Outcomes

Twenty-seven of 30 patients (90%) were in a morphologic complete remission at the first assessment 1 month after the infusion of CTL019. A test to detect minimal residual disease by means of multiparametric flow cytometry was negative in 22 patients, positive in 3 patients (the levels of minimal residual disease were 0.1% [and subsequently negative at 3 months], 0.09%, and 0.22%, respectively), and not performed in 2 patients. A complete remission was achieved in 2 of 3 patients who had previously been exposed to blinatumomab. The 2 patients in whom blast cells were detected in the cerebrospinal fluid at the time of infusion subsequently had no detectable central nervous system (CNS) leukemia as of the most recent follow-up (6 months), and no CNS relapses were observed.

Seven patients who had a complete remission subsequently had a relapse between 6 weeks and 8.5 months after infusion of CTL019 cells. Three relapses developed after early loss of CTL019-modified T cells at 2 weeks to 3 months, and in these patients the relapsed ALL remained CD19-positive. After recovery of normal B cells at 2 to 3 months, one relapse occurred rapidly at 3 months and two relapses were delayed (they occurred at 6 and 8.5 months). Patient 9, who had minimal residual disease (0.22%) at 1 month, had a relapse with CD19-positive ALL at 6 weeks. The disease rapidly progressed, and the patient died from ALL. This patient had highly refractory disease that was in the fourth relapse at the time of infusion and was not eligible for stem-cell transplantation because of coexisting conditions. In three patients, the loss of the expression of CD19 in leukemia cells resulted in a relapse; one of these patients (Patient 2) had received prior blinatumomab therapy. In these patients, CTL019 cells were not lost at the time of relapse.

Figure 1. Figure 1. Probability of Event-free and Overall Survival at 6 Months. Panel A shows the time to an event after infusion of CTL019. Events were relapse (in seven patients), no response (in three patients), and the myelodysplastic syndrome (in one patient). Tick marks indicate the time of data censoring at the last follow-up or on the date of initiation of alternative therapy (in four patients). The curve in Panel B shows overall survival. Data were censored at the time of the last follow-up. In both panels, dashed lines represent 95% confidence intervals.

Of the 27 patients who had a complete remission, 19 remained in remission: 15 patients received no further therapy, and 4 patients withdrew from the study to receive other therapy. In Patient 11, the myelodysplastic syndrome developed during ALL remission, and the patient withdrew from the study to receive other therapy. The median follow-up was 7 months (range, 1 to 24). No deaths were related to the study treatment. Seven patients died after disease progression or relapse, including 1 patient who died from the myelodysplastic syndrome. At 6 months, the event-free survival rate was 67% (95% confidence interval [CI], 51 to 88) and the overall survival rate was 78% (95% CI, 65 to 95) (Figure 1A and 1B).

In Vivo Expansion and Persistence of CTL019

Figure 2. Figure 2. Persistence of CTL019. Panel A shows the results of detection of CTL019-positive T cells detected by means of flow cytometry in peripheral-blood samples. “Confirmed negative” was defined as the first of two consecutive negative measurements (<0.1% CTL019-positive cells in CD3-positive cells). Patients 1 through 25 were participants in the pediatric trial (which included children and young adults 5 to 22 years of age), and Patients 26 through 30 were participants in the adult trial (which included patients 26 to 60 years of age). CTL019-modified T cells were also detected in the cerebrospinal fluid of 17 of 19 patients with specimens that could be evaluated. Panel B shows the Kaplan–Meier curve of the time to the first confirmed negative measurement in peripheral blood and bone marrow. Data were censored at the time of the last follow-up. Dashed lines represent 95% confidence intervals. Panel C shows measurements of CTL019 gene-modified T cells in peripheral blood as assessed by means of quantitative real-time polymerase-chain-reaction (PCR) assay. Genomic DNA was isolated from samples of whole blood obtained at serial time points before and after infusion of CTL019. The horizontal line at 5 copies per microgram of DNA represents the lower limit of quantification of this assay. Data on patients who did not have a response are shown in red. In general, the levels of CTL019 detected by means of quantitative PCR correlated well with the level of CTL019-positive cells detected by means of flow cytometry, with the exception of the levels in 3 patients who did not have a response and whose peak levels measured by means of quantitative PCR (6066, 5982, and 178,481 copies per microgram of genomic DNA, respectively) did not correspond with detection of CTL019 cells by means of flow cytometry or the induction of B-cell aplasia. CTL019 sequences were detected (23 copies of CTL019 cells per microgram of DNA) at month 24 by means of quantitative PCR in the 1 patient who remained in complete remission at the 2-year follow-up. Data at the first time point were obtained before infusion of CTL019 cells. Doses of cells were determined according to the total amount of cells available after manufacturing. The manufacturing goal of 1.5×107 to 5×109 total cells (3×105 to 1×108 cells per kilogram of body weight) was achieved in all treated patients (see Table S2 in the Supplementary Appendix). A split-dose strategy was used to determine safety with 0.1×108 to 1×108 cells per kilogram infused over 1 to 3 days (5×108 to 50×108 cells in patients who weighed 50 kg or more). The transduction efficiency ranged from 5.5 to 45.3%; this yielded a dose of 0.76 to 20.6×106 CTL019 cells per kilogram.

