Table 2.
Summary of nanobodies in treatment of SARS-CoV-2 variants.
| Nanobody name | Nanobody target | Nanobody format | Source | Screening method | Expression system | Affinity to target (KD) |
Neutralizing pseudovirus (IC50) |
Neutralizing SARS-CoV-2 (IC50) |
Mechanism | Virus variants | Features | References |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NB1A2, NB2B4 | Mpro | Monovalent | Mpro-immunized camel |
Phage display | Top10F’ cells | 2.43 nM, 0.46 nM | – | – | Targeting Mpro dimer and dissociate it into monomers, engaging the catalytic and substrate binding sites of Mpro |
– | – | [76] |
| Nb15-NbH-Nb15 | S protein | Heterotrimeric | Alpaca immunized with the extracellular domain of S protein (S1 + S2 ECD, S) | Phage display |
293F cells | 0.541 nM | 0.0004 ± 0 μg/ml | – | – | Wild type, Alpha, Delta, Epsilon, B.1.429 | Nb15-NbH-Nb15 formatting; intranasal administration |
[119] |
| See original literature | S protein | Monomeric, dimeric, trimeric | Llamas immunized with S1 and S2 Fc fusion proteins | Combining in vivo antibody affinity maturation and proteomics | Arctic Express (DE3) cells | See original literature | See original literature | See original literature | Multiple modes (see original literature) |
Alpha, Beta, Gamma | Combining in vivo antibody affinity maturation and proteomics, synergistic viral-neutralizing effects analysis | [96] |
| See original literature | S protein (RBD, S2, or NTD) |
Monovalent, bivalent (Fc fusion) | Llamas immunized with S protein (and boosted with RBD) | Phage display |
E. coli (Monovalent) HEK293-6E cells (Fc fusion) |
See original literature | See original literature | See original literature | See original literature | Wild type, Alpha, Beta, Omicron |
Targets besides RBD | [92] |
| saRBD-1 | RBD of S protein | Monovalent, bivalent | Spike RBD immunized alpaca |
Phage display | Bacteria, 293F cells | 750 pM (monovalent), 302 pM (bivalent) | 4.26 nM (monovalent), 100 pM (bivalent) | – | Competitive binding to RBD of the S protein | Wild type, Alpha, Beta, Gamma, Delta, Epsilon | – | [120] |
| Sb#15, Sb#68 | RBD of S protein | Monovalent, bivalent | Synthetic sybody libraries | Ribosome display + phage display | E. coli MC1061 cells | 12 nM (Sb#15), 9 nM (Sb#68), 0.3 nM (GS4), | 2.3 µg/ml (Sb#15), 2.3 µg/ml (Sb#68), 0.02 µg/ml (G4S), 0.01 µg/ml (Tripod-GS4r) | – | Interfering with ACE2 interaction | Wild type, Alpha, Beta, Delta, | – | [69] |
| VHH72 | RBD of S protein | Bivalent, VHH72-Fc | Llama immunized with SARS-CoV-2 S glycoprotein | Phage display | HEK293 cells, Pichia pastoris | 23 nM | 0.2 µg/mL | – | Disrupting RBD dynamics and receptor binding | Wild type, Delta, Beta, Kappa | Extracellular vesicles-targeting platform | [118], [121] |
| 1B-3F-Fc, 3F-1B-2A-Fc, 1B-3F-2A-Fc | RBD of S protein | Bispecific, trispecific | Naïve and synthetic humanized llama VHH libraries | Phage display | Expi293 cells | 0.25 nM (1B-3F-Fc), 46.8 pM (3F-1B-2A-Fc), 94.7 pM (1B-3F-2A-Fc) | 3.00 nM (3F-1B-2A-Fc), 6.44 nM (1B-3F-2A-Fc) | – | Interfering with ACE2 interaction | Wild type, Alpha, Beta, Delta, Lambda, Omicron |
