Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022-501725-21-00).

A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level.

CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro- and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D µComputer tomography bioluminescence tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (± IL-2) or control BDCA-2 CAR T cell (± IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D µCT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D µCT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D µCT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

ACSA-2 and GLAST classify subpopulations of multipotent and glial-restricted cerebellar precursors.

The formation of the cerebellum is highly coordinated to obtain its characteristic morphology and all cerebellar cell types. During mouse postnatal development, cerebellar progenitors with astroglial-like characteristics generate mainly astrocytes and oligodendrocytes. However, a subset of astroglial-like progenitors found in the prospective white matter (PWM) produces astroglia and interneurons. Characterizing these cerebellar astroglia-like progenitors and distinguishing their developmental fates is still elusive. Here, we reveal that astrocyte cell surface antigen-2 (ACSA-2), lately identified as ATPase, Na+/K+ transporting, beta 2 polypeptide, is expressed by glial precursors throughout postnatal cerebellar development. In contrast to common astrocyte markers, ACSA-2 appears on PWM cells but is absent on Bergmann glia (BG) precursors. In the adult cerebellum, ACSA-2 is broadly expressed extending to velate astrocytes in the granular layer, white matter astrocytes, and to a lesser extent to BG. Cell transplantation and transcriptomic analysis revealed that marker staining discriminates two postnatal progenitor pools. One subset is defined by the co-expression of ACSA-2 and GLAST and the expression of markers typical of parenchymal astrocytes. These are PWM precursors that are exclusively gliogenic. They produce predominantly white matter and granular layer astrocytes. Another subset is constituted by GLAST positive/ACSA-2 negative precursors that express neurogenic and BG-like progenitor genes. This population displays multipotency and gives rise to interneurons besides all glial types, including BG. In conclusion, this work reports about ACSA-2, a marker that in combination with GLAST enables for the discrimination and isolation of multipotent and glia-committed progenitors, which generate different types of cerebellar astrocytes.

Enrichment and isolation of neurons from adult mouse brain for ex vivo analysis.

BACKGROUND: Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. NEW METHODS: Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. RESULTS: Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. COMPARISON WITH EXISTING METHODS: Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. CONCLUSION: These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS.

Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes.

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.

Isolation and characterization of living primary astroglial cells using the new GLAST-specific monoclonal antibody ACSA-1.

Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA-1 was cross-reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA-1. Using an immunomagnetic approach, we established a method for the isolation of GLAST-positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST-positive cells neither interfered with cellular glutamate transport nor compromised astrocyte viability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes.

Triangular neuronal networks on microelectrode arrays: an approach to improve the properties of low-density networks for extracellular recording.

Multi-unit recording from neuronal networks cultured on microelectrode arrays (MEAs) is a widely used approach to achieve basic understanding of network properties, as well as the realization of cell-based biosensors. However, network formation is random under primary culture conditions, and the cellular arrangement often performs an insufficient fit to the electrode positions. This results in the successful recording of only a small fraction of cells. One possible approach to overcome this limitation is to raise the number of cells on the MEA, thereby accepting an increased complexity of the network. In this study, we followed an alternative strategy to increase the portion of neurons located at the electrodes by designing a network in confined geometries. Guided settlement and outgrowth of neurons is accomplished by taking control over the adhesive properties of the MEA surface. Using microcontact printing a triangular two-dimensional pattern of the adhesion promoter poly-D-lysine was applied to the MEA offering a meshwork that at the same time provides adhesion points for cell bodies matching the electrode positions and gives frequent branching points for dendrites and axons. Low density neocortical networks cultivated under this condition displayed similar properties to random networks with respect to the cellular morphology but had a threefold higher electrode coverage. Electrical activity was dominated by periodic burst firing that could pharmacologically be modulated. Geometry of the network and electrical properties of the patterned cultures were reproducible and displayed long-term stability making the combination of surface structuring and multi-site recording a promising tool for biosensor applications.

Standardized preparation of single-cell suspensions from mouse lung tissue using the gentleMACS Dissociator.

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Preparation of single-cell suspensions from mouse spleen with the gentleMACS Dissociator.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture. To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions. The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety. This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.