Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

Abstract

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

“…Here, we report several nanobodies that bind to the SARS-CoV-2 spike protein RBD and block spike protein interaction with the ACE2 receptor. The affinity of NIH-CoVnb-112 in monomeric form is substantially better than the monomeric form of previously reported candidate nanobody therapeutics for SARS-CoV-2: 39 nM for monomeric VHH72 24 , ~ 50 nM for monomeric Ty1 23 , 30.7 nM for monomeric Sb#14 21 , 10 nM for monomeric Sb23 22 , and incompletely reported but likely lower than 1B and 3F 27 . The affinity and blocking potency of NIH-CoVnb-112 will likely be even higher when formulated into dimeric, trimeric, or other protein engineered constructs 27,30,32,33 .…”

“…Blue highlights indicate sequence diversity with NIH-CoVnb-112, highlighted in gray, set as the reference sequence for comparison. For comparison, seven previously reported nanobody sequences have clearly distinct sequences: Ty1 23 , VHH72 24 , H11-D4 19 , MR3 20 , Sb#14 21 , Sb23 22 , and W25UACh 25 and possess shorter CDR3 domains (represented in NIH-CoVnb-112 by amino acids 99–120). …”

Structural Analysis of Neutralizing Epitopes of the SARS-CoV-2 Spike to Guide Therapy and Vaccine Design Strategies

“…RBM-specific Class II members (mAbs P2B-2F6 [ 72 ], BD-368-2 [ 62 ], CV07-270 [ 70 ], S2H13 [ 69 ], C002 [ 63 ], C104 [ 63 ], C119 [ 63 ], and C121 [ 63 ] and VHHs H11-D4 [ 73 ], H11-H4 [ 73 ], Ty1 [ 75 ], and Sb23 [ 74 ]) have epitopes that overlap with those of ACE2 even less than Class I members ( Figure 3 A). They recognize an epitope found within the crevice formed by the RBM β-hairpin, and many of them overlap with Y449, a residue also recognized by ACE2 [ 22 ].…”

“…Thus, most nanobodies, when connected by a linker and fused to the Fc domain of human IgG1, displayed an increase in neutralization potency against SARS-CoV-2 pseudotyped viruses, with similar IC 50 values as mAb IgGs [ 73 , 75 , 85 , 108 ]. These increases in neutralization were significant for some of the nanobodies, such as Sb23 (RBM-specific Class II), which had ~100-fold improved neutralization as an Fc-fusion, bringing its IC 50 down to 7 ng/mL [ 74 ]. Generating a trivalent construct was demonstrated to have even greater effects in the case of Nb6 (RBM-specific Class III).…”

COVID-19: disease, or no disease? - that is the question. It’s the dose stupid!

“…One approach to diminishing the viral yield after infection, so far untried, that we favor is to develop single domain antibodies (sdAbs) of camelid origin [ 25 , 26 ]. Such sdAbs are less immunogenic, can be produced in abundance in usable formats, remain soluble and stable even at elevated temperatures thereby enhancing their durability in functioning [ 25 , 26 ]. Furthermore, such antibodies can be injected via intranasal routes to neutralize the virus limit its infecting dose and will cause no antibody dependent enhancement of infection.…”