Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants - Puchol Tarazona - 2020 - Biotechnology Journal - Wiley Online LibraryBiotechnology JournalVolume 15, Issue 3 1900308 Research Article Open Access Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti-HIV-1 Antibodies 2F5, 2G12, and PG9 in Plants Alejandro A. Puchol Tarazona, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorELISAbeth Lobner, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, AustriaSearch for more papers by this authorYvonne Taubenschmid, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorMelanie Paireder, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorJuan A. Torres Acosta, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorKathrin Göritzer, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorHerta Steinkellner, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorLukas Mach, Corresponding Author lukas.mach@boku.ac.at Alejandro A. Puchol Tarazona, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorElisabeth Lobner, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, AustriaSearch for more papers by this authorYvonne Taubenschmid, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorMelanie Paireder, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorJuan A. Torres Acosta, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorKathrin Göritzer, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorHerta Steinkellner, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, AustriaSearch for more papers by this authorLukas Mach, Corresponding Author lukas.mach@boku.ac.at Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat 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. Abstract Broadly neutralizing antibodies (bNAbs) hold promise for the treatment of HIV-positive individuals. bNAb production in plant-based expression platforms is frequently accompanied by their proteolytic inactivation. In Nicotiana benthamiana, bNAb proteolysis appears to be mediated by endogenous subtilisin-like serine proteases. The high sequence diversity of HIV poses a major challenge for the human immune system. Nevertheless, some long-term survivors develop antibodies capable of neutralizing a large fraction of the HIV-1 strains circulating in the population. These broadly neutralizing antibodies (bNAbs) have attracted much interest due to their potential to reduce viremia in HIV-1-infected humans upon passive administration and thus provide a modality for the prevention, therapy, and cure of HIV-1 infections.1 Several bNAbs display a unique global shape due to asymmetric positioning of their fragment antigen-binding (Fab) arms, which relates to their virus-neutralizing activities.2 Remarkably, various bNAbs carry sulfated tyrosine residues in their antigen-binding sites.3, 4 This unconventional post-translational modification is critical for high-affinity binding of the respective antibodies to the HIV-1 envelope, which complicates the production of such bNAbs in heterologous expression platforms.5 The surface of HIV-1 virions is covered by a dense glycan shield which hides many antigenic sites of the envelope glycoprotein. To gain access to their peptide epitopes, many bNAbs have therefore evolved unusually long complementarity-determining region (CDR) H3 loops.3, 4 With 28 residues, PG9 features one of the longest CDR H3 loops known to date.6 2F5 and 4E10 are also characterized by extended CDR H3 loops consisting of 22 and 18 amino acids, respectively.7, 8 By contrast, the CDR H3 loop of the anti-glycan bNAb 2G12 (14 residues) matches the average length of human CDR H3 domains.9 For high-affinity binding to the HIV-1 envelope glycoprotein, 2G12 relies on its unique domain-swapped structure with a third antigen-binding site formed at the novel VH/VH\' interface.10 It has been shown that a single point mutation in the framework 1 region of the heavy chain, Ile19 → Arg (I19R), is sufficient to disrupt the VH/VH\' interface and thus prevent the intramolecular VH domain exchange of 2G12, thereby disabling the capacity of the antibody to neutralize HIV-1.11 Although commercial production of bNAbs and other therapeutic monoclonal antibodies (mAbs) is performed mostly in mammalian cell factories such as Chinese hamster ovary (CHO) cells, plants are emerging as versatile alternative bioreactor systems.12, 13 The advantages of plant-based expression platforms include their comparatively low upfront investment costs, making them particularly attractive for deployment