Optimization of a locomotion-based zebrafish seizure model.

BACKGROUND: Locomotor assays in zebrafish have emerged as a screening test in early drug discovery for antiseizure compounds. However, parameters differ considerably between published studies, which may explain some discrepant results with (candidate) antiseizure medications. NEW METHOD: We optimized a locomotor-based seizure assay in zebrafish with pentylenetetrazol (PTZ) as the pharmacological proconvulsant to generate a therapeutic window in which proconvulsant-treated zebrafish larvae could be discriminated from a non-treated control. To generate a reliable control, exposure time and concentration of valproate (VPA, anticonvulsant) was optimized. RESULTS: Wells with one or three larvae show a similar PTZ dose-dependent increase in locomotion with less variability in motility for the latter. Zebrafish immersed in 10 mM PTZ showed a significant increase in movement with a sustained effect, without any indication of toxicity. Animals treated with 3 mM VPA showed the strongest reduction of PTZ-induced movement without toxicity. The decrease in PTZ-induced locomotion was greater after 18 h versus 2 h. COMPARISON WITH EXISTING METHOD(S): For the larval zebrafish PTZ-induced seizure model, varying experimental parameters have been reported in literature. Our results show that PTZ is often used at toxic concentrations, and we provide instead reliable conditions to quantify convulsant behaviour using an infrared-beam motility assay. CONCLUSIONS: We recommend using three zebrafish larvae per well to quantify locomotion in 96-multiwell plates. Larvae should preferably be exposed to 10 mM PTZ for 1 h, consisting of 30 min acclimation and 30 min subsequent recording. As positive control for anticonvulsant activity, we recommend exposure to 3 mM VPA for 18 h before administration of PTZ.

Two for one: targeting BCMA and CD19 in B-cell malignancies with off-the-shelf dual-CAR NK-92 cells.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has proven to be a valuable new treatment option for patients with B-cell malignancies. However, by applying selective pressure, outgrowth of antigen-negative tumor cells can occur, eventually resulting in relapse. Subsequent rescue by administration of CAR-T cells with different antigen-specificity indicates that those tumor cells are still sensitive to CAR-T treatment and points towards a multi-target strategy. Due to their natural tumor sensitivity and highly cytotoxic nature, natural killer (NK) cells are a compelling alternative to T cells, especially considering the availability of an off-the-shelf unlimited supply in the form of the clinically validated NK-92 cell line. METHODS: Given our goal to develop a flexible system whereby the CAR expression repertoire of the effector cells can be rapidly adapted to the changing antigen expression profile of the target cells, electrotransfection with CD19-/BCMA-CAR mRNA was chosen as CAR loading method in this study. We evaluated the functionality of mRNA-engineered dual-CAR NK-92 against tumor B-cell lines and primary patient samples. In order to test the clinical applicability of the proposed cell therapy product, the effect of irradiation on the proliferative rate and functionality of dual-CAR NK-92 cells was investigated. RESULTS: Co-electroporation of CD19 and BMCA CAR mRNA was highly efficient, resulting in 88.1% dual-CAR NK-92 cells. In terms of CD107a degranulation, and secretion of interferon (IFN)-γ and granzyme B, dual-CAR NK-92 significantly outperformed single-CAR NK-92. More importantly, the killing capacity of dual-CAR NK-92 exceeded 60% of single and dual antigen-expressing cell lines, as well as primary tumor cells, in a 4h co-culture assay at low effector to target ratios, matching that of single-CAR counterparts. Furthermore, our results confirm that dual-CAR NK-92 irradiated with 10 Gy cease to proliferate and are gradually cleared while maintaining their killing capacity. CONCLUSIONS: Here, using the clinically validated NK-92 cell line as a therapeutic cell source, we established a readily accessible and flexible platform for the generation of highly functional dual-targeted CAR-NK cells.