Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells. METHODS: We used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency. RESULTS: Results indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells. CONCLUSIONS: These studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.

Glycomaterials to Investigate the Functional Role of Aberrant Glycosylation in Glioblastoma.

Glioblastoma (GBM) is a stage IV astrocytoma that carries a dismal survival rate of ≈10 months postdiagnosis and treatment. The highly invasive capacity of GBM and its ability to escape therapeutic challenges are key factors contributing to the poor overall survival rate. While current treatments aim to target the cancer cell itself, they fail to consider the significant role that the GBM tumor microenvironment (TME) plays in promoting tumor progression and therapeutic resistance. The GBM tumor glycocalyx and glycan-rich extracellular matrix (ECM), which are important constituents of the TME have received little attention as therapeutic targets. A wide array of aberrantly modified glycans in the GBM TME mediate tumor growth, invasion, therapeutic resistance, and immunosuppression. Here, an overview of the landscape of aberrant glycan modifications in GBM is provided, and the design and utility of 3D glycomaterials are discussed as a tool to evaluate glycan-mediated GBM progression and therapeutic efficacy. The development of alternative strategies to target glycans in the TME can potentially unveil broader mechanisms of restricting tumor growth and enhancing the efficacy of tumor-targeting therapeutics.

Quantitative determinations using portable Raman spectroscopy.

A portable Raman spectrometer was used to develop chemometric models to determine percent (%) drug release and potency for 500mg ciprofloxacin HCl tablets. Parallel dissolution and chromatographic experiments were conducted alongside Raman experiments to assess and compare the performance and capabilities of portable Raman instruments in determining critical drug attributes. All batches tested passed the 30min dissolution specification and the Raman model for drug release was able to essentially reproduce the dissolution profiles obtained by ultraviolet spectroscopy at 276nm for all five batches of the 500mg ciprofloxacin tablets. The five batches of 500mg ciprofloxacin tablets also passed the potency (assay) specification and the % label claim for the entire set of tablets run were nearly identical, 99.4±5.1 for the portable Raman method and 99.2±1.2 for the chromatographic method. The results indicate that portable Raman spectrometers can be used to perform quantitative analysis of critical product attributes of finished drug products. The findings of this study indicate that portable Raman may have applications in the areas of process analytical technology and rapid pharmaceutical surveillance.

Screening of unapproved drugs using portable Raman spectroscopy.

We present a four-step screening approach for unapproved drugs. The screening approach is both qualitative and quantitative in design in order to determine if the sample under study contains the correct and acceptable amount of the declared active pharmaceutical ingredient. Four commercially-available unapproved antibiotic and antiviral drugs were used in this study. Out of the 40 individual samples tested, 100% of the samples matched for the declared active pharmaceutical ingredient with no false positives. Following this qualitative identification step, a quantitative assay was used to determine the potency of the product. The assay involves dissolving the sample in water and using a partial least squares model to predict the potency of the product. The average Raman potency results for the four products tested were compared with chromatographic reference methods and the spectroscopic data were found to be within ∼1-6% of those obtained with the reference method for the four products tested. The results indicate that aqueous-based Raman assays may be a suitable field-deployable alternative to traditional techniques run in a laboratory environment.