in developing countries.14 Moreover, plants are highly amenable to controlled manufacturing of desirable post-translational modifications, thus enabling the generation of mAb variants featuring tailored functional properties with unprecedented precision.15 Since bNAbs require the effector functions of the fragment crystallizable (Fc) domain for in vivo activity,16 expression of these antibodies in glycoengineered plants holds great promise for the production of anti-HIV-1 therapeutics with increased potencies. This feat has been already achieved for the bNAbs 2G12, 4E10, and PG9.5, 17, 18 It is of note that a phase I clinical trial has demonstrated the safety of plant-derived 2G12 in humans.12 It is well known that antibodies frequently suffer from proteolytic degradation when expressed in plants.19 We have previously observed that the bNAbs 2F5, 2G12, and PG9 differ in their susceptibility to attack by endogenous plant proteases. In the case of 2F5 and PG9, all detected cleavage events occurred within or close to their CDR H3 loops.20 Intriguingly, this pronounced preference for cleavage in the CDR H3 region was not noticed for other antibodies undergoing fragmentation in plant-based expression platforms.21, 22 In this report, we demonstrate that CDR H3 proteolysis abrogates the antigen-binding activities of 2F5 and PG9. This was also observed for 2G12-I19R, a mutant version of 2G12 displaying a canonical Y-shaped antibody structure.11 Furthermore, we provide evidence that the compact folds of wild-type 2G12 (2G12-wt) and 4E10 render the CDR H3 regions of these antibodies less susceptible to plant proteases. Finally, our data indicate that at least two different types of subtilisin-like serine proteases are involved in bNAb degradation in the interstitial spaces of plant tissues. 2G12-wt and 2G12-I19R were produced in Nicotiana benthamiana as reported previously.23 Briefly, N. benthamiana ∆XT/FT plants lacking plant-specific α1,3-fucosylation and β1,2-xylosylation24 were grown for 4–5 weeks at 24 °C with a 16-h light:8-h dark photoperiod prior to infiltration with agrobacteria carrying the respective mAb expression vectors. Infiltrated N. benthamiana leaves were harvested after 4–5 days. Antibody extraction and purification was then performed as reported previously.5, 23, 24 2G12-wt was also produced in CHO cells, as were all other bNAbs used in this study (provided by Polymun Scientific, Austria). Size-exclusion chromatography (SEC) combined with multi-angle light scattering (MALS) analysis was used to determine the native molecular masses of the mAbs used in this study. Analyses were performed on a Prominence LC-20A HPLC system equipped with the refractive index detector RID-10A, the photodiode array detector SPD-M20A (all from Shimadzu, Japan) and the MALS detector DAWN Heleos 8+ with QELS (Wyatt Technology, USA). Samples (50µg) were filtered through a 0.1µm Ultrafree-MC filter (Merck, Germany) and then fractionated on a Superdex 200 10/300 column (GE Healthcare, UK) equilibrated with PBS supplemented with 200mm NaCl. Chromatographic separation was carried out at a flow rate of 0.75mL min−1 at 25 °C and analyzed using ASTRA 6 software (Wyatt Technology). The accuracy of the MALS detector was verified with a sample of BSA. Differential scanning calorimetry (DSC) was performed on a MicroCal VP-Capillary DSC (Malvern Panalytical, UK) or a MicroCal PEAQ-DSC Automated (Malvern Panalytical), both equipped with a 96-well plate autosampler. mAbs (0.5mg mL−1 in PBS) were heated from 20 to 100 °C with a heating rate of 1 °C min−1 in high feedback mode. In all cases, rescans showed irreversible unfolding of the proteins under study and were therefore used for baseline correction. Data were normalized for protein concentration and fitted with a non-two-state thermal unfolding model using MicroCal PEAQ-DSC software version 1.4 (Malvern Panalytical). Heat capacity (Cp) was expressed in kJ mol−1°C−1. mAbs (50µg mL−1) were treated with apoplastic fluid of ∆XT/FT plants (5–200µg mL−1) prepared as described20 in 100mm sodium acetate (pH 5.5) or 20mm sodium citrate and 40mm sodium phosphate (pH 4.0–7.0) at 37 °C in the absence or presence of selected protease inhibitors (Sigma-Aldrich, USA or Bachem, Switzerland; FP-biotin was from Santa Cruz Biotechnology, USA). After incubation for up to 16h, reactions were stopped by the addition of SDS-PAGE sample buffer. SDS-PAGE, western blotting, and cleavage site analysis by Edman degradation were then performed