Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-β. TGF-β plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-β within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-β, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-β and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-β.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with antiCD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

Here we report the use of the MACSima™ imaging platform to perform high-content imaging for the pre-clinical validation of tumor target expression on tumor and healthy human tissues indicative for potential on-target/off-tumor toxicity in vivo. Major advances have been achieved in cancer therapy in the past decade. In particular, targeted-immunotherapy has progressed and clinical benefits for patients have been achieved. However, on-target/off-tumor toxicity is a potential threat which has been shown to be more pronounced in solid than in liquid tumors. These findings highlight the need for better pre-clinical assays to improve the safety profile of immunotherapies. On-target/off-tumor toxicity is mainly based on the expression of tumor-associated antigens (TAA) in healthy tissues under physiological conditions. Currently, most prediction methods for on-target/off-tumor expression are based on bulk mRNA expression data of healthy tissue. These prediction models, however, have limitations, mainly poor predictable relation between RNA and protein level. Moreover, it is frequently unclear which cell types are affected by on-target/off-tumor effects. To overcome these limitations we employ multi-parameter imaging to analyze the expression of TAAs directly at the protein level. Additionally, we gain spatial information about tissue and cellular distribution of TAAs. Notably, our novel high-content imaging technology potentially allows for the analysis of hundreds of markers in a single experiment, paving the way for high-dimensional characterization of cells within complex solid tissues. We performed high-content imaging with the MACSima™ platform to validate the expression of known TAAs across tumor and healthy tissue samples. Subsequently, we employed unbiased cluster analysis revealing correlation patterns within the datasets to identify cell types at risk. In detail, we analyzed several high-grade serous ovarian carcinoma and pancreatic ductal adenocarcinoma for the expression of known TAAs, described tumor markers, and tissue lineage markers. Additionally, we evaluated the expression of these TAAs across several healthy human tissues. Next we performed pixel- and object-based data analysis for unbiased cluster analysis. Thereby we identified cell clusters that express TAAs in ovarian and pancreatic cancers, as well as in healthy tissues. We found that primarily epithelial cells express the analyzed TAAs in different tissues and that TAA expression shows inter- and intra-tumor heterogeneity. Taken together, we established a novel workflow for multi-parameter characterization of tissues employing the MACSima™ imaging platform. Our work enables efficient identification of potential tissues for on-target/off-tumor toxicity, paving the way for improved pre-clinical evaluation of TAAs as new targets. Citation Format: Christoph Herbel, Vera Dittmer, Manuel Martinez-Osuna, Laura Nadine Kuester, Daniel Schaefer, Jan Drewes, Jutta Kollet, Werner Mueller, Michael Mallmann, Peter Mallmann, Philipp Stroebel, Olaf Hardt, Dominik Eckardt, Andreas Bosio. Evaluation of tumor-associated antigen expression with the MACSimaTM high-content imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4694.

TO THE EDITOR: Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent preleukemic chromosomal translocations that emerge frequently in utero.[1][1] The most common translocation t(12;21) occurs in 25% of B-cell precursor ALL and results in the formation of the chimeric

AbstractETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365–77. ©2017 AACR.

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. Pre-leukaemic clones carrying ETV6-RUNX1 oncogenic lesions are frequently found in neonatal cord blood, but only few ETV6-RUNX1 carriers develop pB-ALL. The highly demanding and pending challenge is to reveal the multistep natural history of ETV6-RUNX1 pB-ALL, because it can offer non-toxic prophylactic interventions to preleukemic carriers. However, the lack of a genetically engineered ETV6-RUNX1 mouse model mimicking the human pB-ALL has hampered our understanding of the pathogenesis of this disease. This rule has now been broken in a study of the effect of the ETV6-RUNX1 oncogene in cancer development in a mouse model in which oncogene expression is restricted to the stem cell compartment. In this article, we review the different attempts to model this disease, including the recent representative success stories and we discuss its potential application to both identify etiologic factors of childhood ETV6-RUNX1 pB-ALL and prevent the conversion of a preleukemic clone in an irreversible transformed state.

Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency.

Es wurde ein dreidimensionales Polymerfasernetzwerk aufgebaut, charakterisiert und anschließend daran das Enzym Pyrrolochinolinchinon-abhängige Glukosedehydrogenase (PQQ)GDH gebunden. Das Polymerfasernetzwerk wurde durch Elektrospinnen einer Mischung des Polymers Polyacrylnitril und verschiedener leitfähiger Polymere der Polyanilin-Familie auf Indium-Zinn-Oxid-Elektroden aufgebracht. Die so hergestellten Fasermatten erwiesen sich bei mikroskopischen Untersuchungen gleichförmig präpariert und die Faserdurchmesser bewegten sich im Bereich weniger hundert Nanometer. Das Redoxpaar Kaliumhexacyanoferrat (II/III) zeigte an diesen Polymer-Elektrodenstrukturen eine quasi-reversible Elektrochemie. Bei weitergehenden Untersuchungen an den enzymmodifizierten Fasern ((PQQ)GDH) konnten unter Substratzugabe (Glukose) bioelektrokatalytische Ströme nachgewiesen werden. Das Fasernetzwerk fungiert hier nicht nur als Immobilisierungsmatrix, sondern als auch als Teil des Signalwandlers.

