Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike

Abstract

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.

Challenges and opportunities for antiviral monoclonal antibodies as COVID-19 therapy

“…Linksy et al [ 133 ] and Cao et al [ 134 ] developed molecules by de novo design using helices capable of binding RBDs on S proteins; the best molecule, LCB1, had IC 50 of 24 pM (0.16 ng/mL) in live SARS-CoV-2 infection assays [ 134 ]. Human VH domain based molecules (VH-Fc) capable of neutralizing SARS-CoV-2 infection have also been developed by various groups [ 133 , 135 , 136 ]. Ab8 had an IC 50 of 40 ng/mL for neutralization of live SARS-CoV-2 [ 133 ].…”

Highlighting membrane protein structure and function: A celebration of the Protein Data Bank

“…The focus has been rightfully placed on replicases, polymerases, and receptors that enable viruses to pursue their dichotomic life cycle transitioning from a "lifeless" state as a viral particle to a prolific replicator when hijacking the host-cell machinery. In the particular case of membraneenveloped viruses, the viral proteins essential to the ingress of the virus into the host cells are MPs that are important drug targets, as demonstrated with the spike protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov2) (441)(442)(443). However, there is a class of membrane-embedded proteins termed VPs that are essential for virulence and infectivity (444)(445)(446) and have proven to be a valuable but yet elusive targets for therapies.…”

Coronavirus disease 2019 and the revival of passive immunization: Antibody therapy for inhibiting severe acute respiratory syndrome coronavirus 2 and preventing host cell infection: IUPHAR review 31

“…Their accumulation in the lung when administered systemically is negligible, and they are not suitable for alternative treatments based on aerosolized local pulmonary delivery (Hart et al, 2001). Antibody fragments, such as human VH single domains or Camelidae and synthetic nanobodies, represent a valid alternative source of clonal binders to study viruses and potentially inhibit their cell invasion (Bracken et al, 2021; de Marco, 2020; Sun et al, 2020). In particular, the unique biophysical properties of nanobodies, including small size and thermostability, simplify their manufacturing, whereas their in vitro selection can be exploited to recover binders with exclusive characteristics in terms of epitope specificity or resistance to chemical and physical conditions (Harmsen et al, 2007; Olichon et al, 2007) that are instrumental for the preparation of inhalation‐stable compounds for local treatment (Van Heeke et al, 2017).…”

“…In the context of single‐domain antibodies, human VHs have the advantage of minimizing the immunoreaction risk that repeated treatments with nanobodies (see next paragraph) could induce. A human VH phage library has been successfully exploited to isolate 85 neutralizing binders directed against two non‐overlapping epitopes of RBD, both simultaneously involved in ACE2 contact (Bracken et al, 2021). The availability of such independent pools of VHs enabled the preparation of multivalent and bi‐paratopic constructs with significantly higher affinity and neutralizing potency, compared with the monomeric forms (Figure 2).…”