The CTL019 cells were easily detectable by means of flow cytometry, thus reflecting high in vivo proliferation. In 27 patients who had a response, high peak proportions of CTL019-modified T cells were detected by this method (median, 39.8% of CTL019-positive cells in CD3-positive cells; range, 4.4 to 69.3), whereas in the 3 patients who did not have a response, 0.2%, 0.6%, and 8.2% of CD3-positive cells, respectively, were CTL019-positive at peak levels. CTL019 cells were detectable in the blood by means of flow cytometry for up to 11 months (Figure 2A and 2B).

Figure 3. Figure 3. B-Cell Aplasia. Panel A shows the results of testing to detect the percentage of CD19-positive lymphocytes in peripheral-blood samples by means of flow cytometry. Patients 1 through 25 were participants in the pediatric trial (which included children and young adults 5 to 22 years of age), and Patients 26 through 30 were participants in the adult trial (which included patients 26 to 60 years of age). Negative results were defined as less than 3% of lymphocytes that were positive for CD19. An outlier sample (Patient 16 at month 3) with 4% CD19-positive lymphocytes was discrepant with the measurements on clinical flow cytometry (<1% CD19-positive cells), bone marrow measurements at the same time point, and four subsequent monthly evaluations and was, therefore, considered to be negative. Panel B shows a Kaplan–Meier curve of the time to either CD19 positivity or relapse. Data on patients in remission were censored at the time of the last follow-up (indicated by tick marks). Dashed lines represent 95% confidence intervals. All patients required intravenous immunoglobulin replacement, and no serious infectious complications were observed as a result of B-cell aplasia; however, bronchitis (in one patient), acute otitis media (in two patients), salmonella infection (in one patient), and recurrent urinary tract infections (in one patient) were observed.

The probability of persistence of CTL019 at 6 months was 68% (95% CI, 50 to 92). CTL019 sequences remained detectable by means of quantitative polymerase-chain-reaction (PCR) assay in patients with sustained remissions until 2 years (Figure 2C and data not shown). This assay showed very high levels of proliferation of CTL019 cells; all patients had peak levels greater than 5000 copies per microgram of genomic DNA, and 26 patients had peak levels greater than 15,000 copies per microgram of genomic DNA. One patient (Patient 17) received infusions again at 3 months and 6 months because of early loss of CTL019 cells with B-cell recovery, and this patient subsequently had persistence of CTL019. In the patient with the longest remission (2 years), B-cell aplasia (absence of CD19-positive cells) (Figure 3) continued for a year after the loss of CTL019 cells detectable by flow cytometry, suggesting functional persistence of CTL019 cells below the limits of detection by flow cytometry, whereas CTL019 remained detectable by means of quantitative PCR. The probability of relapse-free B-cell aplasia at 6 months was 73% (95% CI, 57 to 94).

CTL019 for Relapse after Allogeneic Stem-Cell Transplantation

In the 18 patients who were treated for relapse of disease after allogeneic stem-cell transplantation, the median donor chimerism at the time of leukapheresis was 100% (range, 68 to 100). No graft-versus-host disease was observed after infusion of CTL019. Event-free survival and overall survival did not differ significantly between the patients who had previously undergone stem-cell transplantation and those who had not undergone stem-cell transplantation (P=0.21 for event-free survival and P=0.24 for overall survival).

Therapy after Administration of CTL019

Five patients withdrew from the study after the administration of CTL019 to receive other therapy; three of these patients underwent allogeneic stem-cell transplantation while their disease was in remission, and the disease remained in remission 7 to 12 months after the infusion of CTL019. Patient 12, who had undergone a previous stem-cell transplantation, had a post-transplantation relapse of T-cell ALL that aberrantly expressed CD19, was refractory to two intensive reinduction regimens, and entered a morphologic remission after the infusion of CTL019, but the patient had minimal residual disease (0.09%). She subsequently received bortezomib and an infusion of donor lymphocytes, and the disease remained in remission without minimal residual disease at 11 months. In Patient 11, the myelodysplastic syndrome developed and led to overt acute myeloid leukemia with a monosomy 8 clone that also shared cytogenetic features with the original B-cell ALL.