The creation of electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting approach within the field of bioelectrocatalysis. In this study, we investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cytochrome c (cyt c). Two different pH optima were found for the reaction in acidic and neutral solutions. When cyt c was adsorbed on an electrode surface while the enzyme remained in solution, ET proceeded efficiently in media of neutral pH. Interprotein ET was also observed in acidic media; however, it appeared to be less efficient. These findings suggest that two different ET pathways between the enzyme and cyt c may occur. Moreover, cyt c and FDH were immobilized in multiple layers on an electrode surface by means of another biomacromolecule: DNA (double stranded) using the layer-by-layer technique. The biprotein multilayer architecture showed a catalytic response in dependence on the fructose concentration, indicating that the ET reaction between both proteins is feasible even in the immobilized state. The electrode showed a defined response to fructose and a good storage stability. Our results contribute to the better understanding of the ET reaction between FDH and cyt c and provide the basis for the creation of all-biomolecule based fructose sensors the sensitivity of which can be controlled by the layer preparation.

Electrospinning is known as a fabrication technique for electrode architectures that serve as immobilization matrices for biomolecules. The current work demonstrates a novel approach to construct a conductive polymeric platform, capable not only of immobilization, but also of electrical connection of the biomolecule with the electrode. It is produced upon electrospinning from mixtures of three different highly conductive sulfonated polyanilines and polyacrylonitrile on ITO electrodes. The resulting fiber mats are with a well-retained conductivity. After coupling the enzyme pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) to polymeric structures and addition of the substrate glucose an efficient bioelectrocatalysis is demonstrated. Depending on the choice of the sulfonated polyanilline mediatorless bioelectrocatalysis starts at low potentials; no large overpotential is needed to drive the reaction. Thus, the electrospun conductive immobilization matrix acts here as a transducing element, representing a promising strategy to use 3D polymeric scaffolds as wiring agents for active enzymes. In addition, the mild and well reproducible fabrication process and the active role of the polymer film in withdrawing electrons from the reduced PQQ-GDH lead to a system with high stability. This could provide access to a larger group of enzymes for bioelectrochemical applications including biosensors and biofuel cells.

The nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1) is a master regulator of immune and inflammatory responses.[1][1],[2][2] NF-κB1 belongs to the NF-κB/Rel family of transcription factors that consists of five members in humans: NF-κB1 (p105/p50), NF-κB2 (p100/p52

