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

Elisabeth Lobner,Benjamin Salzer,Patrick Mayrhofer,Johannes B Huppa,Manfred Lehner,Anna Wachernig,Renate Kunert,Venugopal Gudipati

doi:10.3389/fbioe.2020.00049

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.

Highlights

Cluster of differentiation 19 (CD19) functions in B cells as the dominant signaling component alongside CD21, CD81 and CD225 (Tedder et al, 1997; Tedder, 2009; Wang et al, 2012)
From day 5 on cells were incubated with chemical chaperones which caused in general a significant reduction in cell growth, whereby the highest cell density of about 3 × 107 cells mL−1 could only be reached in the absence of a chemical chaperone
Data represented as CD19-AD2 titers versus the integral of viable cell density (IVCD) clearly show that treatment with valproic acid (VPA) and phenylbutyric acid (PBA) leads to higher productivities within a shorter time period than treatment with DMSO or the control without any supplementation (Figure 1B)

Summary

Introduction

Cluster of differentiation 19 (CD19) functions in B cells as the dominant signaling component alongside CD21, CD81 and CD225 (Tedder et al, 1997; Tedder, 2009; Wang et al, 2012). In case of CD19-ECD this was illustrated by the formation of disulfide bonded oligomeric aggregates (Chang et al, 2018) and the fact that 40 L of cell culture supernatant were required for providing 14 mg protein to resolve the X-ray structure of an N-glycosylation mutant of CD19-ECD (Teplyakov et al, 2018). In contrast to the formerly suggested two independent Ig-like domains separated by a disulfide bonded non-Ig-like domain (Tedder and Isaacs, 1989; Zhou et al, 1991) the crystal structure of the glycan mutant of CD19-ECD revealed a novel fold in which the Ig-like domain of one Ig fold is inserted into the other as shown in Figure 1A (Teplyakov et al, 2018)

Methods

Results

Conclusion