokol beskriver metoden til ikke-invasiv sporing af T-celler, der er genetisk manipuleret til at udtrykke kimære antigenreceptorer in vivo med en klinisk tilgængelig platform.

Abstract

T-celler, der er genetisk manipuleret til at udtrykke kimære antigenreceptorer (CAR), har vist hidtil usete resultater i pivotale kliniske forsøg for patienter med B-cellemaligniteter eller myelomatose (MM). Imidlertid begrænser mange hindringer effektiviteten og forbyder den udbredte anvendelse af CAR T-celleterapier på grund af dårlig handel og infiltration i tumorsteder samt manglende vedholdenhed in vivo. Desuden er livstruende toksiciteter, såsom cytokinfrigivelsessyndrom eller neurotoksicitet, store bekymringer. Effektiv og følsom billeddannelse og sporing af CAR T-celler muliggør evaluering af T-cellehandel, ekspansion og in vivo-karakterisering og muliggør udvikling af strategier til at overvinde de nuværende begrænsninger ved CAR T-celleterapi. Dette papir beskriver metoden til inkorporering af natriumiodidsymporteren (NIS) i CAR T-celler og til CAR T-cellebilleddannelse ved hjælp af [18F] tetrafluorborat-positronemissionstomografi ([18F] TFB-PET) i prækliniske modeller. De metoder, der er beskrevet i denne protokol, kan anvendes på andre CAR-konstruktioner og målgener ud over dem, der anvendes til denne undersøgelse.

Introduction

Kimær antigenreceptor T (CAR T) celleterapi er en hurtigt voksende og potentielt helbredende tilgang i hæmatologiske maligniteter1,2,3,4,5,6. Ekstraordinære kliniske resultater blev rapporteret efter CD19-rettet CAR T (CART19) eller B-cellemodningsantigen (BCMA) CAR T-celleterapi2. Dette førte til, at US Food and Drug Administration (FDA) godkendte CART19-celler til aggressivt B-cellelymfom (axicabtagen ciloleucel (Axi-Cel)4, tisagenlecleucel (Tisa-Cel)3 og lisocabtagene maraleucel)7, akut lymfoblastær leukæmi (Tisa-Cel)5,8, mantelcellelymfom (brexucabtageneautoleuce)9 og follikulært lymfom (Axi-Cel)10 . Senest har FDA godkendt BCMA-rettet CAR T-celleterapi til patienter med myelomatose (MM) (idecabtagene vicleucel)11. Desuden er CAR T-celleterapi til kronisk lymfatisk leukæmi (CLL) i sen klinisk udvikling og forventes at modtage FDA-godkendelse inden for de næste tre år1.

På trods af de hidtil usete resultater af CAR T-celleterapi er dens udbredte anvendelse begrænset af 1) utilstrækkelig in vivo CAR T-celleudvidelse eller dårlig handel med tumorsteder, hvilket fører til lavere satser for varig respons12,13 og 2) udvikling af livstruende bivirkninger, herunder cytokinfrigivelsessyndrom (CRS)14,15 . Kendetegnene ved CRS omfatter ikke kun immunaktivering, hvilket resulterer i forhøjede niveauer af inflammatoriske cytokiner / kemokiner, men også massiv T-celleproliferation efter CAR T-celleinfusion15,16. Udviklingen af en valideret strategi af klinisk kvalitet til at afbilde CAR T-celler in vivo vil således gøre det muligt for 1) CAR T-cellesporing i realtid in vivo at overvåge deres handel til tumorsteder og afdække potentielle resistensmekanismer og 2) overvågning af CAR T-celleudvidelse og potentielt forudsigelse af deres toksiciteter såsom udvikling af CRS.