as reported previously.20 For isolation of mAb degradation products, antibodies (250µg) were digested with apoplastic fluid in 500µL 100mm sodium acetate (pH 5.5) to completion. After the addition of 10µL settled rProtein A Sepharose 4 Fast Flow beads (GE Healthcare), the samples were incubated for 2h at 4 °C under constant agitation. The beads were collected by centrifugation and washed four times with 500µL PBS prior to elution of the bound antibody fragments with 100µL 100mm glycine/HCl (pH 3.0). The eluate was immediately neutralized with 10µL 1 m Tris/HCl (pH 8.0). This elution/neutralization cycle was repeated four times. All eluate fractions were combined and concentrated by ultrafiltration after buffer exchange into PBS containing 0.02% (w/v) NaN3. To assess the antigen-binding properties of bNAb 2F5 and its degradation products, 96-well enzyme-linked immunosorbent assay (ELISA) plates were coated with 100ng per well of gp41 peptide GGGLELDKWASL (Polymun Scientific) or Fc-specific goat anti-human IgG F(ab\')2 fragments (Sigma-Aldrich) in 50mm sodium carbonate/bicarbonate buffer (pH 9.6) for 16h at 4 °C. The wells were then washed with PBST and subsequently incubated with bNAb samples (starting concentration: 1–8µg mL−1) serially diluted (1:2) in PBST containing 1% BSA (dilution buffer) for 1h at 23 °C. After washing, bound antibodies were detected with 0.03µg mL−1 γ-chain-specific goat anti-human IgG peroxidase conjugate (Sigma-Aldrich) in dilution buffer. After 1h at 23 °C, plates were washed and then developed with 0.1mg mL−1 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) and 0.006% H2O2 in 35mm citric acid/65mm sodium phosphate (pH 5.0) for 15min at 23 °C. Reactions were quenched by the addition of 90mm H2SO4 prior to analysis by spectrophotometry at 450nm. The ELISA procedures for the analysis of 2G12-wt, 2G12-I19R, and PG9 samples had been reported previously.5, 23 We have previously observed that 2F5 and PG9 are more prone to proteolysis than 2G12 when expressed in plants.20 Since all detected cleavage events were located in the CDR H3 regions of the antibodies, we reasoned that the steric accessibility of the CDR H3 loop could be a major determinant of the proteolytic vulnerability of bNAbs. To test this hypothesis, we have used site-directed mutagenesis to generate 2G12-I19R, a mutant version of 2G12 displaying a conventional Y-shaped quaternary structure.11 2G12-I19R and its wild-type counterpart were produced by agrobacterium-mediated transient expression in N. benthamiana leaves and then isolated by means of protein A affinity chromatography. The purified proteins were characterized comprehensively by a combination of biochemical and biophysical methods. First, SDS-PAGE was performed under reducing and non-reducing conditions to estimate the approximate molecular masses of the fully assembled antibodies and their subunits (Figure S1, Supporting Information). Upon reduction and SDS-mediated denaturation, both 2G12-wt and 2G12-I19R dissociated into two polypeptides of 54 and 24kDa, in good agreement with the calculated molecular masses of their heavy and light chains (heavy chain: 50.9kDa; light chain: 23.3kDa). When the reduction step was omitted, both antibodies migrated as single bands of ≥170kDa, as already observed for wild-type 2G12 in other studies.24 Next, size-exclusion chromatography combined with multi-angle light scattering analysis (SEC-MALS) was performed to determine the native molecular masses of 2G12-wt and 2G12-I19R. SEC-MALS of wild-type 2G12 resulted in two peaks of 150 and 306kDa, corresponding to monomeric (60%; theoretical mass: 148.4kDa) and dimeric (30%; nominal mass: 296.8kDa) forms of the antibody. It has been shown before that batches of wild-type 2G12 produced in mammalian cell factories can contain up to 30% dimers prior to SEC purification, which has been attributed to intermolecular VH domain exchange.25 By contrast, 2G12-I19R eluted as a single monomeric peak ( 90%) of 149kDa. This confirms that the I19R mutation interferes with 2G12 domain swapping and thereby promotes the synthesis of antibody molecules displaying a less compact canonical structural fold,11 which probably accounts for the observed small difference between the SEC retention times of 2G12-wt and 2G12-I19R (Figure1). Figure 1Open in figure viewerPowerPoint Characterization of 2G12-wt and 2G12-I19R (50µg) produced in N. benthamiana by size-exclusion chromatography combined with multi-angle light scattering analysis. The elution positions of the molecular mass standards thyroglobulin (670kDa), IgG (158kDa), ovalbumin (44kDa), myoglobin (17kDa), and vitamin B12 (1.35kDa) are indicated (dotted lines). The small peaks ( 10%) eluting at retention times of 11–13min correspond to oligomeric aggregates of the antibodies. The elution times of the monomeric 2G12-wt and 2G12-I19R peaks were 15.6 and 15.2min, respectively. Data are presented as mean ± SEM of at least two independent experiments. a.u., arbitrary units. 