Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9

Abstract

The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.

Keywords: CRISPR/Cas9; Nicotiana benthamiana; genome editing; human immunodeficiency virus; immunoglobulin G; plant biotechnology; proteases.

Figure 1

SDS-PAGE analysis of the production time profile of CAP256-VRC26.25 and its susceptibility to protease degradation. Samples were analyzed on a Bolt™ 4–12% Bis-Tris Plus gel (A) and thereafter assayed by Western blotting (B). CAP256-VRC26.25 production in N. benthamiana (ΔXTFT) were assessed over 11 days, with 23.33 μg of protein assessed from each of the N. benthamiana (ΔXTFT) clarified extracts. M represents the molecular weight marker (PageRuler™ Plus Prestained Protein Ladder).

Figure 2

Fold change in the expression of NbCysP6, NbVPE1a and NbVPE1b. Fluctuations in gene expression of NbCysP6 (blue), NbVPE1a (maroon), and NbVPE1a (green) under all tested conditions were analyzed by RT-qPCR. Error bars indicate standard error of the mean (SEM) for three biological replicates. The table indicates the fold change in the expression of NbCysP6, NbVPE1a, and NbVPE1b on three dpi and seven dpi. Experimental groups were normalized to reference genes and related to the uninfiltrated control set at one.

Figure 3

SDS-PAGE and Western blot analyses of the production time profile of the coexpression of CAP256-VRC26.25 with Cas9 and both sgRNA:NbCysP6 and sgRNA:NbVPE1a/b. Samples were analyzed on a Bolt™ 4–12% Bis-Tris Plus gel (A) and thereafter assayed by western blotting (B). CAP256-VRC26.25 production with the coexpression of CRISPR/Cas9 and both sgRNA:NbCysP6 and sgRNA:NbVPE1a/b in N. benthamiana (ΔXTFT) was assessed over 11 days, with 23.33 μg of protein assessed from each of the N. benthamiana (ΔXTFT) clarified extracts. M represents the molecular weight marker (PageRuler™ Plus Prestained Protein Ladder).

Figure 4

ELISA-determined production levels of CAP256-VRC26 mAbs produced N. benthamiana (ΔXTFT). The production of CAP256-VRC26.25 mAbs in N. benthamiana (ΔXTFT) was assessed over an 11-day period.

Figure 5

Cathepsin L-like protease activities of leaf samples with and without genome editing mediated disruption of proteases. Three dpi samples are in blue whereas seven dpi samples are in maroon. The y-axis represents the mean activities expressed as fluorescence units (dF) per min per μg protein. Mean activities of three biological replicates are shown within bars. Error bars indicate standard error of the mean (SEM).

Figure 6

Legumain protease activities of leaf samples with and without genome editing mediated disruption of proteases. Three dpi samples are in blue whereas seven dpi samples are in maroon. The y-axis represents the mean activities expressed as fluorescence units (dF) per min per μg protein. Mean activities of three biological replicates are shown within bars. Error bars indicate standard error of the mean (SEM).

Figure 7

Effects of cysteine protease disruption on total soluble protein. Three dpi samples are in blue whereas seven dpi samples are in maroon. Error bars indicate standard error of the mean (SEM). The table indicates the mean TSP concentration on three dpi and seven dpi.

Figure 8

SDS-PAGE and Western Blot analysis of the purification of CAP256-VRC26 mAbs produced in N. benthamiana (ΔXTFT) with and without genome editing vectors. (A,B) SDS-PAGE and Western blot analysis of the purification of CAP256-VRC26.25 without genome editing vectors. (C,D) SDS-PAGE and Western blot analysis of the purification of CAP256-VRC26.25 produced with genome editing vectors. M represents the molecular weight marker (PageRuler™ Plus Prestained Protein Ladder).

Figure 9

Overlaid chromatogram of the HPLC-SEC analysis of the CAP256-VRC26 mAbs. HPLC-SEC chromatogram of the CAP256-VRC26.25 mAbs analyzed under non-reducing conditions. Chromatogram of CAP256-VRC26.25 mAbs produced in HEK293 (blue). CAP256-VRC26.25 coexpressed with (green) and without (red) CRISPR/Cas9 and sgRNA:NbCysP6 and sgRNA:NbVPE1a/b in N. benthamiana (ΔXTFT) and CAP256-VRC26.25 produced in HEK293 (blue).

Figure 10

Far-UV CD spectra of CAP256-VRC26.25 mAbs. Far-UV CD spectra of CAP256-VRC26.25 produced with (maroon) and without (green) CRISPR/Cas9 and sgRNA:NbCysP6 and sgRNA:NbVPE1a/b coexpression in N. benthamiana (ΔXTFT) and of CAP256-VRC26.25 produced in HEK293 (blue).

Figure 11

Fluorescence emission spectra of CAP256-VRC26.25 mAbs excited at 280 nm. Fluorescence emission spectra of CAP256-VRC26.25 produced with (maroon) and without (green) CRISPR/Cas9 and sgRNA:NbCysP6 and sgRNA:NbVPE1a/b coexpression in N. benthamiana (ΔXTFT) and of CAP256-VRC26.25 produced in HEK293 (blue).

Figure 12

Fluorescence emission spectra of CAP256-VRC26.25 mAbs excited at 295 nm. Fluorescence emission spectra of CAP256-VRC26.25 produced with (maroon) and without (green) CRISPR/Cas9 and sgRNA:NbCysP6 and sgRNA:NbVPE1a/b coexpression in N. benthamiana (ΔXTFT) and of CAP256-VRC26.25 produced in HEK293 (blue).