Creation of the First Non-Human Primate Model That Faithfully Recapitulates Chimeric Antigen Receptor (CAR) T Cell-Mediated Cytokine Release Syndrome (CRS) and Neurologic Toxicity Following B Cell-Directed CAR-T Cell Therapy
Authors: Taraseviciute Agne , Kean Leslie , Jensen Michael C 
Published: October 2019

Abstract
The advent ofadoptive T-cell therapy using CD19 Chimeric Antigen Receptor (CAR) T cells has revolutionized the treatment of relapsed and refractory acute lymphoblastic leukemia (ALL). CAR T cells have shown encouraging results in clinical trials, with complete remissions in 90% of patients with refractory B-cell ALL. However, CD19 CAR T cell therapy is associated with significant side effects, including cytokine release syndrome (CRS), encompassing fevers, myalgias, hypotension, respiratory distress, coagulopathy as well as neurologic toxicity, ranging from headaches to hallucinations, aphasia, seizures and fatal cerebral edema. Our understanding of CRS and neurologic toxicity has been significantly limited by the lack of animal models that faithfully recapitulate these symptoms.
We chose the non-human primate (NHP), Macaca mulatta, given that it closely recapitulates the human immune system, to create an animal model of B-cell-directed CAR T cell therapy targeting CD20. Rhesus macaques (n=3) were treated with 30-40mg/kg cyclophosphamide followed 3-6 days later by an infusion of CAR T cells at a dose of 1x107 transduced cells/kg. Recipient animals were monitored for clinical signs and symptoms of CRS and neurotoxicity, and data were collected longitudinally to determine CAR T cell expansion and persistence, B cell aplasia, as well as clinical labs of CRS and cytokine levels.
Prior to testing the CD20 CAR T cells, we performed a control experiment, in which 1x107/kg control T cells, transduced to express GFP only (without a CAR construct), were infused following cyclophosphamide conditioning. This infusion resulted in short-lived persistence of the adoptive cellular therapy, with disappearance of the cells from the peripheral blood by Day +14 (Figure 1, green traces) and no clinical signs of CRS (Figure 2) or neurologic toxicities. In contrast, recipients of 1x107 cells/kg CD20 CAR-expressing T cells (n = 3) demonstrated significant expansion of the CAR T cells, and persistence for as long as 43days post-infusion, which corresponded to concurrent B cell aplasia (Figure 1). These recipients also developed clinical signs and symptoms of CRS as well as neurologic toxicity which was manifested by behavioral abnormalities and extremity tremors, beginning between days 5 to 7 following CAR T cell infusion, with the onset of clinical symptoms coinciding with maximum CAR T cell expansion and activation. The neurologic symptoms were responsive to treatment with the anti-epileptic medicationlevetiracetam. The clinical syndrome was accompanied by elevations in CRP, Ferritin, LDH and serum cytokines, including IL-6, IL-8 and ITAC (Figure 2 A and B), recapitulating data from clinical trials using CD19 CAR T cells. An expansion of CD20 CAR T cells on day 7 following infusion was also observed in the CSF in the animals, and coincided with the onset of neurotoxicity. Strikingly, we also detected CD20 CAR T cells in multiple regions of the brain via flow cytometry, including the frontal, parietal, and occipital lobes, as well as the cerebellum, and demonstrated an increased number of infiltrating T cells by immunofluorescence in the brains of animals treated with CD20 CAR T cells when compared to healthy controls.
These data demonstrate the successful establishment of a large animal model of B-cell directed CAR T cell therapy that recapitulates the most significant toxicities of CAR T cell therapy, including CRS and neurotoxicity. This model will permit a detailed interrogation of the mechanisms driving these toxicities as well as the pre-clinical evaluation of therapies designed to prevent or abort them after CAR T cell infusion.
Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion. Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion.

Disclosures
Kean: Juno Therapeutics, Inc: Research Funding. Jensen:Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.