2G12-wt and 2G12-I19R were also characterized by differential scanning calorimetry (DSC), a sensitive biophysical method for the assessment of the thermal stability of antibodies and their subdomains.26 Three major thermal transitions can be discriminated. The first midpoint of transition (Tm1) is due to the unfolding of the Fab domain, whereas the second and third midpoints of transitions (Tm2 and Tm3) reflect the thermal denaturation of the CH2 and CH3 domains, respectively.27, 28 In the case of 2G12-wt produced in N. benthamiana (Figure2), Tm1 (68.6 °C) was only slightly lower than Tm2 (70.5 °C). This probably reflects the reduced flexibility of the Fab arms in the domain-swapped arrangement of the wild-type molecule due to additional interactions between the antibody chains at the VH/VH\' interface. Essentially the same results were obtained with SEC-purified monomeric wild-type 2G12 produced in CHO cells (Tm1: 68.6 °C; Tm2: 70.6 °C; Figures S2 and S3, Supporting Information), thus demonstrating that the high Tm1 of 2G12-wt is not due to the presence of dimers. As expected, the CH2 and CH3 domains of 2G12-I19R displayed virtually the same thermal stability as observed for the wild-type variants of the antibody. However, Tm1 (58.1 °C) was more than 10 °C lower than for the wild-type protein (Figure2). This reveals that the replacement of germline Arg19 with Ile was not only required for increasing the avidity and selectivity of 2G12 for the HIV-1 envelope, but also necessary for biophysical stabilization of the maturated antibody. Like 2G12-wt, the proteolysis-resistant bNAb 4E10 is characterized by a particularly compact fold.2 This is consistent with a much higher Tm1 (70.2 °C; Figure S3, Supporting Information) than observed for the protease-sensitive antibodies 2F5 (64.4 °C) and PG9 (64.8 °C; Figure S4, Supporting Information). Figure 2Open in figure viewerPowerPoint Analysis of 2G12-wt and 2G12-I19R (150µg) produced in N. benthamiana by differential scanning calorimetry. Raw data (black) were fitted using a non-two-state thermal unfolding model (grey). Data are presented as mean ± SEM of two independent experiments. Incubation of the bNAb 2F5 with apoplastic fluid isolated from untreated and agroinfiltrated N. benthamiana leaves leads to equally rapid proteolysis of its heavy chain, indicating constitutive expression of the responsible proteases.20 Hence, we have now tested 4E10, PG9, and 2G12-I19R for their sensitivity to apoplastic extracts obtained from untreated leaves. Like 2G12-wt,20 4E10 proved resistant to proteolytic attack when incubated with apoplastic fluid for up to 4h at pH 5.5 (Figure3). By contrast, the heavy chains of PG9 and 2G12-I19R were cleaved by apoplastic proteases in a similar manner as observed for 2F5, yielding a characteristic 40kDa heavy-chain fragment. The 2F5, PG9, and 2G12-I19R degradation products were purified by protein A affinity chromatography and then subjected to N-terminal sequencing analysis to identify the cleavage sites in their heavy chains. Edman degradation of the 40kDa bands revealed that cleavage occurred invariably within or adjacent to the CDR H3 loops of the antibodies (Table S1 and Figure S5, Supporting Information). The 40-kDa degradation product of 2F5 was found to arise from hydrolysis of the Phe107-Gly108 bond in the heavy chain (TLF↓GVPIA). The same N-terminus has been detected previously on 2F5 fragments isolated from plant tissues.20 Two cleavage events were detected for 2G12-I19R: GSD102↓R103LSDN and GTV118↓V119TVSP. D102↓R103 agrees well with the location of the cleavage site leading to the formation of a 40-kDa 2G12 degradation product in maize seeds.29 V118↓V119 is one residue upstream of the N-terminus of a 2G12 fragment isolated from tobacco leaves.21 In the case of PG9, proteolysis occurred at NYY111↓D112FYDG, YYD112↓F113YDGY, and HYM123↓D124VWGK. Processing at Y111↓D112 and D112↓F113 had been noted previously for plant-derived PG9.20 Figure 3Open in figure viewerPowerPoint Processing