Antibody proteolysis: a common picture emerging from plants

Donini, Marcello; Lombardi, Raffaele; Lonoce, Chiara; Carli, Mariasole Di; Marusic, Carla; Morea, Veronica; Micco, Patrizio Di

doi:10.1080/21655979.2015.1067740

Cited by 19 publications

(17 citation statements)

References 15 publications

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“…In particular, we showed that both bands derive from a proteolytic cleavage in the hinge region connecting constant domains (CH 2 ‐CH 3 ) to scFv. This is not surprising since it was demonstrated that the hinge region of IgG1 antibodies is particularly susceptible to proteolysis in plants . Previous studies also demonstrated the occurrence of several endopeptidases both in the rhyzosecretome and the extracellular medium of plants, in particular of a subtilisin‐like serine protease .…”

Section: Discussionmentioning

confidence: 82%

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures

et al. 2018

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Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study is to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, the authors produce Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4-dichlorophenoxyacetic acid (2,4-D) is found to induce high accumulation levels (334 mg L ) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumor-targeting, CD20-specific 2B8-FcΔXF recombinant antibody from glyco-engineered ΔXTFT N. benthamiana HR cultures. The addition of 2,4-D determine a 28-fold increase of the accumulation of fully functional 2B8-FcΔXF in the culture medium, at levels of ≈16 mg L . Antibody N-glycosylation profiling reveal the prominent occurrence of GnGn structures and low levels of xylose- and fucose-containing counterparts. This result is the first example of the expression of an engineered anti-CD20 antibody with a scFv-Fc format at high levels in HR.

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Section: Discussionmentioning

confidence: 82%

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures

et al. 2018

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“…A practical challenge at present to further confirm the potential of plants as bio-factories for therapeutic IgGs is to improve their overall stability by a better control of endogenous proteolysis in host tissues. Plants engineered to express mammalian antibodies generally produce considerable amounts of these proteins on a leaf weight basis, but unintended proteolysis in the cell secretory pathway or later on in the apoplast often reduces the overall yield and quality of the resulting protein products [11]. Human blood-typing antibody C5-1 was reported for instance to undergo restricted proteolysis in N .…”

Section: Discussionmentioning

confidence: 99%

“…A major drawback of this approach, however, is the presence of non-assembled antibody fragments as a result of proteolytic processing in planta [11]. Endogenous proteases are involved in many biological processes, and hundreds of genes coding for these enzymes have been identified in plant genomes [12,13].…”

Section: Introductionmentioning

confidence: 99%

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An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

et al. 2016

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The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

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“…Degradation profile of mAb H10 has been extensively studied in different plant species as well as production systems. This molecule was shown to be particularly susceptible to proteolytic cleavage in the proximity of the interdomain antibody sequences [33]. In particular, a previous study from our group demonstrated that mAb H10 purified from leaves of the transgenic tobacco line 7.6 was almost completely degraded leading to the formation of Fab fragments [13].…”

Section: Characterization Of Antibody Degradation Profiles In Hrsmentioning

confidence: 95%

Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

Lonoce

Salem

Marusic

et al. 2016

Biotechnology Journal

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Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2-3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of 'next generation' human therapeutic antibodies.

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Antibody proteolysis: a common picture emerging from plants

Cited by 19 publications

References 15 publications

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

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