Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a major therapeutic regimen for hematological malignancies, ~28%–49% of the treated patients relapse or progress after allo-HSCT.1,2 For the relapsed patients, salvage therapies generally consists of donor lymphocyte infusion (DLI),3 chemotherapy and a second allo-HSCT.4 Particularly, DLI has been established as a standard clinical therapy for relapsed chronic myeloid leukemia since 1990,3 and accumulating evidence has proved its antileukemic effect in B-cell acute lymphoblastic leukemia (B-ALL).5,6 However, only 0%–57.1% of the relapsed B-ALL patients showed secondary remission after DLI treatment.5–9 In addition, graft versus host disease (GVHD) has been a major risk after DLI, with observed incidence rate of 12%–50%.7–14
In recent years, chimeric antigen receptor-modified T cells (CAR-T) immunotherapy has achieved remarkable success in relapsed or refractory hematological malignancies.15 CAR involves a fusion protein, which recognizes specific antigen due to an antibody single chain variable fragment component and a signal transduction component containing 1 or 2 costimulator molecules (ie, CD28 or 4-1BB). The most widely used CAR-T cells are autologous CD19-CAR-T cells. Early studies in patients with relapsed or refractory B-ALL have demonstrated a response rate of 93% after CD19-CAR-T treatment.16 In 2013, Kochenderfer et al15 successfully used CAR-T cells for the first time in lymphoma patients who relapsed after allo-HSCT. Since then, 6 other clinical trials have been conducted with allogeneic CAR-T cells to treat relapsed hematological malignancies, with response rates varying from 44%–100%.15,17–22 However, there is little data that directly compare CAR-modified DLI (CAR-DLI) and DLI therapy in patients who relapsed after allo-HSCT.
In this report, 5 B-ALL patients who relapsed after allo-HSCT received CAR-DLI therapy, and we compared their survival outcome with 27 patients who had received DLI therapy. The median complete remission (CR) duration (CRD) of CAR-DLI group was significantly (P=0.020) longer when compared with DLI group: 9 months (range, 2–29) versus 3.2 months (range, 0–17.4). In addition, patients receiving CAR-DLI showed significant (P=0.049) survival advantage over DLI group, with median overall survival (OS) of 12 months (range, 3–29) and 3.7 months (range, 0–65), respectively. Our study suggest that CAR-DLI may represent a more effective therapy for relapsed B-ALL patients.
MATERIALS AND METHODS
Study Design and Participants
The clinical trial was designed to evaluate the safety and efficacy of CAR-T-cell immunotherapy on relapsed or refractory B-ALL patients. A written informed consent was obtained from each patient according to the Declaration of Helsinki protocol. The trial was conducted at the Changhai Hospital and the study was approved by the institutional review boards of Changhai Hospital. The characteristics of patients in the 2 groups were summarized in Table 1.
For CAR-DLI, the conditioning therapy consists of intravenous fludarabine (30 mg/m2) and cyclophosphamide (500 mg/m2, every 12 h) for 3 days. CAR-DLI was performed for 5 days after the conditioning therapy.
The conditioning therapy for DLI consists of VDCPL (vincristine, daunorubicin, cyclophosphamide, dexamethasone and pegaspargase) or hyper-CVAD (cyclophosphamide, vincristine, daunorubicin, dexamethasone). DLI was also performed 5 days postconditioning therapy.
Disease and Toxicity Assessment
Disease status was assessed with minimal residue disease (MRD) and donor chimerism rate (the donor cell to patient cell ratio) using bone marrow (BM) samples before and after conditioning therapy, and samples were collected 15 days, 1, 2, 3, 6, 9 months, 1 year and 2 years after CAR-DLI or DLI. MRD was evaluated by morphology and multiparameter flow cytometry (MPFC). The cut-off value of MRD-negative was 0.01%. Donor chimerism rate was evaluated with single nucleotide polymorphism polymerase chain reaction. The cut-off value of full donor chimerism was defined as 95%. Toxicity assessment was based on the NCI Common Terminology Criteria for Adverse Events, 4.02.23 Cytokine release syndrome (CRS) assessment was performed using University of Pennsylvania Cytokine Release Syndrome Grading System.24 Tocilizumab was incorporated into the management of grades III–IV CRS.
