Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe aggravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process, however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. We first isolated a MERS-CoV neutralizing antibody that is reactive to the receptor-binding domain (RBD) of spike (S) glycoprotein. To increase stability, we introduced mutations into the complementarity-determining regions (CDRs) of the antibody. In the HCDRs (excluding HCDR3) in this clone, two hydrophobic residues were replaced with Glu, two residues were replaced with Asp, and four residues were replaced with positively charged amino acids. In LCDRs, only two Leu residues were replaced with Val. These modifications successfully generated a clone with significantly greater stability and equivalent reactivity and neutralizing activity following nebulization compared to the original clone. In summary, we generated a MERS-CoV neutralizing human antibody that is reactive to recombinant MERS-CoV S RBD protein for delivery via a pulmonary route by introducing stabilizing mutations into five CDRs.
Highlights
Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 from a patient who suffered acute pneumonia and subsequent renal failure [1]
After the third and fourth rounds of biopanning against recombinant MERS-CoV S receptor-binding domain (RBD) protein, the scFv clones were retrieved in a high-throughput manner as described previously [39]
A total of 36 unique scFv clones were highly reactive to recombinant MERS-CoV S RBD protein
Summary
Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 from a patient who suffered acute pneumonia and subsequent renal failure [1]. In response to the ongoing epidemic, several groups have developed anti-MERS-CoV neutralizing monoclonal or polyclonal antibodies that target RBD [15,16] These antibodies were generated from B cells derived from convalescent patients, nonimmune human antibody phage-display libraries, fully humanized mice, transchromosomic bovines, or hybridomas from mice that were immunized with MERS-CoV S. These antibodies potently inhibit RBD binding to the DPP4 receptor [17,18,19,20,21,22,23]. MERS-CoV neutralizing antibody may accumulate at higher concentrations following delivery via a pulmonary route, suggesting higher efficacy. We reduced the number of hydrophobic residues and introduced solubilizing mutations within the complementarity-determining regions (CDRs), generating an antibody that is resistant to aggregation during nebulization and retains its MERS-CoV neutralizing activity
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