This work was partly supported through the National Institutes of Health (R37CA266344, 1K99CA273304), Department of Defense (CA201127), Mayo Clinic K2R pipeline (S.S.K.), the Mayo Clinic Center for Individualized Medicine (S.S.K.), and the Predolin Foundation (R.L.S.). In addition, we would like to thank the Mayo Clinic NMR Core Facility staff. Figure 1 was created in BioRender.com

This protocol follows the guidelines of Mayo Clinic&#39;s Institutional Review Board (IRB), Institutional Animal Care and Use Committee (IACUC A00001767), and Institutional Biosafety Committee (IBC, Bios00000006.04).
NOTE: All the materials used to work with mice must be sterile.
1. Injection of busulfan to NSG mice
<ol>
	<li>Obtain male, 8-12 weeks old, immunocompromised, NOD-SCID IL2r&#947;null (NSG) mice, and weigh them prior to injection.<br /...

S.S.K. is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis) and to Mettaforge (through Mayo Clinic). R.L.S. and S.S.K. are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen. S.S.K. receives research funding from Kite, Gilead, Juno, Celgene, Novartis, Humanigen, MorphoSys, Tolero, Sunesis, Leahlabs, and Lentigen.

In this report, a methodology to assess CART cell-associated toxicities using an ALL PDX model has been described. More specifically, this model seeks to mimic two life-threatening toxicities, CRS and NI, that patients often experience after the infusion of CART cells. It recapitulates many hallmarks of CART toxicities observed in the clinic: weight loss, motor dysfunction, neuroinflammation, inflammatory cytokine and chemokine production, and the infiltration of different effector cells into the central nervous system<s...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64535">Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model</a>. The Authors section was updated from:
Claudia Manriquez Roman1,2,3,4,5 
R. Leo Sakemura1,2 
Fang Jin6 
Roman H. Khadka6 
Mohamad M. Adada1,2 
Elizabeth L. Siegler1,2 
Aaron J. Johnson6 
Saad S. Kenderian1,2,6 
1T Cell Engineering Laboratory, Mayo Clinic, Rochester, 
2Division of Hematology, Mayo Clinic, Rochester, 
3Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, 
4Department of Molecular Medicine, Mayo Clinic, Rochester, 
5Regenerative Sciences PhD Program, Mayo Clinic, Rochester, 
6Department of Immunology, Mayo Clinic, Rochester
to:
Claudia Manriquez Roman1,2,3,4,5 
R. Leo Sakemura1,2 
Brooke L. Kimball1,2 
Fang Jin6 
Roman H. Khadka6 
Mohamad M. Adada1,2 
Elizabeth L. Siegler1,2 
Aaron J. Johnson6 
Saad S. Kenderian1,2,6 
1T Cell Engineering Laboratory, Mayo Clinic, Rochester, 
2Division of Hematology, Mayo Clinic, Rochester, 
3Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, 
4Department of Molecular Medicine, Mayo Clinic, Rochester, 
5Regenerative Sciences PhD Program, Mayo Clinic, Rochester, 
6Department of Immunology, Mayo Clinic, Rochester

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64535">Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model</a>. The Authors section was updated.