Peripheral blood mononuclear cells were collected from the healthy transplant donors by leukapheresis. Enriched peripheral blood mononuclear cells were activated using CD3 (1 µg/mL) and CD28 (1 µg/ml) microbeads (Metenyi Biotec, 130-111-160, Bergisch Gladbach, Germany). The α-CD19-specific single chain variable fragment25,26 was designed by HuaDao Biopharma Limited Corporation (Shanghai, China). Activated T cells were then transduced with the lentiviral vector encoding CD19-4-1BB transgene and expanded in the presence of interleukin (IL)-2 (400 IU/mL, 200-02; Pepro Tech, 200-02, NJ) for 14 days before infusion. Sample processing, CAR-T cells manufacturing and laboratory analysis were mainly performed at HuaDao Biopharma Limited Corporation.
CAR-T Subset Analysis
CAR-T-cell immunophenotypes were validated by MPFC (Beckman Coulter, Navios, CA). The fluorochrome-conjugated monoclonal antibodies for CD3, CD4, CD8,CD45RA, CCR7, and CD62L were all purchased from BD Biosciences (NJ), and APC-anti-CD19-CAR antibody was prepared by HuaDao Biopharma Limited Corporation.
CAR-T Expansion and Persistence Assay
CAR-T-cell expansion and in vivo persistence were confirmed by quantitative polymerase chain reaction method using cobas z480 (Roche Diagnostic system, Branchburg, NJ). TIANamp genomic DNA kit (dp304-03, Beijing, China) was used to extract DNA from peripheral blood, BM, and cerebral spinal fluid. TAKARA SYBR Premix EX Tag II (RR820A, Tokyo, Japan) was used to determine the CAR transgene copies.
The cytotoxicity of CAR-T cells was assessed using lactic acid dehydrogenase (LDH) release assay. CAR-T cells were coincubated with target antigen positive (CD19-K562) cell line at 37°C, 5% CO2 for 16 hours, and the supernatant was collected for detection of LDH release (CK12; Dojindo, Kumamoto, Japan).
The in vitro cytokine secretion by CAR-T cells was examined using human TH1/TH2 cytokine CBA kit II (BD Bioscience, 551809, NJ). The CAR-T cells were coincubated with target cell line (CD19-K562) in vitro for 16 hours, and the supernatants were collected for cytokine analysis. In addition, the treated patients’ serum samples were also analyzed for cytokines on days 0, 2, 4, 6, 8, 10, 12, and 14 using Immulite 1000 Immunoassay System (Siemens, Berlin, Germany) in Changhai hospital.
CR was defined as the presence of <5% BM blast cells as assessed by morphological assessment, and no >0.01% as detected by MPFC analysis, with hematopoietic recovery without any signs of extramendullary leukemia. CRD was defined as the length of time from the first CAR-DLI or DLI infusion to relapse, death or the last follow-up point from CR. OS represents the length of time from the first CAR-DLI or DLI infusion to death due to any cause or the last follow-up point.
The survival probabilities of OS and CRD were analyzed using the Kaplan-Meier method and were estimated with log-rank test. A P-value of <0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 5.01.
CAR-T-Cell Characterization and Antitumor Activity
We first characterized CAR-T cells and assessed their antitumor activity. The median transduction efficiency of CAR-T was 38.5% (range, 12.55%–69.8%). The CD4/CD8 proportion and naïve/memory T-cell composition of CAR-T was evaluated in each patient (Figs. 1A, B). In order to confirm the ability for CAR-T cells to recognize the target cells, in vitro cytokine secretion and tumor cytotoxicity assays were performed. The results showed that CAR-T cells robustly secreted IL-2, IFN-γ, and tumor necrosis factor-α (TNF-α) (Figs. 1C–E). In addition, LDH release analysis revealed that the tumor cell lysis increased with the increasing effector-to-target cell ratio for each of the 5 patients (Figs. 1F–J). Together, these data indicate that the CAR-T cells can specifically recognize and kill target tumor cells.