Cytokine-Release Syndrome

A major toxic effect associated with CTL019 is the cytokine-release syndrome, a systemic inflammatory response that is produced by elevated levels of cytokines; these elevations are associated with T-cell activation and proliferation. The cytokine-release syndrome ranges from mild and self-limiting, with high temperatures and myalgias, to severe and life-threatening, with a clinical course that also includes vascular leak, hypotension, respiratory and renal insufficiency, cytopenias, and coagulopathy. Several aspects of the cytokine-release syndrome mirror those of the macrophage activation syndrome.8 All the patients in our studies had the cytokine-release syndrome, which was mild to moderate in 22 of the 30 patients. These patients required hospitalization for febrile neutropenia and received broad-spectrum antibiotics and pain medications. Severe cytokine-release syndrome, which required intensive care with varying degrees of respiratory support (from placement of a nasal cannula to mechanical ventilation), developed in 8 patients (27%), and all these patients required vasopressor support for hypotension. Coagulopathy, with elevated prothrombin and partial-thromboplastin times as well as severe hypofibrinogenemia, was observed in patients who had severe cytokine-release syndrome, although clinical bleeding was rare (observed in 3 patients).

Figure 4. Figure 4. Correlates of the Cytokine-Release Syndrome. Panel A shows peak levels of interleukin-6 in the first 28 days after infusion of CTL019 cells in patients with severe cytokine-release syndrome as compared with patients with cytokine-release syndrome that was not severe. Severe cytokine-release syndrome was defined as hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation. Panel B shows the severity of cytokine-release syndrome according to the baseline disease burden in bone marrow after chemotherapy and before infusion (in the pediatric trial only). Solid circles indicate complete remission, open circles no response, and horizontal lines medians.

Severe cytokine-release syndrome started a median of 1 day after infusion, whereas cytokine-release syndrome that was not severe started a median of 4 days after infusion (P=0.005). Laboratory markers of systemic inflammation, including C-reactive protein and ferritin levels, were elevated in all the patients. Patients who had severe cytokine-release syndrome had higher peak levels of interleukin-6 than did patients who did not have severe cytokine-release syndrome (P<0.001) (Figure 4A); they also had higher peak levels of C-reactive protein (P=0.02), ferritin (P=0.005), interferon-γ (P<0.001), and soluble interleukin-2 receptor (P<0.001) (Fig. S2 in the Supplementary Appendix). The baseline disease burden (the percentage of blast cells in bone marrow before infusion) correlated with the severity of the cytokine-release syndrome; a higher disease burden was significantly associated with severe cytokine-release syndrome (P=0.002) (Figure 4B). Patients with severe cytokine-release syndrome also had higher levels of CTL019-positive CD8 cells (P=0.012) and CTL019-positive CD3 cells (P=0.026).

We previously found marked elevation of interleukin-6 levels after CTL019 therapy and rapid reversal of severe cytokine-release syndrome with the interleukin-6–receptor blocking antibody tocilizumab8; therefore, tocilizumab was incorporated into the management of severe cytokine-release syndrome in this study. Nine patients received tocilizumab, which resulted in rapid defervescence and stabilization of blood pressure, with improvement (weaning from vasopressor support) over a period of 1 to 3 days. Six patients also received short courses of glucocorticoids, and four patients received a second dose of tocilizumab for recrudescence of the cytokine-release syndrome after transient improvement with the first dose. All the patients recovered fully, and there was a complete reversal of symptoms and a normalization of laboratory results. Relapses occurred in two of the nine patients who received immunosuppressive therapy for the cytokine-release syndrome.

Encephalopathy

Thirteen patients had neurologic toxic effects, which ranged from delirium during the period of high temperatures to global encephalopathy with one or more of the following: aphasia, confusion, delirium, and hallucinations. Six patients had delayed encephalopathy that occurred after high temperatures had resolved and was independent of the severity of the cytokine-release syndrome and whether the patient had received prior tocilizumab therapy. Symptoms were self-limiting (lasting 2 to 3 days and resolving over 2 to 3 days), and they resolved fully without further intervention or apparent long-term sequelae. One patient with encephalopathy had two seizures that may have been caused by concomitant electrolyte abnormalities. Several patients had normal computed tomographic or magnetic resonance imaging of the head and lumbar puncture that was negative for infection or leukemia.

B-Cell Aplasia

We performed flow cytometry to detect CD19-positive B cells in order to monitor patients for the development of B-cell aplasia, which can be used as a pharmacodynamic measure of CTL019 function (Figure 3). B-cell aplasia occurred in all the patients who had a response and persisted for up to 1 year after CTL019 cells were no longer detectable by means of flow cytometry. Patients with B-cell aplasia received immunoglobulin replacement to maintain IgG levels greater than 500 mg per deciliter.