Chimeric antigen receptor-modified T cells for acute lymphoid leukemia

Abstract

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

Figure 1. Clinical Responses to CTL019 Infusion in Two Children with Relapsed, Chemotherapy-Refractory Acute Lymphoblastic Leukemia (ALL)

The two children, both of whom had CD19+ B-cell– precursor ALL, received infusions of CTL019 cells on day 0. Panel A shows changes in serum lactate dehydrogenase (LDH) levels and body temperature after CTL019 infusion, with the maximum temperature per 24-hour period indicated by the circles. Patient 1 was given methylprednisolone starting on day 5 at a dose of 2 mg per kilogram of body weight per day, tapered to 0 by day 12. On the morning of day 7, etanercept was given at a dose of 0.8 mg per kilogram. At 6 p.m. on day 7, tocilizumab was given at a dose of 8 mg per kilogram. A transient improvement in pyrexia occurred after the administration of glucocorticoids on day 5, with complete resolution of fevers occurring after the administration of cytokine-directed therapy. Panel B shows serum levels of cytokines and inflammatory markers measured at the indicated time points after CTL019 infusion. Cytokine values are shown with the use of a semilogarithmic plot indicating change from baseline. Baseline values (on day 0 before infusion) in Patient 1 and Patient 2, respectively, were as follows: interleukin-1β, 0.9 and 0.2 pg per milliliter; interleukin-6, 4.3 and 1.9 pg per milliliter; interferon-γ, 0.08 and 0.23 pg per milliliter; tumor necrosis factor α (TNF-α), 1.5 and 0.4 pg per milliliter; interleukin-2 receptor α, 418.8 and 205.7 pg per milliliter; interleukin-2, 0.7 and 0.4 pg per milliliter; interleukin-10, 9.9 and 2.3 pg per milliliter; and interleukin-1 receptor α, 43.9 and 27.9 pg per milliliter. Pronounced elevations in a number of cytokines and cytokine receptors developed in both patients, including soluble interleukin-1 receptor α; interleukin-2 receptor; interleukin-2, 6, and 10; TNF-α; and inter fer on-γ. Panel C shows changes in the circulating absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and white-cell count. The increase in the ALC was primarily from activated CTL019 T lymphocytes.

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 log₁₀ 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-cyto-metric detection of CTL019 cells in CSF from Patients 1 and 2. FMO denotes fluorescence minus one. Both the x and y axes are log₁₀ scales. Panel D shows activated large granular lymphocytes in Wright-stained smears of the peripheral blood and cytospin preparations of CSF from Patient 2.

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.