CAR-DLI Therapy and DLI Therapy
Five patients, who relapsed at a median time of 8 months (range, 1.4–15.2 mo) after allo-HSCT and had received several courses of chemotherapy, received CAR-DLI therapy. A median CAR+ cell number of 1.6×107 (range, 0.43×107–5.5×107) per kg of the body weight was infused. The CAR gene copies increased to a peak level within 8–10 days after infusion, and detectable CAR gene copies lasted for a median of 41 days (range, 10–117 d) in peripheral blood (Fig. 2A). Patient 3 with only central nervous system (CNS) relapse after allo-HSCT displayed a cell count increase in the cerebrospinal fluid (CSF), a week after CAR-DLI infusion and lasted for 3 months. Additional examination of CSF by MPFC showed MRD-negative in CNS (Fig. 2B). In control group, DLI was administered at a median of 4.2 months (range, 1.9–17) after allo-HSCT. The median CD3+ cell number of 4.8×107 (range, 1.1×107–2.05×108) per kg of body weight was infused. Fourteen patients were infused only once, and 13 patients received repeated infusions. Oral cyclosporine was administered to prevent acute GVHD (aGVHD) 2 days before DLI and continued for 2 weeks. Cyclosporine dose adjustment was based on its trough concentration (200–300 ng/mL).
Patient Response, Survival, and aGVHD
The MRD levels and donor chimerism rates of 5 patients in CAR-DLI group has been shown in Figures 3A–E. The CR rate of CAR-DLI and DLI patients were 100% and 62.9%, respectively (P=0.144). In CAR-DLI group, patients 1 and 5 obtained sustained remission, and the MRD of patients 2 and 3 displayed a sharp decrease in 3 and 8 months after CAR-DLI, and patient 4 showed CNS remission and BM remission during the period of relapse. Except for patient 4, other patients also revealed donor chimerism rate decrease during relapse and recovered from remission after CAR-DLI. In DLI group, 6 of 27 (22.2%) patients demonstrated CR by conditioning therapy and 11 of 27 (40.7%) patients obtained CR due to DLI. The CRD and OS of patients receiving CAR-DLI and DLI are displayed in Figures 4A and B. Patients who received CAR-DLI therapy had a median CRD of 9 months (range, 2–29), while median CRD of DLI patients was 3.2 months (range, 0–17.4) (P=0.020). Similarly, the median OS of CAR-DLI patients was 12 months (range, 3–29), while it was 3.7 months (range, 0–65) (P=0.049) in DLI-treated patients.
In CAR-DLI group, no apparent evidence of aGVHD was observed in any of the 5 recipients. However, in DLI group, 2 (7%) and 4 (14.8%) patients developed grades I–II and grades III–IV aGVHD, respectively. The median time between DLI administration and the onset of aGVHD syndrome was 8 days (range, 4–30). The targeted organs of aGVHD involved skin (n=2), gut (n=3), concomitant skin, and liver (n=1). All these patients received methylprednisolone treatment at a dose ranging from 1 to 2 mg/kg, and 2 patients restarted cyclosporine. Overall, 3 patients in the DLI group died of grade IV gut aGVHD.
Cytokine Release Syndrome
The serum cytokine levels of 5 patients were monitored for 2 weeks after CAR-DLI. The levels of IL-2R, IL-6, IL-8, IL-10, and TNF-α increased to different extents, and peaked values were recorded within 2–6 days after CAR-DLI, and then returned to lower levels (Figs. 5A–E). Among the 5 patients, patient 3 developed grade I CRS; patients 2, 4, and 5 developed grade II CRS and patient 1 developed grade III CRS with high fever up to 40°C for 3 days and elevated serum IL-6 and TNF-α cytokine levels. This patient was treated with intravenous tocilizumab (8 mg/kg) 3 days after infusion and exhibited complete recovery in the next 2 days, along with the disappearance of CRS symptoms and lower levels of cytokines.
DLI has been a crucial salvage therapy for B-ALL patients who relapsed after allo-HSCT. In the present study, we observed median CRD and OS of 9 and 12 months, respectively in CAR-DLI group, which were significantly longer than CRD (P=0.020) and OS (P=0.049) observed from patients in DLI group. Thus, CAR-DLI may represent an alternative and more effective therapy for B-ALL patients with relapsed diseases after allo-HSCT.
