Table 1.

Representative high avidity and ultrapotent anti-SARS-CoV-1 and −2 nanobodies.

Nanobody Name	Source and method	Virus type	Binding affinity (KD)	Neutralizing activity (IC50)	Mechanism of neutralization/inhibition	Protective efficacy	Ref
VHH-72 Bivalent VHH-72-Fc	Immune library + phage display, IgG Fc fusion	SARS-CoV-1 MERS-CoV SARS-CoV-2 RBD	36.8 nM	13.3 nM	Recognize epitope residues (Trp100, Tyr356/494, Cys366, Phe 364,Ser358,Arg 426) on spike RBD, Blocks RBD-ACE2 interaction, Neutralized SARS-CoV-2 pseudoviruses	N/A	[62]
H11-D4 H11-H4 H11-D4-Fc H11-H4-Fc	Naïve library, IgG Fc fusions	SARS-CoV-2 RBD	39 nM 12 nM N/A N/A	N/A N/A 4–6 nM 18 nM	H11-H4 recognize RBD epitope via hydrogen bonding (residues; Lys 449-Phe456, Gly482 – Ser 494), van der Waals interaction and salt bridge contact; Blocks RBD–ACE2 interaction; Neutralized SARS-CoV-2 pseudoviruses	N/A	[132]
Ty1 Ty1-Fc Fu2 Fu2-Fc Fu2-Ty1 homodimer/ heterodimer	Immune library + phage display, IgG Fc fusion, Chemical linkage (Sortase A labelling and Cu-free click-chemistry)	SARS-CoV SARS-CoV-2 SARS-CoV-2 Variant (B.1.351)	5–10 nM 0.12 nM	54 nM 1 nM 7 nM 0.75 nM 0.8 nM 140 pM	Ty1 recognize RBD epitope residues (T470, V483-E484, Y449, F490, Q493); Fu2 recognize RBD epitope residues (381 – S375) via anti-parallel β-strand hydrogen bond; Fu2 induced the formation of spike trimer-dimers; Block RBD-ACE2 interaction, Neutralized pseudotyped and live SARS-CoV-1, −2 and variant (B.1.351)	Fu2-Ty1 prophylactically and therapeutically mice from SARS-CoV-2 challenge	[61], [95]
Nb-15,Nb17, Nb19 Nb56; Nb-30,Nb-12 Bivalent format Trimer format	Immune (llama and Nanomouse) library, IgG Fc fusion	SARS-CoV-2 Variants (B.1.1.7, B.1.351, P.1) SARS-CoV-1	Monomer 30–3 nM Multimeric 0.8 nM − 2.4pM	Monomer 11.7 nM Multimeric (65–9 pM)	Recognize conserved epitope residues different from RBD; Focus and block RBD – ACE2 interaction; Neutralized pseudotyped and live SARS-CoV-1, −2 and variant (B.1.351)	N/A	[74]
Sb23 Bivalent Sb23-Fc	Synthetic library + Phage display IgG Fc fusion	SARS-CoV-2	10 nM 225 pM	0.6 µg/ml 0.007 µg/ml	Recognize spike RBD residues Block RBD – ACE2 interaction Neutralized pseudotyped SARS-CoV-2 virus	N/A	[133]
sdAb Bivalent sdAb-Fc	Humanized Synthetic library + phage display IgG Fc fusion	SARS-CoV-2	0.99–35.5 nM	0.0009–0.07 µg/ml 0.001–0.043 µg/ml	Recognize spike RBD residues Block RBD – ACE2 association Neutralized pseudotyped and authentic SARS-CoV-2 virus	N/A	[134]
Nb91, Nb3 Homodimer homotrimer Heterodimer	Naïve library IgG Fc fusion	SARS-CoV-2	N/A	N/A 32.36–54.07 nM 4.7 – 4.89 nM 1.54 nM	Recognize spike RBD residues Block RBD – ACE2 association Neutralized pseudotyped SARS-CoV-2 virus	N/A	[77]
WNbFc2,7,15,36 WNbFc mixtures	Immune library + phage display IgG Fc fusion Cocktail	SARS-CoV-2 Variant N501Y D614G	0.25–0.55 nM 0.22– 0.52 nM	0.1 – 3.18 nM	Recognize spike RBD residues (Y109,E484,F486,Q493,N501,K417,R403) Block RBD – ACE2 interactions Neutralized both wild-type SARS-CoV-2 and N501Y D614G variant	Nanobody-Fc mixtures prophylactically prevented Wild-type SARS-CoV-2 and N501Y D614G variant in mice	[90]
1B, 3 F,2A-Fc Bispecific 1B-3 F;1B-3 F-Fc Trispecific 3 F‑1B‑2A-Fc 1B-3 F-2A-Fc	Naïve and synthetic humanized phage libraries IgG Fc fusion Cocktail	SARS-CoV-2	0.82 – 1.6 nM 0.25 nM 95 pM 47 pM	1 nM 0.71 nM 0.74 nM	Bind spike S1 RBD residues Block SARS-CoV-2/ACE2 interaction Neutralized pseudotyped SARS-CoV-2 virus	N/A	[75], [76]
Nbs 20,34,89,95 Nb 21 Tri-Nb 20 Tri-Nb21 (PiN-21)	Immune library + MS proteomic strategy Strategic linking	SARS-CoV-2	0.102/0.133 nM 0.045 nM 4.1 pM 1.3 pM	10.4 – 108 pM < 1 pM < 1 pM < 1 pM	Recognize spike RBD residues and epitope closer to trimmer NTD Block SARS-CoV-2/ACE2 interaction Neutralized pseudotyped and authentic SARS-CoV-2 virus	PiN-21 (Tri-Nb21) prophylactically and therapeutically prevent and treat SARS-CoV-2 infection in Syrian hamster	[64], [94]
Nb 6 mNb 6 mNb6-tri	Synthetic library + yeast surface display Strategic linking	SARS-CoV-2	210 nM 0.45 mM < 1 pM	2 μM 6.3 nM 54–120 pM	Recognize spike RBD residues, bind and lock spike in the inactive state Block SARS-CoV-2/ACE2 interaction Neutralized pseudotyped and authentic SARS-CoV-2 virus	N/A	[63]
VH A01, B01, B02; Biparatopic (VH2A01-B01) Multivalent (VH 3 B01)	Synthetic humanized library + phage library IgG Fc fusion Strategic linking	SARS-CoV-2	23 – 113 nM 0.1 nM 0.12 nM	33.5 nM 26.2 nM 4.0 nM	Out-Competed ACE2 and bind RBD and spike ectodomain Neutralize pseudotyped and authentic SARS-CoV-2 virus	N/A	[93]
VHH E,U,V,W Biparatopic/Bivalent (VE,EV,EE,VV) Trivalent EEE	Immune library + phage library Strategic linking	SARS-CoV-2 Escape mutants	1.86 – 22 nM 84/200 pM N/A	60 nM 0.7 – 1.32 nM 170 pM	VHH V, U and E bind and recognize distinct ACE2 binding epitopes on spike RBD in different orientation; Nanobodies trigger activation of the fusion machinery; Block ACE2 – RBD interaction Neutralize pseudotyped and authentic SARS-CoV-2 virus	N/A	[60]
VHH-72 Bivalent VHH-72-Fc VHH-hFerr	Immune library 24-mer Apoferritin protein cage as scaffold	SARS-CoV-2	N/A 36.8 nM < 1 pM (beyond detection limit)	1.3 µg/ml 0.00011 µg/ml	Recognize spike RBD epitope residues Neutralized pseudotyped SARS-CoV-2	N/A	[130]