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

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.