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Fusion Protein, CAR-T cells, selection, Interferon

Abstract

The commercialization of the first Chimeric Antigen Receptor (CAR) T cell therapies in 2017 proved to be a major advancement in personalized cancer treatment [1-3]. Despite this success there has been a large variation in responses and unpredictable toxicity in patients- which is partially attributed to  inter-patient heterogeneity of CAR-T product infusion [4]. Future development of CAR-T therapies will likely seek to overcome variations in responses and unpredictable toxicity in patients by addressing the interpatient heterogeneity of CAR-T products. An interesting approach for improving clinical  success of CAR-T cell therapies is the selection of specific CAR-T subpopulations. The CAR-T procedure fundamentally alters the cytokine secretion phenotypes of the T cells, with both CD4+ and CD8+ CAR-T cells. Specifically, IFN-γ which is released in large amounts in activated CD8+ T cells, is a  ey moderator of cell-mediated immunity [5]. Thus, a ‘cytokine-optimized’ CAR-T product would balance the levels of cytokine production for optimal activation and efficacy yet avoid the pitfalls of overstimulation. In this research, a novel fusion protein, containing an IFN-γ scFv fused to the CD19  extracellular domain, has been designed and characterized to capture expressed IFN-γ exclusively on the surface of the expressing CAR-T cells.

Size identification of the fusion protein using SDS-PAGE gel electrophoresis confirmed the fusion protein produced was of the estimated molecular mass and correct glycosylation. ELISA assays confirmed the binding of the novel fusion protein to various concentrations of IFN-γ, including in the  presence of competitor mAb’s. ELISA assays also confirmed the binding of the CD19 ECD to the common anti-CD19 scFv, FMC63, through use of an FMC63-Fc protein. Binding of the fusion protein to anti-CD19 CAR expressing T cells was confirmed using flow cytometry and the sensitivity range was  evaluated. This fusion protein can successfully identify individual CAR-T expressing cells and select CAR expressing cells based on their CAR expression levels, and work continues to evaluate this approach in the identification of cell-specific IFN-γ secretion.

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