Erratum: 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 revolutionized the field of cancer immunotherapy. It has proven to be successful in treating relapsed/refractory acute lymphoblastic leukemia (ALL), large B cell lymphoma, mantle cell lymphoma, follicular lymphoma, and multiple myeloma1,2,3,4,5,6,7, leading to recent FDA approvals. Despite the initial success in clinical trials, treatment with CART cell therapy results in toxicities that are often severe and occasionally lethal. The most common toxicities after CART cell therapy include the development of CRS and NI, also referred to as immune effector cell-associated neurotoxicity syndrome (ICANS)8,9. CRS is caused due to the overactivation and massive expansion of CART cells in vivo, leading to the subsequent secretion of multiple inflammatory cytokines, including interferon-&#947;, tumor necrosis factor-&#945;, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6). This results in hypotension, high fevers, capillary leak syndrome, respiratory failure, multi-organ failure, and in some cases, death10,11. CRS develops in 50-100% of cases after CART19 cell therapy11,12,13. ICANS is another unique adverse event associated with CART cell therapy and is characterized by generalized cerebral edema, confusion, obtundation, aphasia, motor weakness, and occasionally, seizures9,14.&#160;Any grade of ICANS occurs in up to 70% of patients, and Grades 3-4 are reported in 20-30% of patients5,10,15,16. Overall, CRS and ICANS are common and can be fatal.
The management of ICANS after CART cell therapy is challenging. Most patients with ICANS also experience CRS17, which can often be treated with the IL-6 receptor antagonist tocilizumab or steroids18. A previous report revealed that early intervention with tocilizumab decreased the rate of severe CRS but did not affect the incidence or severity of ICANS19. Currently, there is no effective treatment or prophylactic agent for ICANS, and it is crucial to investigate preventive strategies20.
Myeloid cells and associated cytokines/chemokines are thought to be the main drivers of the development of CRS and ICANS21. While CRS is directly related to the extreme elevation of cytokines and T cell expansion, the pathophysiology of ICANS is largely unknown22,23. Therefore, it is imperative to establish a mouse model that recapitulates these toxicities after CART cell therapy to study the mechanisms and develop preventive strategies.
There are multiple preclinical animal models currently used to study, optimize, and validate the efficacy of CART cells, as well as to monitor their associated toxicities. These include syngeneic, xenograft, immunocompetent transgenic, humanized transgenic, and patient-derived xenograft mice, in addition to primate models. However, each of these models has drawbacks, and some do not reflect the true efficacy or safety concerns of CART cells24,25. Therefore, it is imperative to carefully choose the best model for the intended goals of the study.
This article seeks to describe the methodology that is used to assess CART cell-associated toxicities, CRS and NI, using an ALL patient-derived xenograft (PDX) in vivo model (Figure 1). Specifically, in the methods described here, CART19 cells generated in the authors&#39; laboratory are used following previously described protocols. Briefly, human T cells are isolated from healthy donor peripheral blood mononuclear cells (PBMCs) via a density gradient technique, stimulated with CD3/CD28 beads on day 0, and lentivirally transduced on day 1 with CARs composed of a CD19-targeted single chain variable fragment fused to 4-1BB and CD3&#950; signaling domains. These CART cells are then expanded, de-beaded on day 6, and cryopreserved on day 826,27,28,29,30. As outlined previously, mice are subjected to lymphodepleting treatment, followed by the administration of patient-derived leukemic blasts (ALL)28. First, tumor engraftment is verified via submandibular blood collection. Following the establishment of an appropriate tumor burden, CART19 cells are administered to the mice. Then, the mice are weighed daily to assess well-being. Small animal magnetic resonance imaging (MRI) is performed to assess NI, along with tail bleeding to assess T cell expansion and cytokine/chemokine production. The techniques described below are highly recommended to be used as a model to study CART cell-associated toxicities in a PDX model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;APC Anti-Human CD19</td>
			<td>Biolegend</td>
			<td>302211</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep Pad</td>
			<td>Wecol</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Analyze 14.0 software</td>
			<td>AnalyzeDirect Inc.</td>
			<td>N/A</td>
			<td>https://analyzedirect.com/analyze14/</td>
		</tr>
		<tr>
			<td>Artificial tears (Mineral oil and petrolatum)</td>
			<td>Akorn</td>
			<td>17478-062-35</td>
			<td>Topical ophtalmic gel to prevent eye dryness</td>
		</tr>
		<tr>
			<td>BD FACS Lysing Solution&#160;</td>
			<td>BD</td>
			<td>349202</td>
			<td>Red blood cells lysing buffer</td>
		</tr>
		<tr>
			<td>BD Micro-Fin IV insulin syringes</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Brillian Violet 421 Anti-Human CD45</td>
			<td>Biolegend</td>
			<td>304032</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Avance II 7 Tesla&#160;</td>
			<td>Bruker Biospin</td>
			<td>N/A</td>
			<td>MRI machine</td>
		</tr>
		<tr>
			<td>Busulfan (NSC-750)</td>
			<td>Selleckchem</td>
			<td>S1692</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright absolute counting beads</td>
			<td>Invitrogen</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX System B4-R2-V2</td>
			<td>Beckman Coulter</td>
			<td>C10343</td>
			<td>flow cytometer</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>ERT Control/Gating Module&#160;</td>
			<td>SA Instruments</td>
			<td>Model 1030</td>
			<td>Small Animal Monitoring Respiratory and Gating System</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Millipore Sigma</td>
			<td>F8067</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Bright-Line</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Human AB Serum; Male Donors; type AB; US</td>
			<td>Corning</td>
			<td>35-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (Liquid)</td>
			<td>Sigma-Aldrich</td>
			<td>792632</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>Microvette 500 Lithium heparin</td>
			<td>Sarstedt</td>
			<td>20.1345.100</td>
			<td>Blood collection tube</td>
		</tr>
		<tr>
			<td>MILLIPLEX Huma/Cytokine/Chemokine Magnetic Beads Panel</td>
			<td>Millipore Sigma</td>
			<td>HCYTMAG-60K-PX38</td>
			<td>Immunology Multiplex Assay to identify cytokines and chemokines</td>
		</tr>
		<tr>
			<td>Omniscan</td>
			<td>Ge Healthcare Inc.</td>
			<td>0407-0690-10</td>
			<td>Gadolinium-based constrast agent</td>
		</tr>
		<tr>
			<td>Pd Anti-Mouse CD45</td>
			<td>Biolegend</td>
			<td>103106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Glutamine (100x), Liquid</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom Polysterene Test tube</td>
			<td>Corning</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide, 5% (w/v)</td>
			<td>Ricca Chemical</td>
			<td>7144.8-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Surgical Blade</td>
			<td>Bard-Parker</td>
			<td>371215</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>

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.

