Total soluble protein (TSP) extracts from XTFT leaves expressing different EPO-Fc variants were analyzed by immunoblotting at 5 days post-infiltration (dpi) using (A) anti-EPO and (B) anti-hIgG antibodies. 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 transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability. Keywords: half-life and Carboxin enhanced erythropoietic bioactivity in comparison with native human EPO (Salgado et al., 2015). The market for recombinant therapeutic proteins is increasing rapidly (Moorkens et al., 2017). Plants have proved to be suitable platforms for the production of biopharmaceuticals, and a growing number of plant-made therapeutic proteins have entered clinical trials (Sack et al., 2015; Lomonossoff and DAoust, 2016). is most suited for the rapid large-scale synthesis of recombinant proteins (Bally et al., 2018), and recent advances in plant glycoengineering have allowed the production of plant-derived protein with tailored human-like glycosylation (Montero-Morales and Steinkellner, 2018). Recombinant expression of EPO-Fc in has been reported previously (Castilho et al., 2011b, 2012, 2013; Nagels et al., 2012). Carboxin Importantly, glycosylation of plant-derived EPO-Fc has been modulated toward the synthesis of multi-antennary (Castilho et al., 2011b; Nagels et al., 2012), sialylated (Castilho et al., 2013), and mucin-type O-glycosylated (Castilho et al., 2012) glycans. Despite the major achievements on glyco-engineering, attempts to transiently express EPO-Fc in showed that the fusion protein is rather unstable and the intact EPO-Fc fusion is expressed at low levels. Besides EPO-Fc, several other Fc-fusion proteins expressed in plants have shown similar instabilities with varying amounts of free Fc being observed (Lu et al., 2012; De Buck et al., 2013; Kim et al., 2017, 2018; Rattanapisit et al., 2019; Xiong et al., 2019; Diamos et al., 2020; Castilho et al., 2021). These studies have used various therapeutic proteins fused to Fc domains expressed in different ways, differing in the gene construction and transformation method, the nature of the signal peptide, the presence or absence of an endoplasmic reticulum (ER) retention sequence, and the host species. In the present investigation, we examine strategies to stabilize the expression of Fc-fusion proteins in plants using the same therapeutic protein and the same plant expression system. We used EPO as a reporter to evaluate our efforts to enhance the expression of full-length EPO-Fc fusion protein targeted to the apoplast of plants. These included modifications in the hinge region of the IgG1 fusion partner, alternative Fc fusion partners (IgG3 and IgG4), and flexible linkers. Our results highlight that the proteolytic degradation of Fc-fusions most probably arises from an instable conformation of the Y-structure mimicking a full-size antibody. We hypothesize that the oligomerization of Fc-fusion leads to physical instability, which can be overcome by disrupting dimerization. Therefore, we engineered the Fc by mutating critical residues located on the dimerization interface. This strategy allowed the expression of fully stable EPO monomeric Fc fusion proteins. Monomeric Fc-fusions can provide several advantages over traditional dimeric Fc-fusion technology. For example, many Fc monomer fusion proteins have enhanced biological activity in comparison with traditional dimeric Fc-fusions, presumably in part due to a reduction of interference between the effector proteins which can occur in dimeric Fc-fusions (Dumont et al., 2006). Importantly, small monomeric Fc can be exploited to expand the Fc-based therapeutic applications, due to its unique receptor binding pattern. Monomeric Fc (i) binds to FcRn and exhibits a Rabbit Polyclonal to DUSP16 similar half-life to wild-type dimeric Fc; (ii) lacks binding to FcRIIIa, which results in the Carboxin absence of Fc-effector functions; and (iii) possesses high-affinity binding to FcRI that can be used for toxin targeting (Ying et al., 2012, 2014; Wang et al., 2017). Materials and Methods Cloning of EPO-Fusions Erythropoietin-fusion sequences were amplified by PCR using as templates the codon-optimized sequences.