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. 
	NOTE: For statistical significance, it is recommended to use at least five mice per group and to repeat this experiment at least once more with female mice.</li>
	<li>Prepare busulfan for intraperitoneal (i.p.) injection. Using a 1 mL insulin syringe, slowly and carefully fill the syringe with exactly 30 mg/kg of busulfan, followed by i.p injection to the mice, and place them back into the cage.</li>
</ol>
2. Injection of ALL patient-derived blasts (CD19+)&#160;to the NSG mice
NOTE: This protocol follows the guidelines of Mayo Clinic&#39;s Institutional Biosafety Committee (IBC, Bios00000006.04).
<ol>
	<li>Collect primary leukemic blasts from the peripheral blood of patients with relapsed and refractory ALL.</li>
	<li>Prepare target cells for intravenous (i.v.) injection.
	<ol>
		<li>Count CD19+ target cells using a hemocytometer, and record the final volume and concentration of cells. 
		NOTE: To determine cell viability, it is strongly recommended to utilize trypan blue while counting the cells.</li>
		<li>Pellet the cells by centrifugation at 300 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the cell pellet in phosphate-buffered saline (PBS) to a final concentration of 50 &#215; 106 cells/mL in a 1.5 mL microcentrifuge tube, and place on ice.</li>
		<li>Finger-vortex the 1.5 mL microcentrifuge tube containing the target cells until the cells are completely homogenized.</li>
		<li>Using a 1 mL insulin syringe, slowly and carefully fill the syringe with exactly 100 &#181;L of the prepared target cells, and flick the syringe to remove bubbles. 
		NOTE: The volume of 100 &#181;L will administer 5 &#215; 106 cells to each mouse.</li>
		<li>Place the mice under warm light for 5-10 min. Once the tail vein becomes engorged and more visible to the naked eye, place the mice in an appropriate confinement/restraining chamber.</li>
		<li>Wipe the tail of the mouse with an alcohol swab. Inject the target cells directly into the tail vein (i.v.), and place the mouse back in the cage. After the inoculation of the target cells, monitor the mice daily or as recommended by the local animal ethics committee.</li>
	</ol>
	</li>
</ol>
3. Tumor engraftment assessment
<ol>
	<li>Starting at 6 weeks after the target cell injection, assess tumor engraftment by using flow cytometry to measure the expression of CD19+ cells in the peripheral blood.</li>
	<li>Peripheral blood assay to assess CD19+ expansion
	<ol>
		<li>Withdraw blood from the mice 6-8 weeks after CART19 cell injection. 
		NOTE: Several methods can be used to withdraw blood. For this protocol, submandibular blood collection is the preferred method of choice.</li>
		<li>Anesthetize the mouse with isoflurane in an induction chamber with 5% isoflurane. Once anesthesia is achieved, maintain at 1-3% isoflurane inhaled through a nose cone. 
		NOTE: It is strongly recommended to apply ophthalmic ointment to prevent eye dryness.</li>
		<li>Remove the mouse from the chamber, and hold the mouse with the non-dominant hand by grasping the loose skin over the neck. Puncture the vein with a lancet slightly behind the mandible. 
		NOTE: Only the tip of the needle (1-2 mm) is required to enter the vein.</li>
		<li>As the blood is dripping, collect at least 50 &#181;L in a heparin-coated tube, and mix well by inverting the tube up and down several times. Transfer 30 &#181;L of blood into a 5 mL round bottom polystyrene tube. 
		NOTE: For the remaining blood, spin down the tube at 17,000 &#215; g for 10 min at 4 &#176;C. Transfer the serum (supernatant layer) to a clean and labeled 1.5 mL microcentrifuge tube, and store it at &#8722;80 &#176;C (for use in the cytokine assay).</li>
		<li>Add 2 mL of lysis buffer (10x ammonium chloride-based lysing buffer diluted to 1x using nuclease-free water) into the 5 mL round bottom tube containing the blood. Mix very well by vortexing the tube. Incubate the tube at room temperature for 20 min to ensure proper lysis of the red blood cells.</li>
		<li>Add 2 mL of flow buffer (PBS, 0.2 g/L of potassium chloride, 0.2 g/L of potassium phosphate monobasic, 8 g/L of sodium chloride, and 1.15 g/L of sodium phosphate dibasic containing 2% fetal bovine serum and 1% sodium azide).</li>
		<li>Centrifuge the tube at 300 &#215; g for 5 min at 4 &#176;C. Decant the supernatant, and add 2 mL of flow buffer. Repeat this washing step 2x.</li>
		<li>Carefully decant the contents of the tube. Observe the small amount of fluid with a pellet remaining in the tube. Add 100 &#181;L of flow buffer, and resuspend very well.</li>
		<li>Transfer all the remaining liquid in the 5 mL flow tube into a 96-well plate. Make sure each sample is properly labeled. Add 100 &#181;L of flow buffer to the corresponding wells and centrifuge the 96-well plate at 650 &#215; g for 3 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Assess the expansion of the CD19+ target cells by flow cytometry (Figure 2).