Kliniske træk ved mild CRS er høj feber, træthed, hovedpine, udslæt, diarré, artralgi, myalgi og utilpashed. Ved mere alvorlig CRS kan patienter udvikle takykardi/hypotension, kapillær lækage, hjertedysfunktion, nyre-/leversvigt og dissemineret intravaskulær koagulation17,18. Generelt har graden af forhøjelse af cytokiner, herunder interferon-gamma, granulocyt-makrofagkolonistimulerende faktor, interleukin (IL)-10 og IL-6, vist sig at korrelere med sværhedsgraden af kliniske symptomer17,19. Den omfattende anvendelse af “realtids” serumcytokinovervågning til forudsigelse af CRS er imidlertid vanskelig på grund af de høje omkostninger og den begrænsede tilgængelighed. For at udnytte de gavnlige egenskaber ved CAR T-celleterapi kan ikke-invasiv billeddannelse af adoptive T-celler potentielt bruges til at forudsige effektiviteten, toksiciteterne og tilbagefaldet efter CAR T-celleinfusion.

Flere forskere har udviklet strategier til at anvende radionuklidbaseret billeddannelse med positronemissionstomografi (PET) eller enkeltfotonemissionscomputertomografi (SPECT), som giver høj opløsning og høj følsomhed20,21,22,23,24,25,26,27,28,29,30 for in vivo visualisering og overvågning af CAR T cellehandel. Blandt disse radionuklidbaserede billeddannelsesstrategier er natriumiodidsymporteren (NIS) udviklet som en følsom modalitet over for billedceller og vira ved hjælp af PET-scanninger31,32. NIS+CAR T-cellebilleddannelse med [18F]TFB-PET er en følsom, effektiv og praktisk teknologi til vurdering og diagnosticering af CAR T-celleudvidelse, -handel og –toksicitet30. Denne protokol beskriver 1) udviklingen af NIS + CAR T-celler gennem dobbelt transduktion med høj effektivitet og 2) en metode til billeddannelse af NIS + CAR T-celler med [18F] TFB-PET-scanning. BCMA-CAR T-celler til MM bruges som en proof-of-concept-model til at beskrive NIS som reporter for CAR T-cellebilleddannelse. Disse metoder kan dog anvendes på enhver anden CAR T-celleterapi.

Protocol

Protokollen følger retningslinjerne fra Mayo Clinic’s Institutional Review Board, Institutional Biosafety Committee og Mayo Clinic’s Institutional Animal Care and Use Committee. 1. NIS+ BCMA-CAR T-celleproduktion BEMÆRK: Denne protokol følger retningslinjerne fra Mayo Clinic’s Institutional Review Board (IRB 17-008762) og Institutional Biosafety Committee (IBC Bios00000006.04). Produktion af BCMA-CAR, NIS og luciferase-grønt fluorescerende p…

Representative Results

Figur 1 repræsenterer trinnene til generering af NIS + BCMA-CAR T-celler. På dag 0 skal du isolere PBMC’er og derefter isolere T-celler ved negativ selektion. Derefter stimuleres T-celler med anti-CD3 / CD28 perler. På dag 1 transduceres T-celler med både NIS og BCMA-CAR lentivirus. På dag 3, 4 og 5 tælles T-celler og fodres med medier for at justere koncentrationen til at være 1,0 × 106 / ml. For NIS-transducerede T-celler tilsættes 1 μg/ml puromycin for at v…

Discussion

Dette papir beskriver en metode til inkorporering af NIS i CAR T-celler og billeddannelse af infunderede CAR T-celler in vivo gennem [18F] TFB-PET. Som bevis på konceptet blev NIS + BCMA-CAR T-celler genereret via dobbelt transduktion. Vi har for nylig rapporteret, at inkorporering af NIS i CAR T-celler ikke forringer CAR T-cellefunktioner og effektivitet in vivo og tillader CAR T-cellehandel og –udvidelse30. Da CAR T-celleterapier fortsætter med at ekspan…

Declarações

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev delvist støttet gennem Mayo Clinic K2R pipeline (SSK), Mayo Clinic Center for Individualized Medicine (SSK) og Predolin Foundation (RS). Figur 1, 2 og 4 blev oprettet med BioRender.com.

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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