Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants

Abstract

Broadly neutralizing antibodies (bNAbs) to human immunodeficiency virus type 1 (HIV‐1) hold great promise for immunoprophylaxis and the suppression of viremia in HIV‐positive individuals. Several studies have demonstrated that plants as Nicotiana benthamiana are suitable hosts for the generation of protective anti‐HIV‐1 antibodies. However, the production of the anti‐HIV‐1 bNAbs 2F5 and PG9 in N. benthamiana is associated with their processing by apoplastic proteases in the complementarity‐determining‐region (CDR) H3 loops of the heavy chains. Here, it is shown that apoplastic proteases can also cleave the CDR H3 loop of the bNAb 2G12 when the unusual domain exchange between its heavy chains is prevented by the replacement of Ile19 with Arg. It is demonstrated that CDR H3 proteolysis leads to a strong reduction of the antigen‐binding potencies of 2F5, PG9, and 2G12‐I19R. Inhibitor profiling experiments indicate that different subtilisin‐like serine proteases account for bNAb fragmentation in the apoplast. Differential scanning calorimetry experiments corroborate that the antigen‐binding domains of wild‐type 2G12 and 4E10 are more compact than those of proteolysis‐sensitive antibodies, thus shielding their CDR H3 regions from proteolytic attack. This suggests that the extent of proteolytic inactivation of bNAbs in plants is primarily dictated by the steric accessibility of their CDR H3 loops.

Identification of two subtilisin‐like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana

“…While in vitro cleavage of 2F5 by apoplastic fluid can be completely prevented by the addition of PMSF or FP‐biotin, processing of PG9 is most effectively inhibited by a combination of aminoethylbenzenesulphonyl fluoride (AEBSF) and acetyl (Ac)‐YVAD‐CMK [31]. We have therefore tested the effects of these inhibitors on the degradation of 2F5 and PG9 by NbSBT1 and NbSBT2 (Fig.…”

“…S5). Hydrolysis after D 112 , D 115 and D 124 was also observed in PG9 isolated from plants or upon incubation of the antibody with apoplastic fluid [11,31].…”

Generation of enzymatically competent SARS‐CoV‐2 decoy receptor ACE2‐Fc in glycoengineered Nicotiana benthamiana

“…SEC‐multi‐angle light scattering (SEC‐MALS) analysis was carried out as described recently. [ 9 ] Production of ACE2‐Fc in HEK293 cells was performed as described previously. [ 5 ] ACE2‐Fc separated by SDS‐PAGE was detected by Coomassie Blue staining or immunoblotting with anti‐human IgG (H+L)‐horseradish peroxidase antibody (Promega).…”

Targeted genome editing of plants and plant cells for biomanufacturing

“…This not only reduces the yields, but can also interfere with DSP and affect product quality because the degradation products are difficult to remove. For example, full-size IgG antibodies, by far the largest class of biopharmaceuticals, frequently suffer from proteolytic degradation when expressed in plants (Donini et al 2015;Puchol Tarazona et al 2020). The most straightforward strategy to avoid proteolysis is to deplete or eliminate the native plant proteases at their source.…”