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

Summary

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.

Abstract

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.

Introduction

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 myeloma^{1,2,3,4,5,6,7}, leading to recent FDA approvals. Despite the initial success in clinical trials, treatment with CART cell therapy results in toxicities that....

Protocol

This protocol follows the guidelines of Mayo Clinic'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

1. Obtain male, 8-12 weeks old, immunocompromised, NOD-SCID IL2rγnull (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 fem....

Results

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 × 10⁶ PBMCs (i.v.) derived from ALL patients. The mice were monitored for engraftment for ~13 weeks via the tail bleeding assay.......

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
APC Anti-Human CD19	Biolegend	302211
Alcohol Prep Pad	Wecol	6818
Analyze 14.0 software	AnalyzeDirect Inc.	N/A	https://analyzedirect.com/analyze14/
Artificial tears (Mineral oil and petrolatum)	Akorn	17478-062-35	Topical ophtalmic gel to prevent eye dryness
BD FACS Lysing Solution	BD	349202	Red blood cells lysing buffer
BD Micro-Fin IV insulin syringes	BD	329461
Brillian Violet 421 Anti-Human CD45	Biolegend	304032
Bruker Avance II 7 Tesla	Bruker Biospin	N/A	MRI machine
Busulfan (NSC-750)	Selleckchem	S1692
CountBright absolute counting beads	Invitrogen	C36950
CytoFLEX System B4-R2-V2	Beckman Coulter	C10343	flow cytometer
Dulbecco's Phosphate-Buffered Saline	Gibco	14190-144
ERT Control/Gating Module	SA Instruments	Model 1030	Small Animal Monitoring Respiratory and Gating System
Fetal bovine serum	Millipore Sigma	F8067
Hemocytometer	Bright-Line	Z359629-1EA
Human AB Serum; Male Donors; type AB; US	Corning	35-060-CI
Isoflurane (Liquid)	Sigma-Aldrich	792632
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation	Invitrogen	L34966
Microvette 500 Lithium heparin	Sarstedt	20.1345.100	Blood collection tube
MILLIPLEX Huma/Cytokine/Chemokine Magnetic Beads Panel	Millipore Sigma	HCYTMAG-60K-PX38	Immunology Multiplex Assay to identify cytokines and chemokines
Omniscan	Ge Healthcare Inc.	0407-0690-10	Gadolinium-based constrast agent
Pd Anti-Mouse CD45	Biolegend	103106
Penicillin-Streptomycin-Glutamine (100x), Liquid	Gibco	10378-016
Round Bottom Polysterene Test tube	Corning	352008
Sodium Azide, 5% (w/v)	Ricca Chemical	7144.8-16
Stainless Steel Surgical Blade	Bard-Parker	371215
X-VIVO 15 Serum-free Hematopoietic Cell Medium	Lonza	04-418Q