Significance During the ongoing COVID-19 pandemic, protein engineering offers a rapid and powerful approach for building therapeutics to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. We use computational design, affinity maturation, and fusion to dimerization domains to engineer “receptor traps” based on wild-type angiotensin-converting enzyme II (ACE2), the target for viral spike-mediated SARS-CoV-2 entry into cells. The optimized ACE2 receptor traps neutralize authentic SARS-CoV-2 infections as effectively as high-affinity antibodies isolated from convalescent patients and also bind viral spike proteins from other coronaviruses known to cause respiratory diseases. ACE2 receptor traps have large binding interfaces and block the entire receptor binding interface, limiting the potential impact of viral escape mutations. An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.
【저자키워드】 SARS-CoV-2, antiviral therapeutics, computational design, yeast display, receptor trap, 【초록키워드】 neutralizing antibody, ACE2, Coronaviruses, coronavirus, antibody, SARS-COV-2 infection, COVID-19 pandemic, mutations, variant, Infection, ACE2 receptor, virus, SARS-CoV-1, respiratory diseases, viral entry, Receptor binding domain, Protein, Convalescent patients, infections, cells, RBD, lentivirus, receptor, mechanism, yeast, convalescent patient, inactivated, Affinity maturation, binding, Angiotensin-converting enzyme, Receptor binding, Viral resistance, host cells, viral spike protein, acute respiratory syndrome, half-maximal inhibitory concentration, receptor protein, SARS-CoV-2 entry, domain, viral escape, authentic SARS-CoV-2 virus, entry receptor, treat, wild-type, backbone, offer, human immunoglobulin, random mutagenesis, approach, Prevent, neutralize, neutralized, therapeutic response, Seven, IC50s, flexible, amino acid change, addition, generate, other coronavirus, faster, the RBD, impair, ACE2 variant, Significance, 【제목키워드】 ACE2 receptor, neutralize,