Mark G. Frattini

CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.

Five adults with chemotherapy-refractory B-ALL were induced into molecular remissions after treatment with CD19 CAR-targeted T cells. CARving a Niche for Cancer Immunotherapy Acute lymphoblastic leukemia (ALL) is a cancer of the white blood cells that fend off infection. It’s most common in children but—as with many diseases that primarily affect children—has a much worse prognosis when it affects adults. Adults with relapsed disease have a very low chance of survival, and new therapies are desperately needed. Now, Brentjens et al. test T cells engineered to target CD19, which is expressed on both healthy B lymphocytes and B-ALL cells, in five chemotherapy-refractory adult B-ALL patients. Here, the authors treat patients with the patients’ own T cells altered to express not only CD19 but also a fusion of the costimulatory molecule CD28 with CD3ζ chain—so-called “second-generation chimeric antigen receptor (CAR) T cells.” All patients treated with these cells achieved tumor eradication and complete remission. These CAR T cells were well tolerated, although there was substantial cytokine release in some patients that correlated to tumor burden. These patients were treated with steroid therapy. Long-term follow-up in four of these patients was not possible because the CAR T cell therapy allowed these patients to be eligible for subsequent hematopoietic stem cell transplant (HSCT), which resulted in restored hematopoiesis. The remaining patient experienced a relapse of CD19+ cells that coincided with the lack of persistence of the CAR T cells from circulation. These data suggest that subsequent transfusions should be considered for patients unable to undergo HSCT. Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD+ disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD− complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell–mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.

Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.

CD19 CAR T cell therapy induces complete remissions in 88% of 16 adult patients with relapsed or refractory acute lymphoblastic leukemia. CARving Out a Niche for CAR T Cell Immunotherapy Relapsed or refractory B acute lymphoblastic leukemia (B-ALL) in adults has a poor prognosis, with an expected median survival of less than 6 months. An emerging therapy for adult B-ALL is through T cells that target tumor cells with chimeric antigen receptors (CARs). Davila et al. now report the results of a phase 1 clinical trial of CAR T cells in 16 relapsed or refractory adult patients. The CD19-targeting CAR T cell therapy resulted in an 88% complete response rate, which allowed most of the patients to transition to allogeneic hematopoietic stem cell transplantation—the current standard of care. Moreover, the authors carefully characterized cytokine release syndrome (CRS), which is a series of toxicities associated with CAR T cell therapy. They found that serum C-reactive protein (CRP) associated with the severity of CRS, which should allow for identification of the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the CRS. This is especially important because treatment for CRS may limit the efficacy of the CAR T cell therapy. These data support the need for further multicenter trials for CAR T cell therapy. We report on 16 patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) that we treated with autologous T cells expressing the 19-28z chimeric antigen receptor (CAR) specific to the CD19 antigen. The overall complete response rate was 88%, which allowed us to transition most of these patients to a standard-of-care allogeneic hematopoietic stem cell transplant (allo-SCT). This therapy was as effective in high-risk patients with Philadelphia chromosome–positive (Ph+) disease as in those with relapsed disease after previous allo-SCT. Through systematic analysis of clinical data and serum cytokine levels over the first 21 days after T cell infusion, we have defined diagnostic criteria for a severe cytokine release syndrome (sCRS), with the goal of better identifying the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the sCRS. Additionally, we found that serum C-reactive protein, a readily available laboratory study, can serve as a reliable indicator for the severity of the CRS. Together, our data provide strong support for conducting a multicenter phase 2 study to further evaluate 19-28z CAR T cells in B-ALL and a road map for patient management at centers now contemplating the use of CAR T cell therapy.

Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias

We report the findings from the first 10 patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) or relapsed B-cell acute lymphoblastic leukemia (ALL) we have enrolled for treatment with autologous T cells modified to express 19-28z, a second-generation chimeric antigen (Ag) receptor specific to the B-cell lineage Ag CD19. Eight of the 9 treated patients tolerated 19-28z(+) T-cell infusions well. Three of 4 evaluable patients with bulky CLL who received prior conditioning with cyclophosphamide exhibited either a significant reduction or a mixed response in lymphadenopathy without concomitant development of B-cell aplasia. In contrast, one patient with relapsed ALL who was treated in remission with a similar T-cell dose developed a predicted B-cell aplasia. The short-term persistence of infused T cells was enhanced by prior cyclophosphamide administration and inversely proportional to the peripheral blood tumor burden. Further analyses showed rapid trafficking of modified T cells to tumor and retained ex vivo cytotoxic potential of CD19-targeted T cells retrieved 8 days after infusion. We conclude that this adoptive T-cell approach is promising and more likely to show clinical benefit in the setting of prior conditioning chemotherapy and low tumor burden or minimal residual disease. These studies are registered at www.clinicaltrials.org as #NCT00466531 (CLL protocol) and #NCT01044069 (B-ALL protocol).