– | [100], [112], [122] |
| NB1A7, NB1B11 | RBD of S protein | Monovalent, multivalent | Recombinant RBD-immunized camel |
Phage display | Periplasm of E. coli BL21 (DE3) (monovalent), HEK293F cells (multivalent) | 0.74 ± 0.003 nM (NB1A7), 6.76 ± 0.027 nM (NB1B11) |
131.3 ± 1.39 nM (NB1A7), 303.9 ± 1.46 nM (NB1B11) | 1.0 ± 1.5 nM (Format I: NB1A7-Fc + NB1B11-Fc), 0.6 ± 1.19 nM (Format II: NB1A7-NB1B11-Fc) |
Blocking the interaction between ACE2 and RBD | – | – | [61] |
| n3113v, n3130v, bn03, n3113.1-Fc (Y58L) |
RBD of S protein | Monovalent, bivalent | Grafted fully human single-domain antibody | Phage-display, in vitro affinity maturation through error-prone PCR | E. coli HB2151 | 0.81–2.54 nM (n3113v); 1.16–6.04 nM (n3130v); ≤1 nM (bn03) | 0.28 µg/mL (bn03) | – | Inducing S trimer to unstable wide-up state, inhibiting S protein mediated membrane fusion |
Wild type, Alpha, Beta, Gamma, Delta, Omicron | Human origin single-domain antibody library | [68], [99], [123] |
| RBD-1-2G | RBD of S protein | Monovalent, bivalent (RBD-1-2G-Fc), trivalent | Synthetic humanized nanobody libraries | Phage-display | Vibrio natriegens (Monovalent); | 9.4 nM (monovalent toward RBD); 1.9 nM (bivalent RBD-1-2G-Fc); 0.1 nM (trivalent) | 490 nM (RBD-1-2G); 88 nM (RBD-1-2G-Fc); 4.1 nM (RBD-1-2G-Tri) | 1211 nM (RBD-1-2G); 255 nM (RBD-1-2G-Fc); 182 nM (RBD-1-2G-Tri) | Interfering with ACE2 interaction | Wild type, Alpha | Synthetic humanized nanobody libraries | [124] |
| P2C5, P2G1, P5F8 | RBD of S protein | Monovalent, bivalent | Bactrian camel immunized with SARS-CoV-2 RBD | Phage-display | E. coli BL21 cells | 3.97 nM (P2C5), 5.36 nM (P2G1) and 1.94 nM (P5F8) | – | – | Competing with ACE2 for RBD binding | SARS-CoV-2B.1.1.1, Alpha, Beta, Gamma, Delta and Omicron | – | [125] |
| NIH-CoVnb-112 | RBD of S protein | Monovalent | Llama immunized with SARS-CoV-2 S1 protein | Phage-display | BL21 (DE3) competent E. coli strain; X-33 Pichia pastoris |
1–5 nM | – | – | Competing with ACE2 for RBD binding | Wild type, Alpha, Beta, Gamma, and Delta. | Nebulized delivery of NIH-CoVnb-112 | [126], [127] |
| Nb1, Nb2 | RBD of S protein | Bivalent (Nb1–Nb2), tetravalent (Nb1–Nb2-Fc) | Synthetic library | Phage-display | Escherichia coli (Nb1–Nb2), Expi293F cells (Nb1–Nb2-Fc) | ≤0.001 nM | Nb1-Nb2 (All variants ≤ 0.0865 nM) | Nb1-Nb2 (WT-1.207 nM, Alpha-0.8149 nM, Beta-1.776 nM, Gamma-13.01 nM, Delta-0.7317 nM); Nb1-Nb2-Fc (WT-0.0168 nM, Alpha-0.0117 nM, Beta-0.0097 nM, Gamma-0.0987 nM, Delta-0.0232 nM, Omicron-1.46 nM) |
Competing with ACE2 for RBD binding | Alpha, Beta, Gamma, Delta, Lambda (C.37), Kappa (B.1.617.1), Mu (B.1.621), and Omicron | – | [128] |
| 1.10, 1.26, 1.29, 2.15 | RBD of S protein | Monospecific and bispecific | Dromedary camels immunized with RBDmFc |
Surface Display in Bacteria | HEK- 293F cells |
Nb - 3.96 nM (1.10), 0.26 nM (1.26), 12.9 nM (1.29), 0.39 nM (2.15); HCAb - 0.77 nM (1.10), 0.08 nM (1.26), 0.63 nM (1.29), 0.08 nM (2.15) |