Abstract Introduction The ETV6-RUNX1 fusion gene,the most common subtype of childhood pB-ALL, is acquired in utero, producing a persistent and hidden preleukemic clone. However, the underlying mechanism explaining how the preleukemic clone evolves to pB-ALL remains to be identified. The lack of genetically engineered human-like ETV6-RUNX1 pB-ALL models has hampered our understanding of the pathogenesis of this disease. Methods We have used a novel experimental approach to generate a murine strain that mimics the human ETV6-RUNX1 pB-ALL. We expressed ETV6-RUNX1 specifically in hematopoietic stem cells (HSC) of C57BL/6 x CBA mice by placing ETV6-RUNX1 under the control of the Sca1 promoter. Two founder mice were obtained for the Sca1-ETV6-RUNX1 transgene, which had normal gestation, were viable and developed normally. Sca1-ETV6-RUNX1 transgenic mice were characterized with respect to clinical, immunephenotypic and genetic characteristics. For the detection of shared secondary genomic alterations we analyzed three murine Sca1-ETV6-RUNX1 and 11 ETV6-RUNX1 positive human pB-ALL and corresponding germline by whole-exome (WES) and whole-genome sequencing using a HiSeq 2500 (Illumina) platform. Results In our transgenic murine model Sca1-ETV6-RUNX1 transgene expression was detected in HSCs, while there was no detectable expression in pro B cells or later stages of B-cell development, which mimics human ETV6-RUNX1 preleukemic biology. Sca1-ETV6-RUNX1 mice developed exclusively pB-ALL at a low penetrance (7.5%; 3 out of 40) with a CD19+ B220+ IgM- cell surface phenotype. Overall survival was not significantly reduced compared to wild-type mice (P value = 0.7901). pB-ALL in Sca1-ETV6-RUNX1 mice manifested with splenomegaly, disruption of splenic architecture, and appearance of blast cells in the peripheral blood (PB). All leukemic cells displayed clonal immature BCR rearrangement. Tumor pro B cells grew independent of IL-7 and were able to propagate the disease when transplanted into sub-lethally irradiated syngeneic recipient mice. Whole-exome sequencing of murine pB-ALL revealed in one mouse a deletion of three amino acids in the B-cell differentiation factor EBF1, which is well known in the context of human ETV6-RUNX1 leukemia. Additionally we found mutations in genesimplicated in histone modification, i.e. in KDM5C causing a premature translation stop. We compared the genomic alterations detected in the mouse model to published genomic data of pediatric ETV6 -RUNX1 pB-ALL and identified multiple copy number variations, which are shared between the murine and human ETV6 -RUNX1 pB-ALL. Among them were copy number gains and losses including i.e. the tumorsuppressor locus CDKN2A/B with a well-known role in human and mouse pB-ALL. A high proportion of genes implicated in histone modification was also mutated in published data of human ETV6-RUNX1 positive pB-ALL. We validated this novel finding of recurrent alterations of histone modifying genes in both the murine model and the human disease using an independent human ETV6-RUNX1 cohort of 11 patients. In this cohort were able to reproduce this finding. Similar to the murine model, we also detected a missense mutation in the methyltransferase KDM5C in one patient of our cohort of ETV6-RUNX1 positive patients. Conclusion In summary, we have characterized a new Sca1-ETV6-RUNX1 mouse model and this is, to our knowledge the first model, which represents a phenocopy of the human pB-ALL. Sca1-ETV6-RUNX1 mice develop exclusively pB-ALL at a very low penetrance as it is the case in human ETV6-RUNX1 positive pB-ALL. The acquisition of secondary mutations in pB-ALL with a high proportion in histone modifying genes confers the second hit for the conversion of a preleukemic clone into the clinically overt ETV6-RUNX1 positive pB-ALL disease. These findings are important for encouraging novel interventions that might help to prevent or treat ETV6-RUNX1 positive childhood leukemias. Disclosures No relevant conflicts of interest to declare.

AbstractEarlier in the past century, infections were regarded as the most likely cause of childhood B-cell precursor acute lymphoblastic leukemia (pB-ALL). However, there is a lack of relevant biologic evidence supporting this hypothesis. We present in vivo genetic evidence mechanistically connecting inherited susceptibility to pB-ALL and postnatal infections by showing that pB-ALL was initiated in Pax5 heterozygous mice only when they were exposed to common pathogens. Strikingly, these murine pB-ALLs closely resemble the human disease. Tumor exome sequencing revealed activating somatic, nonsynonymous mutations of Jak3 as a second hit. Transplantation experiments and deep sequencing suggest that inactivating mutations in Pax5 promote leukemogenesis by creating an aberrant progenitor compartment that is susceptible to malignant transformation through accumulation of secondary Jak3 mutations. Thus, treatment of Pax5+/− leukemic cells with specific JAK1/3 inhibitors resulted in increased apoptosis. These results uncover the causal role of infection in pB-ALL development.Significance: These results demonstrate that delayed infection exposure is a causal factor in pB-ALL. Therefore, these findings have critical implications for the understanding of the pathogenesis of leukemia and for the development of novel therapies for this disease. Cancer Discov; 5(12); 1328–43. ©2015 AACR.See related commentary by Greaves and Müschen, p. 1244.This article is highlighted in the In This Issue feature, p. 1225

Here we present a novel method "Genomic inverse PCR for exploration of ligated breakpoints" (GIPFEL) that allows the sensitive detection of recurrent chromosomal translocations. This technique utilizes limited amounts of DNA as starting material and relies on PCR based quantification of unique DNA sequences that are created by circular ligation of restricted genomic DNA from translocation bearing cells. Because the complete potential breakpoint region is interrogated, a prior knowledge of the individual, specific interchromosomal fusion site is not required. We validated GIPFEL for the five most common gene fusions associated with childhood leukemia (MLL-AF4, MLL-AF9, MLL-ENL, ETV6-RUNX1, and TCF3-PBX1). A workflow of restriction digest, purification, ligation, removal of linear fragments and precipitation enriching for circular DNA was developed. GIPFEL allowed detection of translocation specific signature sequences down to a 10-4 dilution which is close to the theoretical limit. In a blinded proof-of-principle study utilizing DNA from cell lines and 144 children with B-precursor-ALL associated translocations this method was 100% specific with no false positive results. Sensitivity was 83%, 65%, and 24% for t(4;11), t(9;11) and t(11;19) respectively. Translocation t(12;21) was correctly detected in 64% and t(1;19) in 39% of the cases. In contrast to other methods, the characteristics of GIPFEL make it particularly attractive for prospective studies.

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns.