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).&#160;Figures 1, 2, and 4 were created with BioRender.com.

SSK is an inventor on patents in CAR immunotherapy licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis) and Mettaforge (through Mayo Clinic). RS, MJC, and SSK are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen. SSK receives research funding from Kite, Gilead, Juno, Celgene, Novartis, Humanigen, MorphoSys, Tolero, Sunesis, Leahlabs, and Lentigen. Figures were created with BioRender.com.

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 ma...

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&#160;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 &#34;real-time&#34; 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&#160;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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>22 Gauge needle</td>
			<td>Covidien</td>
			<td>8881250206</td>
			<td></td>
		</tr>
		<tr>
			<td>28 gauge insulin syringe</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human (ETNL) NIS</td>
			<td>Imanis</td>
			<td>REA009</td>
			<td>ETNL antibody binds the cytosolic C-terminus of NIS</td>
		</tr>
		<tr>
			<td>Anti-human BCMA, clone 19F2, PE-Cy7</td>
			<td>BioLegend</td>
			<td>357507</td>
			<td>Flow antibody</td>
		</tr>
		<tr>
			<td>Anti-human CD45, clone HI30, BV421</td>
			<td>BioLegend</td>
			<td>304032</td>
			<td>Flow antibody</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45, clone 30-F11, APC-Cy7</td>
			<td>BioLegend</td>
			<td>103116</td>
			<td>Flow antibody</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG</td>
			<td>R&#38;D</td>
			<td>F0110</td>
			<td>Secondary antibody for NIS staining</td>
		</tr>
		<tr>
			<td>BCMA-CAR construct, second generation</td>
			<td>IDT, Coralville, IA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 Monoclonal Antibody (OKT3), PE, eBioscience</td>
			<td>Invitrogen</td>
			<td>12-0037-42</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td>
			<td>Gibco</td>
			<td>40203D</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX System&#160; B5-R3-V5</td>
			<td>Beckman Coulter</td>
			<td>C04652</td>
			<td>flow cytometer</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Millipore Sigma</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Syringes with Luer-Lok Tips</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Gold Biotechnology</td>
			<td>LUCK-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (Dulbecco&#39;s phosphate-buffered saline)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MPC-S (Magnetic Particle Concentrator)</td>
			<td>Applied Biosystems</td>
			<td>A13346</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human T Cell Isolation Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17951</td>
			<td>negative selection magnetic beads; 17951RF includes tips and buffer</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Millipore Sigma</td>
			<td>F8067</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A-21235</td>
			<td></td>
		</tr>
		<tr>
			<td>Inveon Multiple Modality PET/CT scanner</td>
			<td>Siemens Medical Solutions USA, Inc.</td>
			<td>10506989 VFT 000 03</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane liquid</td>
			<td>Piramal Critical Care</td>
			<td>66794-017-10</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina S5 Imaging System</td>
			<td>PerkinElmer</td>
			<td>CLS148588</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS&#174; Spectrum In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>&#160;124262</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>L3000075</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen</td>
			<td>L34966</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>STEMCELL Technologies</td>
			<td>07851</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.22 uM, sterile</td>
			<td>Thermo Scientific</td>
			<td>450-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow 500 mL Vacuum Filter, 0.45 uM, sterile</td>
			<td>Thermo Scientific</td>
			<td>450-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid&#160;Il2rgtm1Wjl/SzJ</td>
			<td>Jackson laboratory</td>
			<td>05557</td>
			<td></td>
		</tr>
		<tr>
			<td>OPM-2</td>
			<td>DSMZ</td>
			<td>CRL-3273</td>
			<td>multiple myeloma cell line</td>
		</tr>
		<tr>
			<td>pBMN(CMV-copGFP-Luc2-Puro)</td>
			<td>Addgene</td>
			<td>80389</td>
			<td>lentiviral vector encoding luciferase-GFP</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Glutamine (100x), Liquid</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>PMOD software</td>
			<td>PMOD</td>
			<td>PBAS and P3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Pooled Human AB Serum Plasma Derived</td>
			<td>Innovative Research</td>
			<td>IPLA-SERAB-H-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin Dihydrochloride</td>
			<td>MP Biomedicals, Inc.</td>
			<td>0210055210</td>
			<td></td>
		</tr>
		<tr>
			<td>RoboSep-S</td>
			<td>STEMCELL Technologies</td>
			<td>21000</td>
			<td>Fully Automated Cell Separator</td>
		</tr>
		<tr>
			<td>RPMI (Roswell Park Memorial Institute (RPMI) 1640 Medium)</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td></td>
		</tr>
		<tr>
			<td>SepMate-50 (IVD)</td>
			<td>STEMCELL Technologies</td>
			<td>85450</td>
			<td>density gradient separation tubes</td>
		</tr>
		<tr>
			<td>Sodium Azide, 5% (w/v)</td>
			<td>Ricca Chemical</td>
			<td>7144.8-16</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 flask</td>
			<td>Corning</td>
			<td>353112</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrell (isoflurane, USP)</td>
			<td>Piramal Critical Care Inc</td>
			<td>66794-019-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Webcol Alcohol Prep</td>
			<td>Covidien</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic imaging of chimeric antigen receptor t cells with [18f]tetrafluoroborate positron emission tomography/computed tomography

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.

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 &#215; 106/mL. For NIS-transduced T cells, add 1 &#956;g/mL of puromycin to select NIS+ cells. On day 6, remove t...

Watch this Scientific Journal Video about Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography at JoVE.com

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

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.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

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3.2K Views.  Mayo Clinic. 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.

Video: Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

Positron emission tomography (PET) combined with X-ray computed tomography (CT) is an important molecular imaging platform that is required for accurate diagnosis and clinical staging of a variety of diseases. The advantage of PET imaging is the ability to visualize and quantify a myriad of biological processes in vivo with high sensitivity and accuracy. However, there are multiple factors that determine image quality and quantitative accuracy of PET images. One of the foremost factors influencing image quality in PET imaging of the thorax and upper abdomen is respiratory motion, resulting in respiration-induced motion blurring of anatomical structures. Correction of these artefacts is required for providing optimal image quality and quantitative accuracy of PET images.
Several respiratory gating techniques have been developed, typically relying on acquisition of a respiratory signal simultaneously with PET data. Based on the respiratory signal acquired, PET data is selected for reconstruction of a motion-free image. Although these methods have been shown to effectively remove respiratory motion artefacts from PET images, the performance is dependent on the quality of the respiratory signal being acquired. In this study, the use of an amplitude-based optimal respiratory gating (ORG) algorithm is discussed. In contrast to many other respiratory gating algorithms, ORG permits the user to have control over image quality versus the amount of rejected motion in the reconstructed PET images. This is achieved by calculating an optimal amplitude range based on the acquired surrogate signal and a user-specified duty cycle (the percentage of PET data used for image reconstruction). The optimal amplitude range is defined as the smallest amplitude range still containing the amount of PET data required for image reconstruction. It was shown that ORG results in effective removal of respiration-induced image blurring in PET imaging of the thorax and upper abdomen, resulting in improved image quality and quantitative accuracy.

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

Amplitude-based optimal respiratory gating (ORG) effectively removes respiratory-induced motion blurring from clinical 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) images. Correction of FDG-PET images for these respiratory motion artefacts improves image quality, diagnostic and quantitative accuracy. Removal of respiratory motion artefacts is important for adequate clinical management of patients using PET.

Positron emission tomography (PET) combined with X-ray computed tomography (CT) is an important molecular imaging platform that is required for accurate ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Evaluating Quantitative Tumor Cell Killing by CAR-Expressing Jurkat Cells

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory 

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...