The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.
It is very intriguing that the panning with SARS-CoV-2 S1 or RBD protein as antigen resulted in a substantially different spectra of antibodies. The antibodies identified from S1 panning were very diverse, covering four distinct epitopes on SARS-CoV RBD (competition groups A, B, D, and E). In contrast, most of the antibodies from RBD panning belonged to competition group A represented by n3021, which was also the most dominant clone after two rounds of panning. Furthermore, group A antibodies showed moderate competition with ACE2 for RBD binding but proved to be ineffective viral neutralizers. This phenomenon is quite different from that of SARS-CoV, in which the dominance of an antigenic loop within RBD made it relatively easy to isolate potent SARS-CoV neutralizing antibodies independent of repertoire, species, quaternary structure, and the technology used to derive the antibodies. Similarly, we previously used MERS-CoV S1 or RBD to isolate antibodies from a naive antibody library, and panning that used either of the two antigens led to dominant enrichment of antibodies that precisely targeted 90% of the receptor binding site within RBD and neutralized the virus potently. It was proposed that viruses like SARS-CoV have not yet evolved such that their membrane glycoproteins could avoid direct immune recognition of a single site critical to virus pathogenesis. It should be pointed out that the difference in the immunogenicity of RBD was observed solely based on in vitro experiments and might not correlate with humoral immune responses in vivo. In this regard, it is imperative to investigate the immunogenic characteristics of SARS-CoV-2 RBD with special attention to potentially antigenic and non-neutralizing epitopes.
It is also striking that the SARS-CoV-2-specific neutralizing antibodies from competition groups D and E are incapable of competing with ACE2 for SARS-CoV-2 RBD binding. This phenomenon was rarely observed in SARS-CoV, confirming the unique immunogenic profile of SARS-CoV-2. Further investigations are needed to understand the underlying mechanisms that govern these diverse sets of neutralizing and non-neutralizing SARS-CoV-2 antibodies, which could have important implications for the development of effective vaccines.
More interestingly, the group D antibodies n3088 and n3130 were found to neutralize SARS-CoV-2 by targeting a “cryptic” epitope located in the spike trimeric interface. The mAb CR3022 also recognizes this epitope but cannot neutralize SARS-CoV-2, implying the advantage of small-size single-domain antibodies to target cryptic eptiopes. Such hidden trimeric interface epitope was also observed recently in the study of influenza antibodies by our group and several other groups. It is probable that such an epitope would also be important in the study of coronaviruses, and n3088 or n3130 could be ideal for synergistically used with other SARS-CoV-2 neutralizing antibodies, especially the ACE2-competing neutralizing antibodies.
Fully human single-domain antibodies offer the potential for prevention and treatment of COVID-19. First, antibodies derived entirely from human sequences would be less immunogenic than camelid or humanized nanobodies, leading to improved safety and efficacy when used in humans. Indeed, despite humanization, caplacizumab, the first nanobody approved by FDA, still contains multiple camelid residues to maintain the antigen binding affinity . Second, small size and favorable biophysical properties allow for large-scale production of single-domain antibodies within a few weeks in prokaryotic expression systems, thus enabling rapid implementation in an outbreak setting. Furthermore, single-domain antibodies could be delivered to the lung via inhalation, which could offer considerable advantages for treatment of COVID-19, including fast onset of action, low systemic exposure, and high concentration of therapeutics at the site of disease. Lastly, single-domain antibodies can be used alone or synergistically with other neutralizing antibodies. Their small size makes them ideal building blocks for generation of bispecific or multi-specific antibodies to prevent the appearance of viral escape mutants. They can also be easily engineered to further increase neutralization activity by increasing binding moieties. For instance, the trivalent nanobody ALX-0171 was found to have 6,000-fold increased neutralization potency against RSV-A and >10,000-fold against RSV-B compared to its monovalent format.
In summary, we report here the development of a versatile platform for rapid isolation of fully human single-domain antibodies and their application for screening of antibodies against SARS-CoV-2. These antibodies could represent promising candidates for prophylaxis and therapy of COVID-19 and serve as reagents to facilitate vaccine development.
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