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| Item Type: | Article |
|---|---|
| Title: | Getting CD19 into shape: expression of natively folded "difficult-to-express" CD19 for staining and stimulation of CAR-T cells |
| Creators Name: | Lobner, E., Wachernig, A., Gudipati, V., Mayrhofer, P., Salzer, B., Lehner, M., Huppa, J.B. and Kunert, R. |
| Abstract: | The transmembrane protein CD19 is exclusively expressed on normal and malignant B cells and therefore constitutes the target of approved CAR-T cell-based cancer immunotherapies. Current efforts to assess CAR-T cell functionality in a quantitative fashion both in vitro and in vivo are hampered by the limited availability of the properly folded recombinant extracellular domain of CD19 (CD19-ECD) considered as "difficult-to-express" (DTE) protein. Here, we successfully expressed a novel fusion construct consisting of the full-length extracellular domain of CD19 and domain 2 of human serum albumin (CD19-AD2), which was integrated into the Rosa26 bacterial artificial chromosome vector backbone for generation of a recombinant CHO-K1 production cell line. Product titers could be further boosted using valproic acid as a chemical chaperone. Purified monomeric CD19-AD2 proved stable as shown by non-reduced SDS-PAGE and SEC-MALS measurements. Moreover, flow cytometric analysis revealed specific binding of CD19-AD2 to CD19-CAR-T cells. Finally, we demonstrate biological activity of our CD19-AD2 fusion construct as we succeeded in stimulating CD19-CAR-T cells effectively with the use of CD19-AD2-decorated planar supported lipid bilayers. |
| Keywords: | CD19, Difficult-to-Express Protein, BAC, Chemical Chaperones, T Cell Activation, CAR-T Cell, TIRF, Animals, Mice |
| Source: | Frontiers in Bioengineering and Biotechnology |
| ISSN: | 2296-4185 |
| Publisher: | Frontiers Media SA |
| Volume: | 8 |
| Page Range: | 49 |
| Date: | 7 February 2020 |
| Official Publication: | https://doi.org/10.3389/fbioe.2020.00049 |
| PubMed: | View item in PubMed |
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