Successful application of anti-CD19 CAR-T therapy with IL-6 knocking down to patients with central nervous system B-cell acute lymphocytic leukemia

Abstract

Few studies have described chimeric antigen receptor-modified T cell (CAR-T) therapy for central nervous system (CNS) B-cell acute lymphocytic leukemia (B-ALL) patients due to life-threatening CAR-T-related encephalopathy (CRES) safety issues. In this study, CAR-Ts targeting CD19 with short hairpin RNA (shRNA)-IL-6 gene silencing technology (ssCART-19s) were prepared. We conducted a phase 1 clinical trial (ClinicalTrials.gov number, NCT03064269). Three patients with relapsed CNS B-ALL were enrolled, conditioned with the fludarabine and cyclophosphamide for lymphocyte depletion and infused with ssCART-19s for three consecutive days. Clinical symptoms and laboratory examinations were monitored. After ssCART-19 treatment, three patients' symptoms resolved almost entirely. Brain leukemic infiltration reduced significantly based on magnetic resonance imaging (MRI), and there were no leukemic blasts in cerebrospinal fluid (CSF), which was confirmed by cytological and molecular examinations. Additionally, increases in the levels of cytokines and immune cells were observed in the CSF of all patients. Only grade 1 cytokine release syndrome (CRS) manifesting as fever was noted in patients. In conclusion, CAR-Ts with shRNA-IL-6 gene knockdown migrated into the CNS, eradicated leukemic cells and elevated cytokines in CSF with mild, acceptable side effects.

Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Fig. 1

The treatment courses of CAR-T therapy and clinical assessments at time points. A Schematic structure of the CD19 CAR vector containing the anti-human CD19 scFv (FMC63) linked to 4-1BB costimulatory domains and a CD3-ζ signaling domain with shRNA against IL-6. B Patients received the intravenous infusion of ssCART-19s at a dose of 5 × 106 cells per kg for 3 consecutive days after the FC regimen. Patients were evaluated by blood, CSF, BM and MRI examinations during hospitalization (shown in bars of various colors). Abbreviations: CBC, complete blood count; PCT, procalcitonin; CRP, C-reactive protein; MRD, minimal residual disease; STR, short tandem repeats.

Fig. 2

The clinical responses before and after ssCART-19 infusions in patients. A (a) The T2-Weighted imaging (T2WI) signal of the pontine parenchyma in patient 1 was slightly high (arrowhead). (b–c) The end of the central aqueduct and four ventricle outlets became narrow (arrows), secondary to mild obstructive hydrocephalus. (d–f) One month after treatment, the pontine dorsal swelling in patient 1 was significantly reduced, the T2WI signal was nearly normal, and the degree of stenosis decreased at the end of the central aqueduct and at the outlet of the fourth ventricle. B (a–b) The fundus showed papillary edema and a flame-shaped hemorrhage on the surface of the optic disc and nearby retina. The height of the optic disc was over the scale of optical coherence tomography (OCT). These photos were taken when patient 1 was admitted. (c–d) The optic disc edema subsided, the hemorrhage was absorbed in the right eye, and all the vessels of the retina became white-line-like. Bleeding clots in the left eye covered the optic disc. These photos were taken one month after infusion in patient 1. C (a–c) T2WI signals showed pituitary tumors in the sellar region (arrows) when patient 3 was enrolled. (d–f) Tumors were significantly reduced and almost disappeared on day +35 after CAR-T therapy (arrows). D (a–b) The optic disc showed edema and was pale. A large amount of punctate bleeding was observed on the retina. These photos were taken when patient 2 was admitted. (c–d) The optic disc edema was alleviated, and the bleeding decreased. These photos were taken one week after infusion in patient 2. E MRD of ALL was evaluated in CSF in patient 3 by flow cytometric analysis on days −5, +1 and +7. F Flow cytometry analysis of PB from patients after CAR-T infusion using antibodies against CD3 and CD19.

Fig. 3

CAR-T expression and cytokine levels in serum and CSF after infusion. A Expansion and persistence of CAR-T cells in PB and CSF were assessed with quantitative real-time polymerase chain reaction (qRT-PCR). B Cells in the CSF of patient 1 were stained by immunofluorescence on days +1 and + 14, and cells in the CSF of patient 4 were stained on day +7 after infusion. CAR-Ts were identified by PE immunofluorescence in the cytoplasm (yellow arrows), and the nucleus was labeled by DAPI. C The lines represent the patients' temperature in degrees centigrade (°C) per 12-hour period. D The trends of IL-6 and IFN-γ concentrations in PB were monitored each day. These circulating inflammatory cytokines in CSF were also tested upon hospitalization and on days +1 and +7 by lumbar puncture. E The bars represent the concentrations of IL-6 and IFN-γ in PB and CSF on day +7.

Supplementary Fig. 1

CBCs, including the concentrations of white blood cells, lymphocytes, neutrophils and platelets, and biochemical and coagulation tests were performed after ssCART-19 infusion. Abbreviations: ALT, glutamic-pyruvic transaminase(U/L); AST, glutamic oxaloacetic transaminase (U/L); GGT, gamma-glutamyl transpeptidase (U/L); LDH, lactate dehydrogenase (U/L); PT, prothrombin time (s); PT-INR, international standardized ratio of prothrombin time; APTT, activated partial thromboplastin time (s); TT, thrombin time (s); DD, D-dimer (μg/mL).