of bNAbs by apoplastic proteases in vitro. bNAbs (100ng) were incubated with apoplastic fluid (400ng) at pH 5.5 for the indicated times and then analyzed by immunoblotting with antibodies to the heavy chain of human IgG. Untreated bNAbs were loaded as controls (Co). The migration positions of selected molecular mass standards are indicated, with their respective masses expressed in kDa. The results shown are representative of at least two independent experiments. hc, full-length heavy chain; *, 40-kDa heavy-chain degradation product. We have also tested the impact of CDR H3 cleavage on the antigen-binding properties of 2F5, PG9, and 2G12-I19R. Proteolytic fragmentation did not affect their binding to anti-Fc antibodies as measured by ELISA, demonstrating that the constant regions of the cleaved mAbs were still intact. However, proteolysis within the CDR H3 loop caused a much weaker interaction of 2F5 with a gp41-derived peptide encompassing its epitope. In the case of PG9 and 2G12-I19R, treatment with apoplastic proteases essentially abolished the binding of either antibody to the respective antigen (Figure4). Figure 4Open in figure viewerPowerPoint Antigen-binding properties of intact and cleaved bNAbs. Intact and cleaved 2F5, PG9, and 2G12-I19R were tested by ELISA for binding to immobilized anti-Fc antibodies or HIV-1 antigens (2F5: gp41 peptide GGGLELDKWASL; PG9: HIV-1 ZM109 gp120; 2G12-I19R: HIV-1 UG37 gp140). The results shown are representative of at least two independent experiments. The effect of CDR H3 proteolysis on the functionality of 2F5 was also assessed by surface plasmon resonance spectroscopy. A Kd value of 6.3±0.2nm was determined for the binding of gp41 peptide to intact 2F5 (kon: 2.1 × 106 m−1 s−1; koff: 1.3 × 10−2 s−1). By contrast, the Kd for the interaction between cleaved 2F5 and its antigen was determined as 163±7nm (kon: 3.8 × 105 m−1 s−1; koff: 6.2 × 10−2 s−1). These experiments demonstrated that cleavage of 2F5 by apoplastic proteases leads to a 25-fold reduction of its affinity to gp41 due to a much lower association rate in combination with faster dissociation kinetics (Figure S6, Supporting Information). We have previously reported that processing of 2F5 by apoplastic fluid is most efficient at pH 5.5, which corresponds to the apoplastic pH in situ.20 This led us to determine the pH profiles of PG9 and 2G12-I19R hydrolysis by apoplastic proteases. Hardly any degradation of either antibody was noticed at pH 6.0 and above (Figure S7, Supporting Information). However, proteolysis of PG9 and 2G12-I19R proceeded rapidly under more acidic conditions, which is in good agreement with data on the pH stability of wild-type 2G12 in N. tabacum extracts.30 It should be pointed out that the pH optimum of PG9 and 2G12-I19R hydrolysis (pH 4.0–4.5) is in either case considerably lower than observed for 2F5.20 Hence, different proteolytic activities could be engaged in the apoplastic processing of 2F5, PG9, and 2G12-I19R. Our previous studies have indicated that serine proteases participate in the proteolysis of 2F5 in N. benthamiana, with phenylmethylsulfonyl fluoride (PMSF) identified as the most efficient inhibitor of the enzymes responsible for this degradation event.20 We have therefore compared the impact of PMSF and a panel of other small-molecule serine protease inhibitors on the processing of 2F5, PG9, and 2G12-I19R by apoplastic fluid (Figure5). In addition to PMSF, conversion of the 2F5 heavy chain into its characteristic 40-kDa degradation product could be also effectively inhibited by aminoethylbenzenesulfonyl fluoride (AEBSF) and the fluorophosphonate-based serine hydrolase probe FP-biotin. The peptide derivatives benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) were less potent. Intriguingly, fragmentation of PG9 and 2G12-I19R was only partially sensitive to PMSF and FP-biotin. However, AEBSF proved more powerful in preventing the degradation of these antibodies, in particular in the case of 2G12-I19R. Noteworthy, Ac-YVAD-CMK was also capable of reducing PG9 and 2G12-I19R degradation. This prompted us to test AEBSF and Ac-YVAD-CMK in combination, which almost completely abrogated PG9 and 2G12-I19R proteolysis. By contrast, a combination of FP-biotin and Ac-YVAD-CMK inhibited the degradation of PG9 more effectively than that of 2G12-I19R. Taken together, these experiments indicate that secreted serine proteases are largely responsible for bNAb degradation in N. benthamiana. Furthermore, our data reveal that at least two different types of serine endopeptidase activities (i.e., one preferentially inhibited by PMSF and FP-biotin, the other one particularly sensitive to AEBSF and Ac-YVAD-CMK) account for the observed cleavages in the CDR H3 loops of 2F5, PG9, and 2G12-I19R. Figure 5Open in figure viewerPowerPoint Effect of protease inhibitors on in vitro processing of bNAbs. 