– | – | Competing with ACE2 for RBD binding | WA1, Alpha, Beta, Gamma, Delta, Kappa, Zeta | – | [129] |
| VHH72 | RBD of S protein | Multivalent (bivalent, tetravalent, hexavalent) | Llama immunized with SARS-CoV-2 S glycoprotein | Phage display | HEK293-6E cells | 23 nM | 3.3 ± 1.8 nM (bivalent), 0.34 ± 0.072 nM (tetravalent), 0.035 ± 0.003 nM (hexavalent) | – | Competing with ACE2 for RBD binding | Wild-type, Alpha, Beta | High valencies formatting of nanobody (hexavalent) | [103], [118] |
| Nb12, Nb15, Nb17, Nb19, Nb30, and Nb56 | RBD of S protein | Monomer, bivalent, trimer | Llama or nanomice immunized with RBD and/or S protein | Phage display |
WK6 cells (ATCC, 47078), Expi293 cells (Fc conjugated nanobodies) | Monomer - 8.15 nM (Nb15), 5.59 nM (Nb17), 4.72 nM (Nb19), 3.26 nM (Nb56), 30.0 nM (Nb12), 6.55 nM (Nb30); Trimer - 16.4 pM (Nb15), <1 pM (Nb17), 35.1 pM (Nb19), 38.2 pM (Nb56), <1 pM (Nb12), 820 pM (Nb30) |
Monomer - 11.7 nM (Nb12), 0.4 nM (Nb15), 0.6 nM (Nb17), 0.3 nM (Nb19), 6.9 nM (Nb30), 0.9 nM (Nb56); Bivalent - 0.7 nM (Nb12-llama), 0.5 nM (Nb12-human), 0.3 nM (Nb15-human), 79 pM (Nb17-human), 66 pM (Nb19-human), 1.9 nM (Nb30-llama), 1.8 nM (Nb30-human), 0.1 nM (Nb56); Trimer - 0.1 nM (Nb12-human), 65 pM (Nb12-llama), 0.1 nM (Nb15-human), 18 pM (Nb17-human), 9 pM (Nb19-human), 55 pM (Nb56-human) |
– | Interfering with ACE2–RBD associations | WA1, Alpha, Beta, Gamma | Creation of nanobody-producing mice - nanomice | [91] |
| RBD1i13, RBD3i17, RBD6id, RBD10i10, RBD10i14, RBD11i12 | RBD of S protein | Monovalent, bivalent (Fc fusion) | A synthetic yeast-displayed library of nanobodies |
Autonomous hypermutation yeast surface display | Expi293 cells | 32.2 nM (RBD1i13), 230 nM (RBD3i17), 263 nM (RBD6id), 2.14 nM (RBD10i10), 0.72 nM (RBD10i14), 316 nM (RBD11i12) | 0.05 μg/ml (RBD1i13), 0.116 μg/ml (RBD3i17), 0.056 μg/ml (RBD6id), 0.19 μg/ml (RBD10i10), 0.42 μg/ml (RBD10i14), 0.04 μg/ml (RBD11i12) | – | – | – | Autonomous hypermutation yeast surface display (AHEAD) | [109] |
| W25 | RBD of S protein | Monovalent (nanobody, monomeric Fc fusion), bivalent (Fc fusion) | Alpaca immunized with full S protein | bacterial display | Priplasmic expression the E. coli wk6 strain (W25) ExpiCHO cells (W25Fc, W25FcM) |
∼ 0.3 nM (RBD) | – | W25 - 9.82 ± 1.92 nM (D614), 5.09 ± 1.09 nM (G614 variant); W25FcM - 27.40 ± 8.38 nM (D614), 12.36 ± 2.84 nM (G614); W25Fc - 7.39 ± 2.39 nM (D614), 3.69 ± 0.96 nM (G614) |
Competing with ACE2 for RBD binding | D614 variant, G614 variant | – | [109], [130] |
| Nb20, Nb21, Nb34, Nb105, Nb95, Nb17, Nb36 |
RBD of S protein | Monovalent, homodimeric, homotrimeric, heterodimeric | Llama immunized with RBD-Fc fusion protein | Proteomic identification of high-affinity RBD-Nbs | BL21 (DE3) cells | 10.4 pM (Nb20), <1 pM (Nb21) | 0.102 nM (Nb20), 0.045 nM (Nb21), 1.991 nM (Nb34), 5.105 nM (Nb105), 10.05 nM (Nb95), 1.106 nM (Nb17), 1.563 nM (Nb36) | 0.048 nM (Nb20), 0.022 nM (Nb21), 1.125 nM (Nb34), 5.070 nM (Nb105), 5.105 nM (Nb95), 1.500 nM (Nb17) | Sterically interfering with ACE2 binding (directly for class I nanobodies, or indirectly for class II nanobodies), lock the spike in an all-RBD-up conformation or destabilize it (for class III nanobodies) | Alpha, Delta | Proteomic identification of high-affinity RBD-Nbs, resist resistance of nanobody to lyophilization and aerosolization | [101], [105] |
