We generated a novel CD19CAR (CAT) with a lower affinity than FMC63, the binder utilised in many clinical studies. CAT CAR T cells showed increased proliferation/cytotoxicity in vitro and enhanced proliferative capacity and anti-tumor activity than FMC63 CAR T cells in a xenograft model. In a clinical study (CARPALL, NCT02443831), 12/14 patients with relapsed/refractory pediatric BALL obtained molecular remission after CAT CAR T cell therapy. CAR T cell expansion compared favourably with published data on other CD19CARs and persistence was demonstrated in 11 of 14 patients at last follow-up. Toxicity was low with no severe cytokine release syndrome. At a median follow up of 14 months, 5/14 patients (37%) remain in molecular CR with circulating CAR T cells.

Identification of molecular pathways essential for cancer stem cells is critical for understanding the underlying biology and designing effective cancer therapeutics. Here, we demonstrated that β-catenin was activated during development of MLL leukemic stem cells (LSCs). Suppression of β-catenin reversed LSCs to a pre-LSC-like stage and significantly reduced the growth of human MLL leukemic cells. Conditional deletion of β-catenin completely abolished the oncogenic potential of MLL-transformed cells. In addition, established MLL LSCs that have acquired resistance against GSK3 inhibitors could be resensitized by suppression of β-catenin expression. These results unveil previously unrecognized multifaceted functions of β-catenin in the establishment and drug-resistant properties of MLL stem cells, highlighting it as a potential therapeutic target for an important subset of AMLs.

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including IntroductionChildren with Down syndrome (DS), characterized by constitutional trisomy 21, have a 50-fold increased risk of developing acute leukemia in the first few years of life. 1,2 This comprises the normally extremely rare subtype acute megakaryoblastic leukemia (AMKL) as well as the common variety (B-cell precursor) of acute lymphoblastic leukemia (ALL). 2-4 Both DS-AMKL and the transient myeloproliferative disorder (TMD) that often precedes it are consistently associated with acquired mutations in the GATA1 gene. 5 Concordant GATA1 mutations in the blast cells of identical twins with TMD 6 and mutations in the neonatal blood spots of DS newborns 7 indicate that this is an early/prenatal event in DS leukemogenesis.Much less is known about the genetic events predisposing to DS-ALL. Candidate genes for activating mutations in DS-ALL include FLT3 and RAS, both mutated in high hyperdiploid ALL (with acquired trisomy 21), 8,9 PTPN11 and BRAF, mutated in B-cell precursor ALL. 10,11 An additional candidate is the JAK2 pseudokinase domain mutation JAK2⌬IREED, reported in a single case of DS-ALL. 12 A different JAK2 pseudokinase domain mutation, JAK2V617F, is frequently found in the myeloproliferative disorders (MPDs) and believed to be an initiating event. [13][14][15] Submicroscopic deletions that involve genes linked functionally to deregulation of cell cycling or B-cell differentiation were also implicated in the molecular pathogenesis of ALL. [16][17][18][19] In this study we undertook both sequencing of candidate genes and high-resolution singlenucleotide polymorphism (SNP) array analysis to identify genetic events associated with ALL in DS. Methods PatientsPatients' samples (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article) were collected, informed consent was obtained in accordance with the Declaration of Helsinki, and the study was carried out with approval of the ethical review committee from all participating institutions. DNA was extracted from archival (frozen cell or cytogenetic-fixed pellet) leukemic samples. SequencingPrimers were designed to amplify the following candidate genes: FLT3 ITD, FLT3 kinase domain, KIT kinase domain, PTPN11, BRAF, NRAS, and KRAS (Table S2A). Exons 12 to 23 of the JAK2 gene were polymerase chain reaction (PCR)-amplified and sequenced using previously described primers. 13 All samples found to be mutated were PCR-amplified and sequenced in a second, independent experiment. Pyrosequencing was carried out in accordance to the manufacturer's instructions (Biotage AB, Uppsala, Sweden) using the primers shown in Table S2B. Ba/F3 prolifer...

Spheroids formed of mesenchymal stem cells (MSCs) exhibit increased cell survival and trophic factor secretion compared with dissociated MSCs, making them therapeutically advantageous for cell therapy. Presently, there is no consensus for the mechanism of action. Many hypothesize that spheroid formation potentiates cell function by generating a hypoxic core within spheroids of sufficiently large diameters. The purpose of this study was to experimentally determine whether a hypoxic core is generated in MSC spheroids by measuring oxygen tension in aggregates of increasing diameter and correlating oxygen tension values with cell function. MSC spheroids were formed with 15 000, 30 000 or 60 000 cells per spheroid, resulting in radii of 176 + 8 mm, 251 + 12 mm and 353 + 18 mm, respectively. Oxygen tension values coupled with mathematical modelling revealed a gradient that varied less than 10% from the outer diameter within the largest spheroids. Despite the modest radial variance in oxygen tension, cellular metabolism from spheroids significantly decreased as the number of cells and resultant spheroid size increased. This may be due to adaptive reductions in matrix deposition and packing density with increases in spheroid diameter, enabling spheroids to avoid the formation of a hypoxic core. Overall, these data provide evidence that the enhanced function of MSC spheroids is not oxygen mediated.

PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I, or with 111In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from 125I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, 111In-labelled and 125I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with 111In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.