Daniel Li

BACKGROUND: T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS: We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS: The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION: Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865617. FUNDING: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

Abstract Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor–modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19+ cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood–brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity. Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404–19. ©2017 AACR. See related commentary by Mackall and Miklos, p. 1371. This article is highlighted in the In This Issue feature, p. 1355

central memory T cells as independent predictors of CRS. Severe CRS was characterized by hemodynamic instability, capillary leak, and consumptive coagulopathy. Angiopoietin-2 and von Willebrand factor, which are biomarkers of endothelial activation, were increased during severe CRS and also before lymphodepletion in patients who subsequently developed CRS. We describe a classification-tree algorithm to guide studies of early intervention after CAR T-cell infusion for patients at high risk of severe CRS. These data provide a framework for early intervention studies to facilitate safer application of effective CD19 CAR T-cell therapy.

CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4(+)/CD8(+) ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin's lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4(+)/CD8(+) ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival.

Abstract CD19-specific chimeric antigen receptor (CAR) T-cell therapy is highly effective against relapsed or refractory acute lymphoblastic leukemia (ALL), but is hindered by neurotoxicity. In 53 adult patients with ALL, we found a significant association of severe neurotoxicity with high pretreatment disease burden, higher peak CAR T-cell expansion, and early and higher elevations of proinflammatory cytokines in blood. Patients with severe neurotoxicity had evidence of blood–cerebrospinal fluid (CSF) barrier disruption correlating with neurotoxicity grade without association with CSF white blood cell count or CAR T-cell quantity in CSF. Proinflammatory cytokines were enriched in CSF during severe neurotoxicity with disproportionately high levels of IL6, IL8, MCP1, and IP10, suggesting central nervous system–specific production. Seizures, seizure-like activity, myoclonus, and neuroimaging characteristics suggested excitatory neurotoxicity, and we found elevated levels of endogenous excitatory agonists in CSF during neurotoxicity. Significance: We detail the neurologic symptoms and blood, CSF, and neuroimaging correlates of neurotoxicity associated with CD19 CAR T cells and identify neurotoxicity risk factors. Our findings implicate cellular components other than T cells and suggest novel links between systemic inflammation and characteristic neurotoxicity symptoms. Cancer Discov; 8(8); 958–71. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 899