Induction of Remission in Both Patients

Figure 2. Figure 2. Expansion and Visualization of CTL019 Cells in Peripheral Blood, Bone Marrow, and Cerebrospinal Fluid (CSF). Panel A shows the results of flow-cytometric analysis of peripheral blood stained with antibodies to detect CD3 and the anti-CD19 chimeric antigen receptor. Both the x and y axes are log 10 scales. Depicted is the percentage of CD3 cells expressing the chimeric antigen receptor in Patients 1 and 2. Panel B shows the presence of CTL019 T cells in peripheral blood, bone marrow, and CSF as assessed by means of a quantitative real-time polymerase-chain-reaction (PCR) assay. Genomic DNA was isolated from samples of whole blood, bone marrow aspirate, and CSF collected at serial time points before and after CTL019 infusion. The 1% marking line represents the number of detected transgene copies that would be expected if 1% of the total cells in the sample contained a single integration of the chimeric antigen receptor transgene. Panel C shows flow-cytometric detection of CTL019 cells in CSF from Patients 1 and 2. FMO denotes fluorescence minus one. Both the x and y axes are log 10 scales. Panel D shows activated large granular lymphocytes in Wright-stained smears of the peripheral blood and cytospin preparations of CSF from Patient 2.

Table 1. Table 1. Induction of Molecular Remission in the Blood and Bone Marrow of the Patients.

Both children had an increase in circulating lymphocytes and neutrophils in the 2 weeks after CTL019 infusion, as shown by plots depicting the total white-cell count, absolute lymphocyte count, and absolute neutrophil count relative to the timing of CTL019 infusion (Figure 1C). Most of the lymphocytes were T cells that expressed the chimeric antigen receptor (Figure 2, and Figure S2 in the Supplementary Appendix). In both children, high-grade, most likely noninfectious fevers were documented, followed by elevations in lactate dehydrogenase (LDH) levels (Figure 1A). The elevations in LDH levels and the high-grade fevers were similar to those previously described in patients with CLL after CTL019 infusion.7,8 Approximately 1 month after infusion, morphologic remission of leukemia (minimal residual disease, <0.01%) was achieved in both children. Results of flow-cytometric assays of minimal residual disease were confirmed by molecular detection of clonal IgH transcripts by means of deep sequencing (Table 1).

The clinical remission in Patient 1 was associated with a molecular remission that had persisted for 9 months as of January 2013. High-throughput DNA sequencing of the IGH locus revealed a pronounced decrease in total rearranged IGH sequence reads on day 23 in blood and marrow specimens. The malignant clone was not detected in the blood or marrow in more than 1 million cell equivalents that were sequenced on day 180 (Table 1). In contrast, rearranged T-cell–receptor sequences were readily detected in blood and marrow, findings that indicate the integrity of the DNA tested at all time points.

Toxicity of CTL019

Table 2. Table 2. Grade 3 or 4 Adverse Events.

Grade 3 and 4 adverse events are summarized in Table 2. Both patients had acute toxic effects, which consisted of fever and a cytokine-release syndrome that evolved into the macrophage activation syndrome. Both patients were monitored and given prophylaxis for the tumor lysis syndrome. Both had substantial elevations in LDH levels, the causes of which were probably multifactorial but could have included the tumor lysis syndrome. Each uric acid value in Patient 1 was either below or within the normal range, and she received allopurinol on days 5 and 6 only. Patient 2 received prophylactic allopurinol on days 0 through 14 and had abnormal uric acid values of 4.8 to 5.7 mg per deciliter (286 to 339 μmol per liter) on days 8 through 10, which were consistent with mild tumor lysis syndrome.

