Transport of Designed Ankyrin Repeat Proteins through reconstituted human bronchial epithelia and protection against SARS-CoV-2

Abstract

Clinical studies have proven antiviral effectiveness of treatment with a Designed Ankyrin Repeat Protein (DARPin) specific against the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More information on transport mechanisms and efficiency to the site of action is desirable. Transepithelial migration through air-liquid interface (ALI) cultures of reconstituted human bronchial epithelia (HBE) was assessed by Enzyme-Linked Immunosorbent Assays and Confocal Laser Scanning Microscopy for different DARPin designs in comparison to a monoclonal antibody. Antiviral efficacy against authentic SARS-CoV-2, applied apically on HBE, was investigated based on viral titers and genome equivalents, after administration of therapeutic candidates on the basal side. Transepithelial translocation of all DARPin candidates and the monoclonal antibody was efficient and dose dependent. Small DARPins and the antibody migrated more efficiently than larger molecules, indicating different transport mechanisms involved. Microscopic analyses support this, demonstrating passive paracellular transport of smaller DARPins and transcellular migration of the larger molecules. All therapeutic candidates applied to the basal side of HBE conferred effective protection against SARS-CoV-2 infection. In summary, we have shown that DARPins specific against SARS-CoV-2 translocate across intact airway epithelia and confer effective protection against infection and viral replication.

Figure 1

Quantification of DARPin molecules and control antibody in cell lysates, apical washes and basal cell culture medium by ELISA. (A) Illustration of the test system used to quantify transepithelial migration of DARPins and control antibody. HBE were reconstituted from cells isolated from a normal donor lung grown on microporous filter inserts at the air–liquid interface. Fully differentiated HBE comprise of ciliated, secretory and basal cells, and produce their own liquid lining layer. The test compounds were added to the basal cell culture medium. Transepithelial migration was assessed by collecting apical washes and lysis of the HBE cell layer after 24 h. (B) Verification of the formulation concentrations (10, 40 and 160 µg/mL) added to the basal cell culture medium at 0 h. (C–E) Concentration of DARPins and control antibody at 24 h in (C) the basal cell medium, (D) the HBE lysates and (E) the apical washes. (F) Concentrations determined for the DARPin candidates ALE058 and MWA010 in apical washes and in HBE lysates at 24 h. Both HSA-binding DARPin candidates were applied to the basal compartment at a concentration of 160 µg/mL in presence or absence of HSA.

Figure 2

Confocal Laser Scanning Microscopy (CLSM) of reconstituted HBE incubated with fluorescence-labeled DARPins or control antibody. Multivalent DARPin ALE058 (A-D), monovalent HSA-binding DARPin domain MWA010 (E–H), negative control DARPin PSC078 (I–L) and REGN10933 antibody as positive control (M–P) were added to the basal cell culture medium together with the nuclear dye Hoechst 33342 and lipophilic membrane dye CellBrite^® Red. Cell cultures were fixed at 24 h with 4% formaldehyde for later imaging. Columns from left to right show nuclei in blue, Atto-488 labeled candidate and control molecules in green, membranes in magenta, and the overlays. The superposition of the green fluorescent signal of the Atto-488 labeled molecules with the red signal of the membrane dye results in white in the composite images (arrow heads). Scale bars: 25 μm.

Figure 3

Antiviral activity of ALE058, REGN10933 and Remdesivir in HBE infected with SARS-CoV-2. HBE cultures were treated with either (i) ALE058 or REGN10933 at 10, 40 or 160 µg/mL, (ii) Remdesivir at 5 µM, or (iii) mock treated from the basolateral side, 2 days before infection with 10⁴ TCID₅₀ SARS-CoV-2 from the apical side. The protective capacity of the compounds was assessed by (A) determining the infectious virus titer (TCID₅₀/mL) in apical washes with a limiting dilution assay at 96 hpi, with the limit of detection (LOD) at 10 infectious particles/mL, and (B) measuring the viral genome equivalents/mL in apical washes by qRT-PCR at 1, 72 and 96 hpi. Results are shown as boxplots of 6 individual cell cultures (all orginating from the same donor lung) from 2 separate experiments. Boxes display interquartile ranges with the central bar indicating the median and whiskers minimal and maximal values. ****p < 0.0001; n.s. not statistically significant, LOD limit of detection.