Similar to DLI, GVHD has been observed to be a complication after CAR-DLI therapy. However, previous reports have shown that the incidence rate of GVHD in allogeneic CAR-T-cell immunotherapy was lower than GVHD incidence rate displayed by DLI.27 This reduced GVHD incidence and can be attributed to 2 main observations: first, the in vivo persistence of allogeneic CAR-DLI cells is usually shorter than DLI, because CAR-DLI cells express both engineered CAR and allogeneic TCR in parallel, and both can be activated by specific antigens.28 Both CAR and TCR activation could accelerate T-cell exhaustion.29,30 Therefore, CAR-DLI cells appear to be eradicated before the induction of GVHD. Second, in some reports, less CAR-T cells were used in comparison with T cells used in the DLI, which might reduce the risk of developing GVHD.15,17,29
There is a possibility of patients developing recurrence even after receiving CAR-DLI therapy. The relapse after CAR-DLI could occur mainly because of 2 factors. First, the exhaustion of CAR-DLI cells in vivo contributes to the relapse of leukemia. Second, the loss of the target antigen endows leukemic cells to escape from CAR-DLI cell recognition and targeting. Consistent with this, earlier studies have also proposed that the alternative splicing of CD19 mRNA31 or ALL transformed into AML would lead to CD19 negative population,32 thus making the CD19-CAR-T cells less effective.
CAR-DLI efficacy appears to be influenced by the origin of CAR-T-cell subsets. A previous study has shown that CD4+ CAR-T cells have greater ability in cytokine production and cell proliferation, while CD8+ CAR-T displays potent tumor cytolytic activity.16 Independent study revealed that while each T-cell subset was sensitive to CAR transduction, they could demonstrate distinct function during therapy.33 Naïve cell-derived CAR-T cells proliferate and differentiate more vigorously, and are more closely correlated to in vivo expansion, survival, and long-term persistence. Memory-derived CAR-T cells show higher antitumor functions and effectively produce cytokines.34 In our present study, patient 5 had a high proportion of CD8+ T cells and memory T cells, and this may explain the potent tumor cell lysis. However, in terms of the composition of naïve/memory CAR-T cells, the results of previous literatures have remained inconsistent. In our study, patient 5 had low proportion of naïve T cells. Nevertheless, the CAR transgene was detected in the peripheral blood after 4 months of CAR-T-cell infusion. Similarly, patient 1 had a low number of naïve CAR-T cells, and the persistence of these CAR-T cells was >3 months, which was significantly longer in patient 3 who possessed the highest naïve CAR-T-cell proportion.16,34,35
We observed that CAR-T cells could be useful in CNS leukemia. Specifically, patient 3 in our present study relapsed after allo-HSCT with CNS involvement. However, leukemic cells in the CSF were eradicated after CAR-DLI therapy, and the CR status lasted for 5 months. Consistent with our observation, a previous study reported that CAR-T cells can migrate into CSF and eliminate leukemic cells.36 The ability of CAR-T cells to penetrate the blood-brain barrier could have important implications for the treatment of CNS leukemia.
The major limitation of the present study was the small sample size, especially for patients who received CAR-DLI therapy. Therefore, these initial results highlight the importance of additional clinical trials enrolling more patients.
In conclusion, the present study suggests that CAR-DLI therapy is advantageous in advanced B-ALL patients with relapsed diseases after allo-HSCT. CAR-DLI therapy demonstrated longer CRD and prolonged OS in comparison with DLI therapy. Our data also emphasize the importance of conducting prospective, randomized and controlled clinical trials to compare CAR-DLI and DLI therapies in patients who relapsed after allo-HSCT. This would eventually help usconclusively establish the potential clinical value of CAR-DLI therapy.
The authors are thankful to Sabine Fürst from the department of hematology, Marseille, France, for the kind review of the manuscript.
Conflicts of Interest and Source of Funding
This work was supported by grants from the National Natural Science Foundation of China (NSFC) (81470322, 81770209) and Science and Technology Commission of Shanghai Municipality (16411970900). This work was also partly supported by the Gillson Longenbaugh Foundation.
J.Y. is currently receiving grants from the National Natural Science Foundation of China (NSFC) (81470322, 81770209). Q.L. and A.E.C. are currently receiving grants from Gillson Longenbaugh Foundation. G.T. is currently receiving a grant Science and Technology Commission of Shanghai Municipality (16411970900). All the remaining authors have declared there are no financial conflicts of interest with regard to this work.
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Keywords:Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
B-cell acute lymphoblastic leukemia; allogeneic hematopoietic stem cell transplantation; chimeric antigen receptor-modified donor lymphocyte infusion; acute graft versus host disease; cytokine release syndrome