Getting CD19 Into Shape: Expression of Natively Folded "Difficult-to- Express" CD19 for Staining and Stimulation of CAR-T Cells

• Published in: Frontiers in bioengineering and biotechnology 2020-02, Vol.8, p.49-49
• Main Authors: Lobner, Elisabeth, Wachernig, Anna, Gudipati, Venugopal, Mayrhofer, Patrick, Salzer, Benjamin, Lehner, Manfred, Huppa, Johannes B, Kunert, Renate
• Format: Article
• Language: English
• Subjects: BAC; Bioengineering and Biotechnology; CAR-T cell; CD19; chemical chaperones; difficult-to-express protein; T cell activation
• ISSN: 2296-4185; 2296-4185
• DOI: 10.3389/fbioe.2020.00049

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 and 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 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.