| WNb 2, WNb 7, WNb 15, WNb 36 | RBD of S protein | Monovalent, bivalent (Fc fusion) | Alpacas immunized with coronavirus spike and receptor-binding domains (RBD) | Phage display | – | 0.14 - 19.49 nM | Wild type - 0.33 nM (WNbFc 2), 3.18 nM (WNbFc 7), 2.55 nM (WNbFc 15), 0.10 nM (WNbFc 36); N501Y D614G variant - 0.30 nM (WNbFc 2), 5.04 nM (WNbFc 7), 4.91 nM (WNbFc 15), 0.11 nM (WNbFc 36) |
Interfering with ACE2–RBD associations | Wild type and the N501Y D614G variant |
– | [131] | |
| C5, H3, C1, F2 | RBD of S protein | Monomeric, dimeric (Fc fusion), trimeric | Llama immunized with RBD, RBDFc, and/or S protein | Phage display | WK6 E. coli strain (monomeric), Expi293® cells (dimeric, trimeric) | RBD - 615 pM (C1), 99 pM (C5), 25 pM (H3), 40 pM (F2), 18 pM (C5 trimer), 0.3 pM (H3 trimer), 53 pM (C1 trimer) |
– | 18 pM (C5 with Victoria), 25 pM (C5 with Alpha) | Interfering with ACE2–RBD associations | Victoria, Alpha, Beta | – | [65] |
| DL28 | RBD of S protein | Monomeric, dimeric (Fc fusion) | Alpaca immunized with RBD | Phage display | E. coli (monomeric), Expi293 suspension cells | 1.56 nM | – | 5.39 nM (Wild type), 4.61 nM (Alpha), 13.95 nM (Beta), 17.16 nM (Gamma), 21.88 nM (Delta), 8.68 nM (Omicron) | Keeping an RBD loop in a conformation incompatible with ACE2-binding |
Wild type, Alpha, Beta, Gamma, Delta, Omicron | – | [65], [66] |
| H7, G12 | RBD of S protein | Dimeric (Fc fusion) | Synthetic humanized VHH library | Phage display, affinity maturation by CDR1 and CDR2 shuffling |
FreeStyle™ 293-F cells |
4.485 nM (H7-Fc), <1 pM (G12 × 3-Fc) |
– | D614G variant - 133.8 ng/ml (H7-Fc), 13.1 ng/ml (G12 × 3-Fc); Delta - 12.3 ng/ml (H7-Fc), 0.9 ng/ml (G12 × 3-Fc); Omicron BA.1 - 106 ng/ml (H7-Fc), 9.6 ng/ml (G12 × 3-Fc) |
– | Wild type, D614G variant, Delta, Omicron BA.1 | – | [66], [132] |
| C5G2 | RBD of S protein | Monovalent | Synthetic nanobody library |
Phage display-screening and affinity maturation |
E. coli BL21 (DE3) |
1.62 nM | 1.59 nM (Wild type), 0.95 nM (Alpha), 0.56 nM (Beta), 1.06 nM (Gamma), 0.3 nM (Omicron) | – | Binding to RBD at a conserved region and the neighboring NTD domain through its CDR3, and inhibit ACE2 binding by FR2 due to steric hindrance | Wild type, Alpha, Beta, Gamma,Omicron | One nanobody wit triple functions | [132], [133] |
| Nb-007 | RBD of S protein | Monomeric, dimeric (Fc fusion) | Alpaca immunized subcutaneously with RBD | Phage Display |
Brevibacillus choshinensis SP3 cells/ E. coli BL21 (DE3) (monomeric), 293 T cells (Fc fusion) |
67.4 pM | Nb-007 - 37.6 nM (Wild type), 8.13 mM (Beta), 1.07 mM (Delta); Nb-007-Fc - 1.64 nM (Wild type), 42.6 nM (Delta) |
– | Competing with ACE2 for RBD binding | Wild type, Beta, Delta | – | [133], [134] |