	<ol>
		<li>Prepare a master mix containing 0.3 &#181;L of live/dead stain, 0.13 &#181;g (2.5 &#181;L) of anti-human CD19 antibody, 0.06 &#181;g (2.5 &#181;L) of anti-human CD45 antibody, 5 &#181;g (2.5 &#181;L) of anti-mouse CD45, and 42.2 &#181;L of flow buffer for a total of 50 &#181;L per sample. Incubate in the dark at room temperature for 15 min.</li>
		<li>Wash with 150 &#181;L of flow buffer followed by centrifugation at 650 &#215; g for 3 min at 4 &#176;C. Decant the supernatant, and resuspend the pellet in 195 &#181;L of flow buffer and 5 &#181;L of counting beads.</li>
		<li>Acquire on a flow cytometer to determine the level of CD19+ cell expression. For gating and analysis purposes, first gate the population of interest (CD19+) based on the forward versus side scatter characteristics, followed by a single gating population, and then live cells. Once the live cells are gated, assess the human CD45 versus mouse CD45 population, and from the human population, gate the CD19+ target population.</li>
	</ol>
	</li>
	<li>Quantify to determine the amount present in 70 &#181;L, and then express in cells/&#181;L using equation (1): 
	Absolute CD19+ cell count = ([CD19+ cells/counting beads] &#215; number of counting beads within 5 &#181;L)/70 
	&#8203;&#8203;NOTE: Repeat the peripheral blood assay every 5 days to assess the CD19+ cell expansion. Collect no more than 200 &#956;L of blood cumulatively every 2 weeks in accordance with local animal ethics committee guidelines. Once the mice have reached a disease burden of &#8805;10 cells/&#181;L, randomize to CART19 cell or control groups (section 4).</li>
</ol>
4. Administration of CART19 cells&#160;&#160;in vivo
<ol>
	<li>Preparation of CART19 cells (~Day 80): 
	NOTE: The generation of CART19 cells has been previously published27,30,31. This procedure must be carried out inside a Biosafety Cabinet Level 2+ using aseptic technique and personal protective equipment.
	<ol>
		<li>Thaw the CART19 cells in a water bath container (37 &#176;C). Then, wash the cells in warm T cell medium (TCM) to remove the dimethyl sulfoxide. 
		NOTE: TCM is made of 10% human AB serum(v/v), 1% penicillin-streptomycin-glutamine (v/v), and serum-free hematopoietic cell medium27,30.</li>
		<li>Centrifuge the CART19 cells at 300 &#215; g for 8 min at 4 &#176;C, and resuspend in warm TCM to a final concentration of 2 &#215; 106 cells/mL. Allow the CART cells to rest in an incubator (37 &#176;C, 5% CO2) overnight but for no more than 16 h.</li>
	</ol>
	</li>
	<li>Administration of CART19 cells via i.v. injection (Day ~80)
	<ol>
		<li>Count the CART19 cells, and spin down at 300 &#215; g for 8 min at 4 &#176;C.</li>
		<li>Resuspend the CART19 cells with 10 mL of PBS, and spin down at 300 &#215; g for 8 min at 4 &#176;C.</li>
		<li>Resuspend the CART19 cells to a final concentration of 40 &#215; 106 cells/mL with PBS, transfer the cells into a sterile 1.5 mL microcentrifuge tube, and place them on ice.</li>
		<li>Perform CART19 cell tail vein injections (as detailed above) by injecting 100 &#181;L of resuspended cells i.v. 
		&#8203;NOTE: A volume of 100 &#181;L will administer 4 &#215; 106 cells to each mouse. Include a group of untreated mice as a negative control.</li>
	</ol>
	</li>
</ol>
5. Assessment of CART19 cell-associated toxicities
<ol>
	<li>After CART19 cell administration, monitor the mice 2x a day to assess any changes in their well-being, such as motor weakness, hunched body, and loss of body weight. 
	NOTE: All these physical changes correlate with CRS symptoms in this model28.</li>
	<li>Once the mice begin to develop motor weakness and weight reduction (10-20% reduction from the baseline), collect peripheral blood to analyze the tumor burden and conduct serum isolation for cytokines as per the methodology described in section 3 (Figure 3).</li>
	<li>Store the serum microcentrifuge tubes at &#8722;80 &#176;C, and use the sera to analyze the chemokines and cytokines using a Multiplex assay (Figure 4). 
	NOTE: If the mice exhibit signs of hind limb paralysis, appear moribund or&#160;lethargic, and the&#160;weight loss is &#62; 20% of their body weight up to the point that limits their ability to reach food and/or water, then they will be subjected to euthanasia.</li>
</ol>
6. MRI imaging
NOTE: A preclinical (for small animals) MRI scanner with a vertical bore magnet was used for in vivo magnetic resonance and image acquisition32,33.
<ol>
	<li>Mix 120 &#181;L of Gadolinium + 880 &#181;L of saline solution, and load into a 1 mL insulin syringe. Inject 100 &#181;L into each mouse (i.p.). 
	NOTE: Gadolinium should be injected 15-20 min prior to the start of the experiment.</li>
	<li>Anesthetize the mouse using isoflurane (2-3%) in an induction chamber for 10-15 min. 
	NOTE: It is highly recommended to apply ophthalmic ointment to prevent eye dryness.</li>
	<li>Place the mouse spine on the rodent-compatible cradle probe. Then, proceed to position the mouse by hooking its teeth on the bite bar.</li>
