The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

Abstract

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

Glyco-engineered pentameric SARS-CoV-2 IgMs show superior activities compared to IgG1 orthologues

“…Importantly, using the glycoengineered ΔXTFT line (Strasser et al ., 2008) enabled the generation of IgG1 and IgM mAbs with a largely homogenous N-glycosylation profile, largely devoid of plant specific glycosylation. Nevertheless, some glycans at GS 1-3 of IgM are fucosylated, a feature also observed for other glycoproteins expressed in this RNAi line (Gattinger et al ., 2021). While each GS carried a highly reproducible single dominant N-glycan species, the pool of N-glycans in mammalian cell-produced mAbs is by far more heterogeneous, with substantial differences depending on the production conditions (Hennicke et al ., 2017; Hennicke et al ., 2019).…”

Improving Protein Quantity and Quality—The Next Level of Plant Molecular Farming

“…A recent study demonstrated the enhanced stability of recombinant human EPO-Fc transiently produced in N. benthamiana by the substitution of four residues crucial to the Fc dimerization interface. The resulting monomeric EPO was stably expressed, maintained its bioactivity, and became resistant to proteolytic degradation [179]. Based on their findings, the authors of this study suggested that the weak point the Fc fusion technology is the exposure of the vulnerable region due to the lack of CL and highlighted the importance of the disulfide bond bridges between the CH1 and CL for the stability of native IgG structure [179].…”

“…The most widely used fusion part of Fc in plant expression systems appears to be that of the human IgG1. It has been used in the production of a number of bioactive vaccines, including a dengue fever vaccine candidate derived from the consensus domain III (cEDIII) of dengue glycoprotein E [174,175], and therapeutic proteins, such as human EPO [176][177][178][179], human osteopontin (OPN) [180], and human angiotensin-converting enzyme 2 (ACE2), against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [181]. Fusion of human transferrin to the incretin hormone glucagon-like peptide-1 (GLP-1) has also led to robust accumulation of the protein and promising bioactivity in both transiently expressed and stable transgenic Nicotiana plants [114].…”

“…The resulting monomeric EPO was stably expressed, maintained its bioactivity, and became resistant to proteolytic degradation [179]. Based on their findings, the authors of this study suggested that the weak point the Fc fusion technology is the exposure of the vulnerable region due to the lack of CL and highlighted the importance of the disulfide bond bridges between the CH1 and CL for the stability of native IgG structure [179]. Nevertheless, it is also possible that the exposure of the vulnerable hinge region is due to the lack of mucin-type O-glycosylation in plant cells, because the O-glycosylation at the hinge region of immunoglobulin heavy chains has been observed to protect against proteolytic digestion [200,201].…”

Sortase A transpeptidation produces seamless, unbranched biotinylated nanobodies for multivalent and multifunctional applications

“…12,15,16 Notably, conformational freedom is critical for efficient molecular function, especially for biomolecules with limited exibility and stringent function-associated orientations. [17][18][19][20] A typical example is native tetrameric (strept)avidin, which exists as a dimer-dimer, with each subunit contributing to the biotinbinding site of the neighboring monomer. [21][22][23] The biotinbinding site is approachable from one terminal end within the tetrameric architecture.…”