Tolerance and efficacy of autologous or donor-derived T cells expressing CD19 chimeric antigen receptors in adult B-ALL with extramedullary leukemia

Abstract

The engineering of T lymphocytes to express chimeric antigen receptors (CARs) aims to establish T cell-mediated tumor immunity rapidly. In this study, we conducted a pilot clinical trial of autologous or donor- derived T cells genetically modified to express a CAR targeting the B-cell antigen CD19 harboring 4-1BB and the CD3ζ moiety. All enrolled patients had relapsed or chemotherapy-refractory B-cell lineage acute lymphocytic leukemia (B-ALL). Of the nine patients, six had definite extramedullary involvement, and the rate of overall survival at 18 weeks was 56%. One of the two patients who received conditioning chemotherapy achieved a three-month durable complete response with partial regression of extramedullary lesions. Four of seven patients who did not receive conditioning chemotherapy achieved dramatic regression or a mixed response in the haematopoietic system and extramedullary tissues for two to nine months. Grade 2-3 graft-versus-host disease (GVHD) was observed in two patients who received substantial donor-derived anti-CD19 CART (chimeric antigen receptor-modified T) cells 3-4 weeks after cell infusions. These results show for the first time that donor-derived anti-CD19 CART cells can cause GVHD and regression of extramedullary B-ALL. This study is registered at www.clinicaltrials.gov as NCT01864889.

Keywords: B-cell acute lymphoblastic leukemia (B-ALL); anti-CD19 chimeric antigen receptor (CAR) T cells; graft-versus-host disease (GVHD); refractory.

Figure 1.

CD19-specific cytotoxic activity of CART-19 cells. (A) CART-19 cells were produced by activating peripheral-blood mononuclear cells (PBMC) with anti-CD3 antibody OKT3 on day 0 and transducing T cells on days 2 and 3. After 10–12 d of culture, cells were released for infusion. (B) Cytotoxic activity of mock-transfected and CART-19 cells against primary CD19⁺ B-ALL blasts, evaluated in a 6 h CFSE-staining assay; results are shown at an effector:target (E:T) ratio of 20:1. (C) Cytotoxic activity of non-transfected, mock-transfected and CART-19 cells obtained from all nine patients against CD19⁺ Raji and NALM6 and CD19⁻ K562 cell lines. The data are presented as the mean of triplicate values from each patients, and error bars represent SD. Arrows indicated the date of lentivirus transfection.

Figure 2.

Copy number of CAR molecules and CD19⁺ cells in the peripheral blood, bone marrow and Cerrebral spinal fluid. (A) Quantitative real-time PCR was performed on genomic DNA harvested from each patient's PBMCs collected before and at serial time points after CART-19 cell infusion, using primers specific for the transgene. CD19⁺ B cells expressed as count change from baseline in the blood after the infusion of CART-19 cells in all nine patients. Patients 2, 3, 4, 5, and 8 died before the last follow-up, the time of all patients after cell infusion range from 15 to 140 d. In all panels, ▴ indicates imatinib (TKI) therapy, ^↓ indicates the time of relapse, ^↑ indicates the time of second infusion, ^↓ indicates the time of chemotherapy, black squares represent the values for CAR copies by Q-PCR, and black circles indicate CD19⁺ B cell counts in PB. The first chemotherapy regimen: Cyclophosphamide Etoposide, Vincristine and Dexamethasone. The second chemotherapy regimen: Vincristine, Daunorubicin, Cyclophosphamide and Prednison (B, C) For patient Bone marrow and cerebralspinal fluid aspirates were obtained at serial time points after CART-19 cell infusion in patient 7. Black squares represent the values of CAR copies by Q-PCR and black circles indicate the detection of bcr/abl transcripts.

Figure 3.

Median values for fold change of cytokines level within the first month after CART-19 cell infusion. Serum was harvested from each patient's PB, collected before and at serial time points after CART-19 cell infusion. Serum cytokines were measured by FACS.

Figure 4.

Clinical response to T cell infusions. (A) Flow cytometry for CD19 and CD10 expression in BM before and after treatment. Cells were gated on CD45⁺7AAD⁻ cells in patient 9. (B) PET-CT scan of patient 9 before treatment and two months after treatment showed partial regression of her extramedullary leukemic lesions. (C) Samples before and after infusion of blood were obtained at the time points indicated on the x-axis and used for WBC counts and blast cell percent. Black squares represent the values of white blood cells (WBC) counts and black circles indicate CD19⁺ cell percent in PB. ↑ indicates the time of second infusion and ↓ indicates hydroxyurea injection. (D) Flow cytometry for CD20 and CD19 expression in PB before and after treatment. Cells were gated on CD45⁺7AAD⁻ cells in patient 4. (E) A CT scan shows regression of cervical lymph nodes in patient 4 after infusion of CART-19 cells. ↑ indicates a lymph node mass that regressed.

Figure 5.

Toxic clinical response to T cell infusion. (A) This panel shows changes in serum lactate dehydrogenase (LDH) levels, IL-6, circulating C reactive protein (CRP) and body temperature before and after CART-19 infusion, with the maximum temperature per 24-h period indicated by the circles in patient 9. (B) Pulmonary CT of patient 1 before and after CART-19 infusion. ↑ indicates the bronchiectasis-like imaging features or ground-glass changes. (C) This panel shows changes in the levels of total bilirubin, direct bilirubin and indirect bilirubin during the period of in which patient 8 developed GVHD. (D) This panel shows chronically aggravated skin damage in patient 9 due to GVHD.