Functional consequences of complementarity-determining region deactivation in a multifunctional anti-nucleic acid antibody

Abstract

Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues. The structure of 3D8 scFv accommodated single complete CDR deactivations. Different functional activities of 3D8 scFv were affected differently depending on which CDR was deactivated. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering.

Keywords: Anti-DNA Antibody; Antibodies; Antibody Engineering; Autoimmunity; CDR Deactivation; Cell Penetration; DNA Hydrolysis; Immunology; Molecular Biology; Multifunctional Antibody.

FIGURE 1.

Production of scFv Abs. SDS-PAGE gels showing the purified 3D8 scFvs. The scFvs (10 μg) were separated on 12% SDS-PAGE gels and visualized by staining with Coomassie Blue.

FIGURE 2.

Characterization of the secondary structures of 3D8 scFv variants by far-UV CD spectroscopy. The far-UV CD spectra of scFv proteins were recorded in a wavelength range from 190 to 250 nm (x axis) and are expressed as the [θ] value, which represents the mean residue ellipticity (y axis). A, spectra of 3D8-Hi series. B, spectra of 3D8-Li series.

FIGURE 3.

The cell-penetrating activity of 3D8 scFv variants. A–C, assays of cell-penetrating activity. HeLa cells were incubated with the 3D8 scFvs (5 μm) for 6 h at 37 °C. The cells were then permeabilized (or not) before staining. After immunofluorescence staining with rabbit IgG and FITC-labeled anti-rabbit IgG, the amount of protein taken up by the cells (permeabilized cells) or bound to the cell surface (non-permeabilized cells) was quantified by flow cytometry (A and B). Data are representative of three independent experiments. Confocal fluorescence images show 3D8 scFvs internalized within the permeabilized cells (C). Data are representative of four independent experiments. The cell nuclei were stained with Hoechst 33342 (blue). Scale bar = 10 μm.

FIGURE 4.

Heparin binding activity of the 3D8 scFv variants. A, ELISA for heparin binding activity of scFvs. Wells coated with heparin (10 μg/ml) were incubated with various concentrations of scFvs. B, competitive ELISA. Wells coated with bio-ss-(dN)₄₀ DNA (1 μm) were incubated with scFvs (2.5 μg/ml) and heparin (varying concentrations). A and B, the scFvs bound to DNA were detected using rabbit IgG (1 μg/ml) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG. Data represent the mean ± S.D. of sextuplicate wells from two independent experiments.

FIGURE 5.

FRET-based DNA cleavage assay. ScFv (1 μm) or a mixture of scFv (1 μm) and heparin (10 μg/ml) were incubated with a double-labeled DNA substrate (250 nm) as described under “Experimental Procedures.” Fluorescence intensity was measured at 5-min intervals over 6 h in real time. RFU, relative fluorescence units; hp, heparin. Data are representative of four independent experiments.

FIGURE 6.

Biochemical properties of 3D8 CDR-derived peptides. A and B, ELISA for DNA and heparin binding activity. Wells coated with 10 μg/ml plasmid DNA (A) or 1 μg/ml heparin (B) were incubated with biotin-labeled peptides. The bound peptides were detected using alkaline phosphatase-conjugated streptavidin. Data represent the mean ± S.D. of triplicate wells and are representative of two independent experiments. C, FRET-based DNA cleavage assay. scFvs (1 μm) or a mixture of scFv (1 μm) and heparin (10 μg/ml) were incubated with a double-labeled single-stranded DNA substrate (500 nm) as described under “Experimental Procedures.” The fluorescence intensity was then measured in real time over 6 h (at 5-min intervals). RFU, relative fluorescence units. Data are representative of three independent experiments. D and E, cell-penetrating activity of the peptides. HeLa cells were incubated with FITC-labeled peptides (5 μm) for 6 h at 37 °C and then analyzed by flow cytometry (D) and confocal microscopy (E). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 10 μm. Data are representative of three independent experiments.

FIGURE 7.

Biochemical properties of HW6/3D8L1. A–C, ELISA of the binding of scFvs to DR5, DNA, and heparin. scFvs were incubated in wells coated with 5 μg/ml DR5 protein (A), 1 μm bio-ss-(dN)₄₀ DNA (B), or 1 μg/ml of heparin (C). The bound proteins were detected using rabbit IgG followed by alkaline phosphatase-conjugated anti-rabbit IgG. Data represent the mean ± S.D. of triplicate wells from two independent experiments. D and E, cell-penetrating activity of scFvs. HeLa cells were incubated with scFvs (5 μm) for 6 h at 37 °C and then either permeabilized or not. The cells were then incubated with rabbit IgG followed by FITC-labeled anti-rabbit IgG and then analyzed by flow cytometry (D, right panel, permeabilized cells; left panel, non-permeabilized cells) and confocal microscopy (E: permeabilized cells). Cell nuclei were stained with Hoechst 33342 (blue). Scale bar = 10 μm. Data are representative of three independent experiments. F, FRET-based DNA cleavage assay. ScFvs (1 μm) or a mixture of scFvs (1 μm) and heparin (10 μg/ml) were incubated with a double-labeled DNA substrate (250 nm) as described under “Experimental Procedures.” The fluorescence intensity was then measured in real time over 6 h (at 5-min intervals). RFU, relative fluorescence units. Data are representative of four independent experiments. G, spectra of HW6/3D8L1 obtained with far-UV CD spectroscopy.