	<li>Pull the head of the mouse into the 25 mm 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. 
	NOTE: If conducting a contrast-enhanced MRI scan, ensure that the gadolinium (in this case) or contrast agent is injected at least 10 min prior to placing the apparatus into the bore magnet.&#160;Throughout the time of the imaging, the mice will be anesthetized by the inhalation of 2-3% isoflurane in air via the nose cone, and their respiratory rate will be simultaneously monitored.</li>
	<li>For breathing assessment, use a breathing/respiratory monitoring device. 
	NOTE: It is important to assess the body temperature as well in order to prevent possible hypothermia due to extended exposure to anesthesia. It is recommended to use heat&#160;pads to control the body temperature.
	<ol>
		<li>Attach the breathing monitor probe close to the diaphragm, and secure it with surgical tape. Keep the respiration rate between 20-60 breaths per min so that the mouse&#39;s condition is stable. 
		NOTE: This rate provides more stable MRI images and prevents motion artifacts in the acquired image. If the respiratory rate is higher than the 20-60 breaths per min range, increase the concentration of isoflurane. If the respiratory rate is less than 20 breaths per min, the anesthesia is too deep; therefore, concentration needs to be decreased.</li>
	</ol>
	</li>
	<li>Insert the animal probe into the small bore within the vertical-bore small-animal MRI system. Adjust the animal&#39;s head at the center of the coil. Secure the apparatus with the lock so it can stand upright, and connect the instrument to the computer.</li>
	<li>Open and use the software (e.g., Paravision) to set up the scan positions and types of scans. Determine the optimal axial and sagittal positions while keeping them consistent for all the experimental groups. 
	NOTE: It is recommended to use the acquisition of a trial scan as a reference prior to the actual full-length MRI scans.</li>
	<li>Proceed with the MRI data collection (as previously described32,34,35,36). Obtain both T1-weighted and T2-weighted MRI images using the suggested setup mentioned below.
	<ol>
		<li>For T1-weighted MRI images, use T1-weighted multislice multiecho (MSME) sequence with a repetition time (TR) of 300 ms, an echo time (TE) of 9.5 ms, a field of view (FOV) of 4&#160;cm x 2&#160;cm x 2&#160;cm, and a matrix of 192 x 96 x 96.</li>
		<li>For T2-weighted MRI images, use a Multislice Multiecho (MSME) sequence with a TR of 300 ms, a TE of 9.5 ms, an FOV of 4&#160;cm x 2&#160;cm x 2&#160;cm, and a matrix of 192 x 96 x 96 was used.</li>
	</ol>
	</li>
	<li>Once the scans are complete, remove the probe from the bore. Gently extract the mouse by removing the teeth off the bite bar. Monitor the animal in a separate cage until it is fully conscious and has regained adequate motor function.</li>
	<li>After the extraction of data from the software, use analysis software for the quantification and 3D volume rendering (Figure 4) of the hyperintensity regions. 
	NOTE: If possible, it is strongly encouraged to have two separate blinded observers for the data analysis.</li>
</ol>

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><tbody><tr><td>&amp;nbsp;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&amp;nbsp;</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&amp;nbsp;</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&#039;s Phosphate-Buffered Saline</td><td>Gibco</td><td>14190-144&amp;nbsp;</td><td></td></tr><tr><td>ERT Control/Gating Module&amp;nbsp;</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 ...

assessment-chimeric-antigen-receptor-t-cell-associated-toxicities

Research

JoVE Journal

Cancer Research

1.3K Views.  Mayo Clinic, Rochester. Dit protocol maakt gebruik van menselijke CAR-T en tumorcellen en toont nauwkeurig de toxiciteiten aan die gepaard gaan met CART19-toediening. Daarom een samenvatting van wat er in de kliniek is waargenomen. Het belangrijkste voordeel van het gebruik van dit platform is dat het een vertaalbaar en klinisch relevant model biedt waarbij menselijke T-cellen en tumorcellen worden gebruikt.Dit model maakt niet alleen een efficiënte beoordeling van CART19-geassocieerde toxiciteiten mogelijk,...

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

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

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 ...

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.

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.

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

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Immunology and Infection

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 ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Bioengineering

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

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

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