Severe cytokine-release syndrome developed in Patient 1. Glucocorticoids were administered to this patient on day 5, with a brief response in the fever curve but without remission of hypotension. A single course of anticytokine therapy, consisting of etanercept and tocilizumab, was given on day 7, with rapid clinical effects: within hours, defervescence occurred, and the patient was weaned from vasoactive medications and ventilatory support as the clinical and radiologic manifestations of the acute respiratory distress syndrome resolved. She did not have laboratory evidence of the tumor lysis syndrome; however, biochemical evidence of the macrophage activation syndrome was noted, with elevation of the ferritin level to 45,529 ng per deciliter on day 11, coagulopathy with an elevated D-dimer level and hypofibrinogenemia, hepatosplenomegaly, and elevated levels of aminotransferases, LDH (Figure 1A), and triglycerides, as well as a cytokine profile that was consistent with the macrophage activation syndrome. Her ferritin level decreased to 2368 ng per deciliter by day 26, and the clinical and laboratory abnormalities of the macrophage activation syndrome resolved.

In Patient 2, although there was no direct evidence of the tumor lysis syndrome other than fever and changes in the LDH level (Figure 1A), features of the macrophage activation syndrome also developed, with elevations in the ferritin level to 33,360 ng per deciliter on day 7, peaking at 74,899 ng per deciliter on day 11; aminotransferase levels that reached grade 4 for 1 day; and an elevated serum D-dimer level. These biochemical changes were reversible: on day 21, the aminotransferase elevations were grade 1, and the ferritin level 3894 ng per deciliter. The patient was discharged from the hospital on day 16.

Both children had prominent elevations in a number of cytokines and cytokine receptors in the serum (Figure 1B). In both, elevations in interferon-γ and interleukin-6 were most prominent. These observations are similar to the pattern observed previously in patients with CLL who also had a remission of leukemia after CTL019 infusion.8 The peak cytokine elevations were temporally correlated with systemic inflammation as determined by changes in core body temperature (Figure 1A and 1B).

In Vivo Expansion of CTL019

Figure 3. Figure 3. CD19 Expression at Baseline and at the Time of Relapse in Patient 2. Bone marrow samples were obtained from Patient 2 before CTL019 infusion and at the time of relapse, 2 months later. Mononuclear cells isolated from marrow samples were stained for CD45, CD34, and CD19 and analyzed on an Accuri C6 flow cytometer. After a gating on live cells, the blast gate (CD45+ side scatter [SSC] low) was subgated on CD34+ cells, and histograms were generated for CD19 expression. The vertical line in each graph represents the threshold for the same gating on isotype controls. Pretherapy blasts (Panel A) have a range of distribution of CD19, with a small population of very dim-staining cells seen as the tail at the left of the histogram at 102 on the x axis. The numbers on the x axis are arbitrary fluorescence intensity units. The sample obtained at the time of relapse (Panel B) does not have any CD19+ blasts. Analysis of CD19 expression on the pretreatment blast population revealed a small population of CD19+dim or CD19− cells. The mean fluorescence intensity of this small population of cells was 187 units, which is similar to that of the anti-CD19–stained blast cells at relapse, 201 units. The pretherapy marrow sample was hypocellular, with 10% blasts, and the marrow sample at relapse was normocellular, with 68% blasts, accounting for differences in the number of events (cells) available for acquisition.

The fraction of CTL019 T cells in circulation progressively increased in vivo to 72% of T cells in Patient 1 and 34% of T cells in Patient 2 (Figure 2A). The initial transduction efficiency was 11.6% and 14.4% for the T cells infused in Patient 1 and Patient 2, respectively. In both children, the absolute lymphocyte count increased substantially (Figure 1C) and the number of CTL019 cells progressively increased from baseline in vivo (Figure 2A, and Figure S2 in the Supplementary Appendix), reflecting a robust and selective expansion of CTL019 cells. The selective increase in T cells expressing CTL019 in both children is consistent with an antileukemic mechanism involving CD19-driven expansion and with the subsequent elimination of cells expressing CD19 (Figure 3, and Figure S3A and S3B in the Supplementary Appendix).