Vaccination with synthetic analog peptides derived from WT1 oncoprotein induces T-cell responses in patients with complete remission from acute myeloid leukemia

A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 microg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-gamma release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-gamma-secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia.

Terminal myeloid differentiation in vivo is induced by FLT3 inhibition in FLT3/ITD AML

A hallmark of cancer is the disruption of differentiation within tumor cells. Internal tandem duplication mutations of the FLT3 kinase (FLT3/ITD) occur commonly in acute myeloid leukemia (AML) and are associated with poor survival, leading to efforts to develop FLT3 kinase inhibitors. However, FLT3 inhibitors have thus far met with limited success, inducing only a clearance of peripheral blasts with minimal BM responses. Quizartinib is a novel potent and selective FLT3 inhibitor currently being studied in clinical trials. In 13 of 14 FLT3/ITD AML patients with normal karyotype treated with quizartinib, we observed terminal myeloid differentiation of BM blasts in association with a clinical differentiation syndrome. The single patient whose blasts failed to differentiate had a preexisting C/EBPα mutation and another developed a C/EBPα mutation at disease progression, suggesting a mechanism of resistance to FLT3 inhibition. In vitro, in primary blasts cocultured with human BM stroma, FLT3 inhibition with quizartinib induced cell-cycle arrest and differentiation rather than apoptosis. The present study is the first description of terminal differentiation of cancer cells in patients treated with a tyrosine kinase inhibitor. These data highlight the importance of the differentiation block in the patho-genesis of AML.

Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: a non-randomised, open-label, phase 1b study

BACKGROUND Elderly patients (aged ≥65 years) with acute myeloid leukaemia have poor outcomes and no effective standard-of-care therapy exists. Treatment with hypomethylating agents such as azacitidine and decitabine is common, but responses are modest and typically short-lived. The oral anti-apoptotic B-cell lymphoma 2 protein inhibitor, venetoclax, has shown promising single-agent activity in patients with relapsed or refractory acute myeloid leukaemia and preclinical data suggested synergy between hypomethylating agents and venetoclax, which led to this combination phase 1b study. METHODS Previously untreated patients aged 65 years and over with acute myeloid leukaemia who were ineligible for standard induction therapy were enrolled into this non-randomised, open-label, phase 1b study. Patients were required to have an Eastern Cooperative Oncology Group performance status of 0-2 and either intermediate-risk or poor-risk cytogenetics. Patients were enrolled into one of three groups for the dose-escalation phase of this study: group A (venetoclax and intravenous decitabine 20 mg/m2 [days 1-5 of each 28-day cycle]), group B (venetoclax and subcutaneous or intravenous azacitidine 75 mg/m2 [days 1-7 of each 28-day cycle]), and group C (a venetoclax and decitabine substudy with the oral CYP3A inhibitor posaconazole, 300 mg twice on cycle 1, day 21, and 300 mg once daily from cycle 1, days 22-28, to assess its effect on venetoclax pharmacokinetics). Dose escalation followed a standard 3 + 3 design with at least three evaluable patients enrolled per cohort; daily target doses of venetoclax for groups A and B were 400 mg (cohort 1), 800 mg (cohorts 2 and 3), and 1200 mg (cohort 4), and 400 mg for group C. The primary endpoints were the safety and pharmacokinetics of venetoclax plus decitabine or azacitidine, and to determine the maximum tolerated dose and recommended phase 2 dose. Secondary endpoints included the preliminary anti-leukaemic activity of venetoclax with decitabine or azacitidine through the analysis of overall response, duration of response, and overall survival. We analysed safety, pharmacokinetics, and anti-leukaemic activity in all patients who received one or more venetoclax doses. The expansion phase of the study is ongoing but is closed to accrual. This trial is registered with ClinicalTrials.gov, number NCT02203773. FINDINGS 57 patients were enrolled in the study. 23 patients in group A and 22 patients in group B were enrolled between Nov 19, 2014, and Dec 15, 2015, and 12 patients in group C were enrolled between June 14, 2015, and Jan 16, 2016. As of data cutoff on June 15, 2016, the most common grade 3-4 treatment-emergent adverse events were thrombocytopenia (27 [47%] of 57 patients; nine in group A, 13 in group B, and five in group C), febrile neutropenia (24 [42%] of 57; 11 in group A, ten in group B, and three in group C), and neutropenia (23 [40%] of 57; 12 in group A, eight in group B, and three in group C). The most common serious treatment-emergent adverse event in groups A and B was febrile neutropenia (seven [30%] of 23 patients vs seven [32%] of 22), whereas in group C it was lung infection (four [33%] of 12 patients). 49 (86%) of 57 patients had treatment-related adverse events; the most common in groups A and B included nausea (12 [52%] patients vs seven [32%] patients), fatigue (six [26%] patients vs seven [32%]), and decreased neutrophil count (six [26%] patients vs six [27%]), whereas in group C the most common were nausea (seven [58%] of 12 patients), leucopenia (six [50%]), vomiting (five [42%]), and decreased platelet count (five [42%]). The maximum tolerated dose was not reached. The recommended phase 2 dose was 400 mg once a day or 800 mg with an interrupted dosing schedule (safety expansion). In total, four (7%) of 57 patients had died within 30 days of the first venetoclax dose caused by sepsis (group B), bacteraemia (group A), lung infection (group C), and respiratory failure (group A). Tumour lysis syndrome was not observed. Decitabine and azacitidine did not substantially affect venetoclax exposures. Overall, 35 (61%; 95% CI 47·6-74·0) of 57 patients achieved complete remission or complete remission with incomplete marrow recovery. In groups A and B, 27 (60%; 95% CI 44·3-74·3) of 45 patients had complete remission or complete remission with incomplete marrow recovery. INTERPRETATION Venetoclax plus hypomethylating agent therapy seems to be a novel, well-tolerated regimen with promising activity in this underserved patient population. Evaluation of expansion cohorts is ongoing at 400 mg and 800 mg doses using both hypomethylating agent combinations. FUNDING AbbVie and Genentech.

Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions

Eukaryotic DNA replication requires the assembly of multiprotein pre-replication complexes (pre-RCs) at chromosomal origins of DNA replication. Here we describe the interactions of highly purified Schizosaccharomyces pombe pre-RC components, SpORC, SpCdc18, and SpCdt1, with each other and with ars1 origin DNA. We show that SpORC binds DNA in at least two steps. The first step likely involves electrostatic interactions between the AT-hook motifs of SpOrc4 and AT tracts in ars1 DNA and results in the formation of a salt-sensitive complex. In the second step, the salt-sensitive complex is slowly converted to a salt-stable complex that involves additional interactions between SpORC and DNA. Binding of SpORC to ars1 DNA is facilitated by negative supercoiling and is accompanied by changes in DNA topology, suggesting that SpORC-DNA complexes contain underwound or negatively writhed DNA. Purified human origin recognition complex (ORC) induces similar topological changes in origin DNA, indicating that this property of ORC is conserved in eukaryotic evolution and plays an important role in ORC function. We also show that SpCdc18 and SpCdt1 form a binary complex that has greater affinity for DNA than either protein alone. In addition, both proteins contribute significantly to the stability of the initial SpORC-DNA complex and enhance the SpORC-dependent topology changes in origin DNA. Thus, the formation of stable protein-DNA complexes at S. pombe origins of replication involves binary interactions among all three proteins, as well as interactions of both SpORC and SpCdt1-SpCdc18 with origin DNA. These findings demonstrate that SpORC is not the sole determinant of origin recognition.

An exo-cell assay for examining real-time γ-secretase activity and inhibition

Abstractγ-Secretase is an aspartyl protease that cleaves multiple substrates that are involved in broad biological processes ranging from stem cell development to neurodegeneration. The investigation of γ-secretase has been limited by currently available assays that require genetic or biochemical manipulation in the form of substrate transfection or membrane preparation. Here we report an exo-cell assay that is capable of characterizing γ-secretase activity in any cellular system without limitation. Using a highly active, recombinant substrate this assay can quickly and easily ascertain the status of γ-secretase activity in cell systems and patient samples. We have applied this method to determine the activity of γ-secretase in primary cell samples where transfection and/or membrane isolation are not viable options. Importantly, it allows for the detection of real time γ-secretase activity after inhibitor or drug treatment. The application of this assay to determine the role of γ-secretase in physiological and pathological conditions will greatly facilitate our characterization of this complex protease and help in the development and evaluation of γ-secretase-targeted therapies in Alzheimers disease or a variety of neoplasms.

Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase.

Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their structure-activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.

Comparison of Luminescence ADP Production Assay and Radiometric Scintillation Proximity Assay for Cdc7 Kinase

Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.