Abstract
We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed ‘(5 + 1)Fc’, that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.
Highlights
Fragment crystallizable (Fc) fusion proteins represent 15% of all recently approved biologics by the United States Food and Drug Administration (FDA) (2011-2016), with significant market increases forecasted for 2016 to 2025 (Lagasse et al, 2017)
We demonstrate that the engineered Fc fusion does not compromise the VHH’s function of binding and neutralizing Escherichia coli (EHEC) strains O26:H11, O111:Hnm, O145:Hnm and O157:H7
In the context of passive immunization there may be some activation of the host immune system that requires the bovine Fc
Summary
Fragment crystallizable (Fc) fusion proteins represent 15% of all recently approved biologics by the United States Food and Drug Administration (FDA) (2011-2016), with significant market increases forecasted for 2016 to 2025 (Lagasse et al, 2017). To develop a stabilized IgA Fc chain that could improve yield of its fusion partner without impairing function, we investigated two rational design strategies, supercharging and introducing de novo disulfide bonds, on a bovine IgA Fc chain. Seven EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), account for over 95% of all EHEC-related illnesses and have been the main focus of North American food surveillance efforts for the past decade (Mathusa et al, 2010; Public Health Agency of Canada, 2015) Towards addressing this problem, we previously isolated camelid-derived single-domain antibodies (VHHs) specific for intimin, an EHEC adhesin required for colonization, fused them to a bovine IgA Fc and demonstrated that these chimeric VHH-Fc fusions could bind and neutralize the adherence of three of the most prevalent EHEC serotypes: O157, O26, and O111 (Saberianfar et al, 2019). This study is notable because it provides proof of concept that an Fc chain may be rationally designed for improved IgA yield in a plant platform without compromising functionality or its ability to assemble into its secretory form
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