Slaying SARS-CoV-2 One (Single-domain) Antibody at a Time

Abstract

Camelid-derived and synthetic single-domain antibodies (sdAbs) are emerging as potent weapons against the novel coronavirus, SARS-CoV-2. sdAbs are small, compact, thermostable immunoglobulin elements capable of binding targets with subnanomolar affinities. By leveraging the power of phage- and yeast surface-display technologies, rare sdAbs can be isolated from highly diverse and complex antibody libraries. Once in hand, sdAbs can be engineered to improve binding affinity, avidity, target specificities, and biodistribution. In this Opinion piece we highlight a series of sophisticated studies describing the identification of ultrapotent sdAbs directed against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. We discuss the possible applications of these antibodies in the global fight against COVID-19.

Figure 1

Structures of Conventional, Heavy-Chain-Only (HCAb), and Single-Domain Antibodies (sdAbs). Conventional IgG antibodies consist of H and L chain pairs that form Y-shaped structures. Camelids (e.g., alpacas, llamas) make a heavy-chain-only (HCAb) class of antibody. The variable elements (V_HH) of HCAbs, when expressed as autonomous units, are known as single-domain antibodies (sdAbs) or nanobodies. The crystal structure of a representative sdAb (PDB ID 6OBC) is shown with the complementarity determining regions (CDRs) 1, 2, and 3 colored blue, yellow, and red, respectively. Image created in BioRender.

Figure 2

Discovery of Receptor-Binding Domain (RBD)-Specific Single-Domain Antibodies (sdAbs). A depiction of a workflow for isolating sdAbs against SARS-CoV-2 Spike and RBD. (A) (1) Isolate peripheral blood mononuclear cells from naïve or immune llamas or alpacas. (2) PCR-amplify and clone V_HH-encoding genes into M13-phage vector to create a library of high complexity. (3) Affinity-enrich for V_HHs that bind to Spike (or RBD). (4) Clone and express individual V_HHs and test for SARS-CoV-2-neutralizing activity. (B) Left, structure of SARS-CoV-2 Spike monomer bound to ACE2 (PDB ID: 6M0J). Right, SARS-CoV-2 RBD (6ZXN) superimposed with VHH72 (6WAQ), H11-H4 (6ZH9), Ty1 (6ZXN), and Nb6 (7KKK). Image created in BioRender.

Figure 3

Engineering Single-Domain Antibodies (sdAbs) for Therapeutic Applications. (A,B) High-affinity monomeric sdAbs engineered onto IgG Fc regions allow enhanced avidity through bivalency, increased serum half-life, and potential Fc-mediated effector functions. (C) Multimerization of sdAbs through flexible linker regions can increase avidity through simultaneous binding of adjacent subunits. (D) Multimeric sdAbs including single bispecific sdAb–Fc molecules generated by knob-into-holes or lock-and-key techniques. (E) sdAbs similarly engineered onto IgA Fc scaffolds would allow for efficient antigen targeting to mucosal compartments, as well as potentially enhanced valency and antigen aggregation. (F) Multimeric sdAb constructs, targeting surface antigens of SARS-CoV-2, particularly the Spike RBD, may allow broad coverage of neutralizing epitopes on the virus, yielding ultra-potent antibody neutralization through multiple mechanisms. Image created in BioRender.