2F5, PG9, and 2G12-I19R (100ng) were incubated with apoplastic fluid (100ng) at pH 5.5 for 3h in the presence of solvent (dimethyl sulfoxide, DMSO) or the indicated protease inhibitors (20mm AEBSF (A); 1mm PMSF; 100µm FP-biotin (FP-biot, F); 100µm Ac-YVAD-CMK (YVAD, Y); 100µm Z-VAD-FMK (VAD)) and then analyzed by immunoblotting with antibodies to the heavy chain of human IgG. Untreated bNAbs were loaded as controls. The migration positions of selected molecular mass standards are indicated, with their respective masses expressed in kDa. The results shown are representative of at least two independent experiments. hc, full-length heavy chain; *, 40-kDa heavy-chain degradation product. Our studies have revealed that the proteolytic susceptibility of bNAbs is largely determined by the steric accessibility of their CDR H3 loops. We have already put forward that the relative resistance of wild-type 2G12 to proteases may be attributed to its unique domain-swapped architecture.20 This could be now confirmed using a mutant variant (2G12-I19R) displaying a standard Y-shaped quaternary structure. 2G12-I19R was efficiently cleaved by apoplastic proteases within its CDR H3 loop, albeit at a somewhat slower rate than 2F5 and PG9. Notably, the thermal stability of the 2G12-I19R Fab domains is far lower than in the case of wild-type 2G12. Hence, the low flexibility of the Fab domains in the context of the wild-type antibody could restrict the accessibility of the CDR H3 loops and thus reduce their proteolytic vulnerability. The bNAb 4E10 showed also hardly any sensitivity to apoplastic proteases under our assay conditions, although protein engineering experiments have indicated the existence of a potential protease cleavage site in its CDR H3 loop.31 Like wild-type 2G12, 4E10 displays a more compact global shape than other bNAbs, with both of its Fab arms adopting a closed conformation.2 This is corroborated by the much higher Fab denaturation temperature of 4E10 as compared to 2F5 and PG9. Based on the criteria defined in this study, we envisage that it should be possible to predict the proteolytic sensitivity of other bNAbs. One of the currently most promising bNAbs is VRC01, an antibody with a remarkable breath ( 90%) of HIV-1 neutralization.32 The flexibility of its rather short CDR H3 loop (12 amino acids) is restrained by a disulfide bridge.33 This could explain the good yield and integrity of VRC01 expressed in N. benthamiana, which is not accompanied by any noticeable generation of the characteristic 40kDa heavy-chain degradation product indicative of CDR H3 cleavage.34 Similar findings would be expected for 3BNC117 (CDR H3 length: ten residues), a related anti-CD4 binding site bNAb with high efficacy in suppressing viremia in HIV-1-infected humans.35 However, the VRC01 variant NIH 45-46 contains an exposed four-residue insertion in the CDR H3 region which contributes significantly to the interaction of the antibody with HIV-1 gp120 and thus to its increased potency.36 This insertion could render the CDR H3 loop of NIH 45-46 more susceptible to proteolysis. Furthermore, the unprecedented potency of bNAbs belonging to the PGT series32 is invariably associated with long CDR H3 loops (18–32 residues) making crucial contacts with the V3 loop of gp120.37 Hence, it is anticipated that PGT bNAbs are prone to inactivation by cleavage within the CDR H3 loop as observed for 2F5 and PG9. Protein degradation by endogenous proteases complicates recombinant protein production in plant-based biofactories, which limits the manufacturing of bNAbs and other biotherapeutic proteins in such expression systems.38-40 Although proteases occur in virtually all compartments of plant cells,41 substantial evidence has been presented that the degradation of bNAbs is largely confined to the apoplast.30 This is in agreement with our observation that bNAb proteolysis can be recapitulated in vitro by treatment with apoplastic fluid at acidic pH, which matches the conditions in the apoplast in situ.20 Interestingly, a highly consistent antibody-specific cleavage pattern was observed when individual bNAbs were