Shannon L. Maude

Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia

BACKGROUND Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor-modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease. METHODS We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×10(6) to 20.6×10(6) CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells. RESULTS A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti-interleukin-6 receptor antibody tocilizumab. CONCLUSIONS Chimeric antigen receptor-modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by Novartis and others; CART19 ClinicalTrials.gov numbers, NCT01626495 and NCT01029366.).

Genetic Alterations Activating Kinase and Cytokine Receptor Signaling in High-Risk Acute Lymphoblastic Leukemia

Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy

Blinatumomab is a CD19/CD3-bispecific T-cell receptor-engaging (BiTE) antibody with efficacy in refractory B-precursor acute lymphoblastic leukemia. Some patients treated with blinatumomab and other T cell-activating therapies develop cytokine release syndrome (CRS). We hypothesized that patients with more severe toxicity may experience abnormal macrophage activation triggered by the release of cytokines by T-cell receptor-activated cytotoxic T cells engaged by BiTE antibodies and leading to hemophagocytic lymphohistiocytosis (HLH). We prospectively monitored a patient during blinatumomab treatment and observed that he developed HLH. He became ill 36 hours into the infusion with fever, respiratory failure, and circulatory collapse. He developed hyperferritinemia, cytopenias, hypofibrinogenemia, and a cytokine profile diagnostic for HLH. The HLH continued to progress after discontinuation of blinatumomab; however, he had rapid improvement after IL-6 receptor-directed therapy with tocilizumab. Patients treated with T cell-activating therapies, including blinatumomab, should be monitored for HLH, and cytokine-directed therapy may be considered in cases of life-threatening CRS. This trial was registered at www.clinicaltrials.gov as #NCT00103285.

CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia

Relapsed and refractory acute lymphoblastic leukemia (ALL) remains difficult to treat, with minimal improvement in outcomes seen in more than 2 decades despite advances in upfront therapy and improved survival for de novo ALL. Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a powerful targeted immunotherapy, showing striking responses in highly refractory populations. Complete remission (CR) rates as high as 90% have been reported in children and adults with relapsed and refractory ALL treated with CAR-modified T cells targeting the B-cell-specific antigen CD19. Distinct CAR designs across several studies have produced similar promising CR rates, an encouraging finding. Even more encouraging are durable remissions observed in some patients without additional therapy. Duration of remission and CAR-modified T-cell persistence require further study and more mature follow-up, but emerging data suggest these factors may distinguish CAR designs. Supraphysiologic T-cell proliferation, a hallmark of this therapy, contributes to both efficacy and the most notable toxicity, cytokine release syndrome (CRS), posing a unique challenge for toxicity management. This review will discuss the current landscape of CD19 CAR clinical trials, CRS pathophysiology and management, and remaining challenges.

Managing cytokine release syndrome associated with novel T cell-engaging therapies.

AbstractChimeric antigen receptor (CAR)-modified T cells and bispecific T cell–engaging antibodies have demonstrated dramatic clinical responses in recent clinical trials. The hallmark of these novel highly active immunotherapies is nonphysiologic T cell activation, which has correlated not only with greatly increased efficacy but also with notable toxicity in some cases. We and others have observed a cytokine release syndrome (CRS), which correlates with both toxicity and efficacy in patients receiving T cell–engaging therapies. In addition to elevations in effector cytokines, such as interferon-&ggr;, cytokines associated with hemophagocytic lymphohistiocytosis or macrophage activation syndrome, such as interleukin (IL)-10 and IL-6, may also be markedly elevated. Whereas corticosteroids may control some of these toxicities, their potential to block T cell activation and abrogate clinical benefit is a concern. Detailed studies of T cell proliferation and the resultant immune activation produced by these novel therapies have led to more targeted approaches that have the potential to provide superior toxicity control without compromising efficacy. One approach we have developed targets IL-6, a prominent cytokine in CRS, using the IL-6R antagonist tocilizumab. We will review the pathophysiology and management options for CRS associated with T cell–engaging therapies.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy

UNLABELLED The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia.

UNLABELLED Chimeric antigen receptor (CAR)-modified T cells with anti-CD19 specificity are a highly effective novel immune therapy for relapsed/refractory acute lymphoblastic leukemia. Cytokine release syndrome (CRS) is the most significant and life-threatening toxicity. To improve understanding of CRS, we measured cytokines and clinical biomarkers in 51 CTL019-treated patients. Peak levels of 24 cytokines, including IFNγ, IL6, sgp130, and sIL6R, in the first month after infusion were highly associated with severe CRS. Using regression modeling, we could accurately predict which patients would develop severe CRS with a signature composed of three cytokines. Results were validated in an independent cohort. Changes in serum biochemical markers, including C-reactive protein and ferritin, were associated with CRS but failed to predict development of severe CRS. These comprehensive profiling data provide novel insights into CRS biology and, importantly, represent the first data that can accurately predict which patients have a high probability of becoming critically ill. SIGNIFICANCE CRS is the most common severe toxicity seen after CAR T-cell treatment. We developed models that can accurately predict which patients are likely to develop severe CRS before they become critically ill, which improves understanding of CRS biology and may guide future cytokine-directed therapy. Cancer Discov; 6(6); 664-79. ©2016 AACR.See related commentary by Rouce and Heslop, p. 579This article is highlighted in the In This Issue feature, p. 561.

Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia

Background In a single‐center phase 1–2a study, the anti‐CD19 chimeric antigen receptor (CAR) T‐cell therapy tisagenlecleucel produced high rates of complete remission and was associated with serious but mainly reversible toxic effects in children and young adults with relapsed or refractory B‐cell acute lymphoblastic leukemia (ALL). Methods We conducted a phase 2, single‐cohort, 25‐center, global study of tisagenlecleucel in pediatric and young adult patients with CD19+ relapsed or refractory B‐cell ALL. The primary end point was the overall remission rate (the rate of complete remission or complete remission with incomplete hematologic recovery) within 3 months. Results For this planned analysis, 75 patients received an infusion of tisagenlecleucel and could be evaluated for efficacy. The overall remission rate within 3 months was 81%, with all patients who had a response to treatment found to be negative for minimal residual disease, as assessed by means of flow cytometry. The rates of event‐free survival and overall survival were 73% (95% confidence interval [CI], 60 to 82) and 90% (95% CI, 81 to 95), respectively, at 6 months and 50% (95% CI, 35 to 64) and 76% (95% CI, 63 to 86) at 12 months. The median duration of remission was not reached. Persistence of tisagenlecleucel in the blood was observed for as long as 20 months. Grade 3 or 4 adverse events that were suspected to be related to tisagenlecleucel occurred in 73% of patients. The cytokine release syndrome occurred in 77% of patients, 48% of whom received tocilizumab. Neurologic events occurred in 40% of patients and were managed with supportive care, and no cerebral edema was reported. Conclusions In this global study of CAR T‐cell therapy, a single infusion of tisagenlecleucel provided durable remission with long‐term persistence in pediatric and young adult patients with relapsed or refractory B‐cell ALL, with transient high‐grade toxic effects. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02435849.)

Efficacy of JAK/STAT pathway inhibition in murine xenograft models of early T-cell precursor (ETP) acute lymphoblastic leukemia.

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed.