Dataset Information

Chimeric Antigen Receptor Library Screening Using a Novel NF-?B/NFAT Reporter Cell Platform.

ABSTRACT: Chimeric antigen receptor (CAR)-T cell immunotherapy is under intense preclinical and clinical investigation, and it involves a rapidly increasing portfolio of novel target antigens and CAR designs. We established a platform that enables rapid and high-throughput CAR-screening campaigns with reporter cells derived from the T cell lymphoma line Jurkat. Reporter cells were equipped with nuclear factor ?B (NF-?B) and nuclear factor of activated T cells (NFAT) reporter genes that generate a duplex output of enhanced CFP (ECFP) and EGFP, respectively. As a proof of concept, we modified reporter cells with CD19-specific and ROR1-specific CARs, and we detected high-level reporter signals that allowed distinguishing functional from non-functional CAR constructs. The reporter data were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6 days) compared to primary CAR-T cells (21 days). We challenged the reporter platform to a large-scale screening campaign on a ROR1-CAR library, and we showed that reporter cells retrieved a functional CAR variant that was present with a frequency of only 6 in 1.05 × 106. The data illustrate the potential to implement this reporter platform into the preclinical development path of novel CAR-T cell products and to inform and accelerate the selection of lead CAR candidates for clinical translation.

SUBMITTER: Rydzek J

PROVIDER: S-EPMC6369451 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Chimeric Antigen Receptor Library Screening Using a Novel NF-κB/NFAT Reporter Cell Platform.

Rydzek Julian J Nerreter Thomas T Peng Haiyong H Jutz Sabrina S Leitner Judith J Steinberger Peter P Einsele Hermann H Rader Christoph C Hudecek Michael M

Molecular therapy : the journal of the American Society of Gene Therapy 20181120 2

Chimeric antigen receptor (CAR)-T cell immunotherapy is under intense preclinical and clinical investigation, and it involves a rapidly increasing portfolio of novel target antigens and CAR designs. We established a platform that enables rapid and high-throughput CAR-screening campaigns with reporter cells derived from the T cell lymphoma line Jurkat. Reporter cells were equipped with nuclear factor κB (NF-κB) and nuclear factor of activated T cells (NFAT) reporter genes that generate a duplex o ...[more]

PMID: 30573301

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Project description:Osteolytic diseases such as osteoporosis and neoplastic bone metastases are caused by the excessive activation of osteoclasts. Inhibiting the excessive activation of osteoclasts is a crucial strategy for treating osteolytic diseases. This study investigated the roles and mechanisms of regorafenib, a tyrosine kinase inhibitor, on osteoclasts and osteolytic diseases. We first identified the potential targets and mechanisms of regorafenib on osteoclast-related osteolytic diseases using network pharmacological analysis and molecular docking techniques. Then, we verified its role and mechanism on osteoclasts via cellular and animal experiments. Network pharmacology analysis identified 89 common targets shared by regorafenib and osteoclast-related osteolytic diseases. Enrichment analysis suggested that regorafenib may act on osteoclast-related osteolytic diseases by modulating targets such as AKT1, CASP3, MMP9, and MAPK3, regulating biological processes such as cell proliferation, apoptosis, and phosphorylation regulation, and influencing signaling pathways such as MAPK, PI3K/AKT, and osteoclast differentiation. The molecular docking results indicated that regorafenib and AKT1, CASP3, MMP9, MAPK3, and MAPK14 were stably docked. Cell experiments demonstrated that regorafenib significantly inhibited osteoclast differentiation and bone resorption in RAW 264.7 cells and bone marrow macrophages in a dose-dependent manner, with up to 50% reduction at 800 nM concentration without exhibiting cytotoxic effects. Furthermore, Western blot and RT-qPCR results demonstrated that regorafenib inhibited osteoclast differentiation by blocking the transduction of RANKL-induced NF-κB, p38, ERK, and NFAT signaling pathways. In vivo studies using an ovariectomized mouse model showed that regorafenib significantly improved bone volume fraction (BV/TV), bone surface to total volume (BS/TV), and number of trabeculae (TB.N), as well as reduced trabecular separation (Tb.Sp) compared to the OVX groups (P < 0.05). TRAcP staining results revealed a reduction in the number of osteoclasts with regorafenib treatment (P < 0.01). These results indicate that regorafenib exerts its protective effects against osteoclast-related osteolytic disease by inhibiting the RANKL-induced NF-κB, NFAT, ERK, and p38 signaling pathways. This study proves that regorafenib may serve as a potential therapeutic agent for osteoclast-related osteolytic diseases.

Dataset Information

Chimeric Antigen Receptor Library Screening Using a Novel NF-?B/NFAT Reporter Cell Platform.

Publications

Chimeric Antigen Receptor Library Screening Using a Novel NF-κB/NFAT Reporter Cell Platform.

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