produced in different plant-based expression systems, with the position of the hydrolyzed bond varying by no more than three residues.20, 21, 42 This suggests that the same or closely related proteases account for bNAb processing in different plants irrespective of the type of tissue or cell line. Although the plant proteases responsible for cleavage within the CDR H3 loops of bNAbs have not yet been identified, previous inhibitor studies have indicated that serine and cysteine proteases could be involved in these processing reactions.20, 42, 43 In proteomic studies of the N. benthamiana secretome, three major families of serine and cysteine proteases with known endopeptidase activities have been identified: papain-like cysteine proteinases (PLCPs), vacuolar processing enzymes (VPEs), and subtilisin-like serine proteases.41 Using activity-based probes, enzymatically active forms of the two PLCPs NbALP and NbCysP6 have been detected in the apoplast of N. benthamiana leaves.41 We have previously shown that recombinant NbALP and NbCysP6 are capable of cleaving the bNAb 2F5 in its CDR H3 loop.44, 45 However, the insensitivity of 2F5 processing to the general PLCP inhibitor E-6420 contradicts a major contribution of NbALP and NbCysP6 to CDR H3 cleavage in planta. VPEs are endopeptidases belonging to a different clan of cysteine proteases which are not sensitive to E-64 and display a strict specificity for cleavage after asparagine or aspartic acid residues.46 Such cleavage events were observed in the case of PG9 and 2G12-I19R processing by apoplastic proteases, and these reactions were partially sensitive to the known VPE inactivators Ac-YVAD-CMK and Z-VAD-FMK.47 However, PG9 degradation by apoplastic fluid was not affected by the addition of human cystatin C (Puchol Tarazona, unpublished observation), whose chicken homologue is a potent VPE inhibitor.48 This argues against an involvement of VPEs in apoplastic antibody proteolysis, in line with the observation that catalytically active VPE forms could not be detected in N. benthamiana apoplastic fluid.41 Several apoplastic proteases with known endopeptidase activities belong to the pyrolysin family of subtilisin-like serine proteases.49 While many plant subtilisin-like serine proteases (SBTs) display a broad substrate specificity,50 others cleave only after aspartic acid residues.51, 52 Intriguingly, many SBTs are relatively resistant to classical synthetic serine protease inhibitors such as PMSF or AEBSF.50 Furthermore, some of these enzymes can be inactivated by Ac-YVAD-CMK and Z-VAD-FMK.51 It is therefore of note that a combination of AEBSF and Ac-YVAD-CMK proved highly efficient in protecting bNAbs against degradation by apoplastic proteases. Hence, our inhibition profiles for the proteolysis of bNAbs by apoplastic fluid are consistent with the participation of two or more SBTs in this process. Further studies will focus on the identification of these enzymes and their targeted inactivation by genome editing approaches, which could culminate in the development of improved plant-based expression platforms for the production of bNAbs and other proteolysis-sensitive recombinant proteins. The expert technical assistance of Michaela Bogner and Barbara Svoboda is gratefully acknowledged. The authors thank Viktor Klimyuk for permission to use the MagnICON expression system, Martina Chang for CHO-derived antibodies and antigens, and Irene Schaffner for assistance with surface plasmon resonance spectroscopy. The authors are also indebted to Duc Bui-Minh, Theresa Henkel, and Andreas Loos for their contributions during the initial stages of this study. Financial support was provided by the Austrian Science Fund (FWF): project W1224-B09; and the Austrian Research Promotion Agency (FFG): grant 822757. This study was also supported by EQ-VIBT (BOKU Core Facility for Biomolecular and Cellular Analysis). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 1P. Mendoza, H. Gruell, L. Nogueira, J. A. Pai, A. L. Butler, K. Millard, C. Lehmann, I. Suarez, T. Y. Oliveira, J. C. C. Lorenzi, Y. Z. Cohen, C. Wyen, T. Kummerle, T. Karagounis, C. L. Lu, L. Handl, C. Unson-O\'Brien, R. Patel, C. Ruping, M. Schlotz, M. Witmer-Pack, I. Shimeliovich, G. Kremer, E. Thomas, K. E. Seaton, J. Horowitz, A. P. West, Jr., P. J. Bjorkman, G. D. Tomaras, R. M. Gulick, N. Pfeifer, G. Fatkenheuer, M. S. Seaman, F. Klein, M. Caskey, M. C. Nussenzweig, Nature 2018, 561, 479. 2A. K. 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