The aim of this protocol is to assess CART cell-associated toxicities using a PDX mice model from tumor cells of patients with ALL (Figure 1). First, NSG mice received i.p. injections of busulfan (30 mg/kg) with the goal of immunosuppressing them and facilitating CART cell engraftment28. The following day, they received ~5 &#215; 106 PBMCs (i.v.) derived from ALL patients. The mice were monitored for engraftment for ~13 weeks via the tail bleeding ...

Watch this Scientific Journal Video about Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model at JoVE.com

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

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 ...

This protocol utilizes human CAR-T and tumor cells and accurately demonstrates the toxicities associated with CART19 administration. Therefore, recapitulating what has been observed in the clinic. The main advantage of using this platform is that it provides a translatable and clinically relevant model where human T-cells and tumor cells are utilized.This model not only allows efficient assessment of CART19 associated toxicities, but also allows us to understand better how we can overcome these toxicities through improved treatment strategies. To begin, monitor the CART19 administered mice twice a day to assess any changes in their wellbeing, such as motor weakness, hunched body, and loss of body weight. Once the mice develop motor weakness and weight reduction, collect their peripheral blood to analyze the tumor burden.And conduct serum isolation for cytokines, as described in the manuscript. After isolation, store the serum micro-centrifuge tubes at 80 degrees Celsius, and use the cera to analyze the chemokines and cytokines using the multiplex assay. For MRI imaging, mix 120 microliters of gadolinium with 880 microliters of saline solution, and load into a one-milliliter insulin syringe.Inject 100 microliters of the prepared solution intraperitoneally into each mouse. Anesthetize the mouse and place it on the rodent-compatible cradle probe. Then hook its teeth on the bite bar.Pull the head of the mouse into the 25-millimeter volume coil, and adjust the nose cone tethered to the isoflurane anesthesia system. Tighten the knob of the bite bar to maintain the position for the duration of the scan. For breathing assessment, attach the probe of a breathing monitoring device close to the diaphragm, and secure it with surgical tape.Keep the respiration rate between 20 to 60 breaths-per-minute so that the mouses condition remains stable. Insert the animal probe into the small bore within the Vertical Bore Small Animal MRI system and adjust the animal's head at the center of the coil. Using the lock, secure the apparatus in the upright position and connect the instrument to the computer.Next, open the ParaVision software to set up the scan positions and types of scans. Determine the optimal sagittal and axial positions while keeping them consistent for all the experimental groups. To collect the T1-weighted MRI data, use T1-weighted multi-slice, multi-echo sequence with a repetition time of 300 milliseconds, an echo time of 9.5 milliseconds, a field of view of 4.00 X 2.00 X 2.00 centimeters, and a matrix of 192 X 96 X 96.For T2-weighted MRI images, use a multi-slice, multi-echo sequence with similar parameters. Once the scans are complete, remove the probe from the bore and gently extract the mouse by removing the teeth off the bite bar. After extracting data from the software, use analysis software for the quantification and 3D volume rendering of the hyperintensity regions CD19+tumor cell populations were compared between mice treated with CART19, and untreated mice using flow cytometry.Significant weight reduction was observed in mice treated with CART19 cells. Weight loss is considered as a symptom of CRS appearance, which is related to CART-cell associated toxicities. A multiplex assay revealed the expression of cytokines and chemokines in NSG mice serum before and after CART19 administration.T2-weighted images revealed evidence of edema and possible inflammatory infiltrate during neuroinflammation in CART19 treated mice. While the T1-weighted MRI showed contrast enhancement within the brain parenchyma, indicating increased vascular permeability. Three-dimensional reconstructions of the rodent brain were assembled based on T1 hyperintensity regions corresponding to vascular permeability, which renders the volume of gadolinium leakage in the brain.It's important to remember that the daily physical monitoring of the mice after CART19 administration, along with peripheral bleeding, will be the best indicator to proceed with MRI for fully assessing any changes in the brain indicative of CRS or neurotoxicity. This technology is widely applicable to additional CAR-T-cell therapies, and will allow scientists to efficiently test potential toxicities arising from novel CAR-T cells. This proposed model is a stepping stone in understanding how CAR-T-cell associated toxicities can be monitored, treated, and used to validate new CAR-T-cell models.

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Cancer Research

1.2K vistas.  Mayo Clinic, Rochester. Este protocolo utiliza células tumorales y CAR-T humanas y demuestra con precisión las toxicidades asociadas con la administración de CART19. Por lo tanto, recapitulando lo observado en la clínica. La principal ventaja de utilizar esta plataforma es que proporciona un modelo traducible y clínicamente relevante en el que se utilizan células T humanas y células tumorales.Este modelo no solo permite una evaluación eficiente de las toxicidades asociadas a CART19, sino que también ...

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