ABSTRACT
COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Neutralizing/genetics , Antibodies, Viral/genetics , Antibody Affinity , Baculoviridae/genetics , Baculoviridae/immunology , Biotechnology , Bombyx/genetics , Bombyx/immunology , Cells, Cultured , Gene Expression , Hemolymph/immunology , Humans , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fragments/biosynthesis , Immunoglobulin G/biosynthesis , Immunoglobulin G/genetics , SARS-CoV-2/genetics , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
Numerous neutralizing antibodies that target SARS-CoV-2 have been reported, and most directly block binding of the viral Spike receptor-binding domain (RBD) to angiotensin-converting enzyme II (ACE2). Here, we deliberately exploit non-neutralizing RBD antibodies, showing they can dramatically assist in neutralization when linked to neutralizing binders. We identified antigen-binding fragments (Fabs) by phage display that bind RBD, but do not block ACE2 or neutralize virus as IgGs. When these non-neutralizing Fabs were assembled into bispecific VH/Fab IgGs with a neutralizing VH domain, we observed a ~ 25-fold potency improvement in neutralizing SARS-CoV-2 compared to the mono-specific bi-valent VH-Fc alone or the cocktail of the VH-Fc and IgG. This effect was epitope-dependent, reflecting the unique geometry of the bispecific antibody toward Spike. Our results show that a bispecific antibody that combines both neutralizing and non-neutralizing epitopes on Spike-RBD is a promising and rapid engineering strategy to improve the potency of SARS-CoV-2 antibodies.
Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Epitopes/immunology , Immunoglobulin Fab Fragments/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Bispecific/genetics , Antibodies, Bispecific/therapeutic use , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/genetics , Antibodies, Viral/therapeutic use , COVID-19/genetics , Epitopes/genetics , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/therapeutic use , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , COVID-19 Drug TreatmentABSTRACT
Most antibodies isolated from individuals with coronavirus disease 2019 (COVID-19) are specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here, we determined a crystal structure of the COVA1-16 antibody fragment (Fab) with the SARS-CoV-2 receptor-binding domain (RBD) and negative-stain electron microscopy reconstructions with the spike glycoprotein trimer to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long complementarity-determining region (CDR) H3, and competes with the angiotensin-converting enzyme 2 (ACE2) receptor because of steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with the structural and functional rationale for epitope conservation, provide insights for development of more universal SARS-like coronavirus vaccines and therapies.