Molecular deep-sequence analysis of T-cell receptors (TCRs) in the peripheral-blood and marrow samples obtained from Patient 1 on day 23, when more than 65% of CD3+ cells in peripheral blood and marrow were shown to be CTL019+ on flow cytometry, revealed the absence of a dominant T-cell TCR clonotype in both compartments, with the 10 most abundant T cells present at frequencies of 0.2 to 0.7% in bone marrow and 0.2 to 0.8% in peripheral blood. Six of the 10 dominant clones were shared between the two compartments. In addition, both CD4 and CD8 chimeric antigen receptor T cells were present. Thus, the chimeric antigen receptor T cells appeared to proliferate after CD19-stimulated expansion and not by means of TCR signals or clone-specific events such as activation by integration of the lentivirus.

Expansion and Morphologic Features of CTL019 Chimeric Antigen Receptor T Cells

In both children, CTL019 cells expanded in the peripheral blood and bone marrow to levels that were more than 1000 times as high as the original engraftment levels (Figure 2A and 2B). The frequency of CTL019 cells increased to more than 10% of circulating T cells by day 20 in both children (Figure S2 in the Supplementary Appendix), with the absolute magnitude of CTL019 expansion similar to that observed in patients with CLL.8 Unexpectedly, cells in the CSF also showed a high degree of CTL019 gene marking and persisted at a high frequency at 6 months (Figure 2B). The presence of CTL019 cells in the CSF was surprising, given that neither child had detectable central nervous system (CNS) leukemia according to analysis of cytospin preparations at the time of infusion or at the evaluation 1 month after treatment. Furthermore, prior studies of chimeric antigen receptor therapy for B-cell cancers have not shown the presence of chimeric antigen receptor T cells in the CNS.4,6,9,11-13 The morphologic features of the lymphocytes in the blood and CSF are shown for both children in Figure 2D. Because more than 70% of lymphocytes in circulation on day 10 were CTL019 cells (Figure 2A and 2B), most of the large granular lymphocytes in the peripheral blood, as shown in Figure 2D, are probably CTL019 cells. Similarly, because many lymphocytes in the CSF obtained from Patient 2 on day 23 were CTL019 cells (Figure 2B and 2C), the CSF lymphocytes shown in Figure 2D most likely represent the morphologic features of CTL019 cells in vivo that have migrated to the CSF.

Induction of B-Cell Aplasia

In both children, CD19+ cells in bone marrow and blood were eliminated within 1 month after CTL019 infusion (Figure 3, and Figure S3A and S3B in the Supplementary Appendix). In Patient 1, a large proportion of cells remaining in the marrow at day 6 after infusion were CD19+CD20+ leukemic blast cells. This population of cells was not detectable by day 23, an effect that has been maintained (Figure S3A in the Supplementary Appendix). Marrow in this patient remained in remission for 9 months, and peripheral-blood counts remained normal for more than 11 months. Patient 1 did not receive chemotherapy in the 6 weeks before CTL019 infusion, which indicates that the CTL019 cells were sufficient to ablate normal and leukemic B cells in this patient.

Emergence of CD19 Escape Variant in Patient 2

Patient 2 had a clinical relapse that was apparent in the peripheral blood 2 months after CTL019 infusion, as evidenced by the reappearance of blast cells in the circulation. These cells were CD45+dim, CD34+ and did not express CD19 (Figure 3). The absence of the original dominant CD34dim+CD34+CD19dim+ cells is consistent with a potent antileukemic selective pressure of the CTL019 chimeric antigen receptor T cells directed to CD19 (Figure S3B in the Supplementary Appendix). Deep sequencing of IGH revealed the presence of the malignant clone in peripheral blood and marrow as early as day 23 (Table 1), despite a clinical assessment of no residual disease by means of flow cytometry at this time point (Figure S1 in the Supplementary Appendix). In addition, deep sequencing of DNA isolated from bone marrow cells obtained at the time of clinical relapse revealed that the CD45+dimCD34+CD19− cells are clonally related to the initial dominant CD45dim+CD34+CD19dim+ cells, since they share the same IGH sequence.