Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2

Abstract

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC 50) of 4.0 nM (180 ng ml −1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.

Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2

“…VH domains that bind outside of the ACE2 binding site were not identified with this selection campaign. 8 Here, we used an in-house Fab-phage library to identify unbiased Fab binders that recognize Spike-RBD. Briefly, for each round of selection, the Fab-phage pool was pre-cleared with biotinylated Fc immobilized on streptavidin (SA)-coated magnetic beads before incubating with SA-beads conjugated with biotinylated Spike-RBD-Fc ( Figure 1a).…”

“…The epitope of VH B01 was determined previously by cryo-EM. 8 In the absence of a high-resolution structure of Fab D01 bound to Spike, we used the structure of CR3022 12 as a surrogate to model how the two arms on Bis4 could engage Spike. We find that in the context of the same RBD, the VH and Fab bind at separate, non-overlapping epitopes ( Supplementary Figure 6a-b).…”

“…SARS-CoV-2 has emerged as a global health concern and effective therapeutics are necessary to curb the COVID-19 pandemic. Many potential therapeutic options for treating COVID-19 have been explored, including small molecules, 1 convalescent patient sera, 2 decoy receptors, [3][4][5][6] neutralizing antibodies, [7][8][9][10][11][12][13][14][15][16][17][18] and other protein scaffolds. [19][20][21] In particular, antibodies are advantageous due to their specific and potent binding, demonstrated pharmacokinetics, and ability to be recombinantly produced and manufactured at scale.…”

“…SARS-CoV-2 antibodies have been derived from several sources, including B-cells of convalescent patients 7,12,13,17 and people with prior coronavirus infections, 14,15 animal immunization, 10,18 and synthetic libraries or de novo design. 8,9,11,16,19 Most of the antibodies reported to date potently target the receptor-binding domain (RBD) in the trimeric Spike protein on the surface of SARS-CoV-2, 7,13,[22][23][24] which is highly immunogenic and is the key protein that mediates cellular entry via interaction with the host angiotensin-converting enzyme II (ACE2) receptor. 25 However, given the widespread global impact of this pandemic and limitations in biologic manufacturing capacities, means to further increase the potency of these antibodies and thereby decrease the dose required will be critical in meeting the global demand for therapeutics.…”

Targeted Cancer Therapy Using Nanoparticles and Antibody Fragments

“…Single, as well as multiple targeting, can be achieved through the use of an engineered antibody. Multivalent antibodies not only block signaling but also overcome resistance through multiple targets [57].…”