We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.
Neutralizing mAbs are promising for prophylaxis and therapy of SARS-CoV-2 infections. Recently, many potent neutralizing antibodies from COVID19 patients were identified that neutralize pseudovirus with IC50s ranging from 1 to 300 ng/mL, and replication-competent SARS-CoV-2 with IC50s from 15 to 500 ng/mL. By comparison, the VH-Fc ab8 reported here exhibited comparable or better neutralizing potency against SARS-CoV-2 pseudovirus and live virus (IC50s of 30 ng/mL and 40 ng/mL, respectively). Of note, IC50s can vary widely between different assays and laboratories because there is no generally accepted standardized assay. In addition, there are many factors that contribute to potency and efficacy in vivo. Animal models are a more comprehensive and likely more reliable predictor of potential efficacy in humans than in vitro neutralization assays.
To our knowledge, VH-Fc ab8 is the first human antibody domain whose activity was validated in two animal models. In the mouse ACE2-adapted SARS-CoV-2 infection model, VH-Fc ab8 significantly decreased infectious virus by 10-fold at 2 dpi even at a very low dose of 2 mg/. It also exhibited both prophylactic and therapeutic efficacy in a hamster model. It not only reduced the viral load in the lung and alleviated pneumonia, but it also reduced shedding in the upper airway (nasal washes and oral swab), which could potentially reduce transmission of SARS-CoV-2. Impressively, VH-Fc ab8 was active therapeutically, even at 3 mg/kg. The finding that VH-Fc ab8 persisted for 4 days post administration at significant levels indicates that the pharmacokinetics of VH-Fc ab8 is comparable to that of a full size antibody; the half-lives of Fc fusion proteins were reported to vary from those of IgG1s and can range from hours to days. The molecular weight of VH-Fc ab8 (80 kDa) is half of that of full-size IgG1 that suggests an advantage in terms of smaller quantities needed to be produced compared to those for IgG1s to reach similar number of molecules and efficacy. In addition, it was shown that decreasing binder’s size exponentially increases its diffusion through normal and tumor tissues. Thus, decreasing the size 2-fold can increase diffusion through tissues by 4-fold. We found that after administration at the same dose, the concentration of VH-Fc ab8 was higher than that of IgG1 ab1 in both hamster sera and lung tissue. This result might suggest that the VH-Fc ab8 diffusion from the peritoneal cavity to the blood and penetration of lung may be faster than that of IgG1 ab1. This may further explain its efficacy at low doses in animals. Although the low dose showed efficacy in the small animal models, it should be noted that in humans higher doses could be required to achieve comparable degree of efficacy. Another caveat is that in the hamster post-exposure experiment, the VH-Fc ab8 was administered at a time (6 h) when the first round of virus replication was likely completed, but before the infection peak at 1–2 days. Because it inhibits infection of new cells, its administration at around the infection peak or after may not be as effective unless it also kills infected cells in vivo, which is under investigation.
Recently, antibody domains including human VH and camelid VHH were reported having varying neutralization potency. Compared to those domains, VH-Fc ab8 is unique in terms of potency, aggregation resistance, and specificity. VH-Fc ab8 exhibited good developability properties including stability at high concentrations and long incubation at 37°C, as well as absence or very low aggregation. In addition, VH-Fc ab8 did not bind to the human cell line 293T even at high concentration (1 μM) which is ~1,754-fold higher than its Kd indicating absence of non-specific binding to many membrane-associated human proteins. A similar result was obtained by the membrane protein array assay showing that VH-Fc ab8 did not bind to any of 5,300 human membrane-associated proteins, indicating its lack of non-specificity and thus low potential for off-target toxicity when used in vivo. Unlike camel VHHs, the VH ab8 sequence is fully human and therefore likely less immunogenic than that of camelid VHHs.
Multiple structures are now available for the SARS-CoV-2 S protein trimer in complex with various neutralizing antibodies, offering insight into antigenic epitopes and inhibitory mechanisms critical for S protein neutralization. Epitopes on the SARS-CoV-2 S protein RBD have emerged as effective targets, as evidenced by the action of several RBD binding antibodies including CR3022, B38, C105, CB6, H014, and S309. While B38, C105, and CB6 directly compete with ACE2 for binding sites on the RBD surface, H014 occupies a position distinct from these binding sites, precluding ACE2 binding via steric inhibition. S309 targets the RBD of the S protein both in closed and open S protein conformations, exhibiting a different mechanism of neutralization. A recent study of the structure of the S protein trimer in complex with the nanobody H11-D4 (PDB: 6Z43) revealed full occupancy of the nanobody on all three RBDs in a “one up and two down” conformation, similar to what we report here. Our structural analysis demonstrates that the location of the VH ab8 bound to the trimeric S ectodomain directly overlaps the region that would be occupied by ACE2 when bound to the S protein. The ACE2 blocking is likely the major mechanism of the VH-Fc ab8 neutralizing activity, which is significantly augmented by avidity effects due to its bivalency. The narrow neutralization concentration range in the live virus neutralization (10–200 ng/mL for 0%–100% neutralization) indicates a plausible cooperative neutralization mechanism, probably due to the synergistic binding of VH molecules in VH-Fc ab8 to RBDs. Due to its small size, VH may facilitate targeting occluded epitopes on RBD that are otherwise inaccessible to full-length IgGs, which is important because the SARS-CoV-2 S protein is conformationally heterogeneous, exposing neutralizing epitopes to varying degrees. The structural analysis shows that VH ab8 is able to simultaneously target all three RBD epitopes in both “up” and “down” conformations, which may provide a structural basis for a unique cooperative neutralization mechanism for VH-Fc ab8. VH-Fc ab8 with a long flexible linker between VH and Fc may allow two VH molecules to bind simultaneously two protomers in the same S trimer or cross-link two different protomers from different S trimers.
The ab8 epitope is distal to the CR3022 epitope, explaining its lack of competition with CR3022. The ab8 contact residue F486 (L472 in SARS-CoV) is not conserved that likely explains its lack of cross-reactivity to SARS-CoV. From the GISAID and NCBI databases, we found nine mutations in RBD with relatively high frequencies in current circulating SARS-COV-2. Six of them are in the core domain (F342L, N354D, N354D/D364Y, V367F, R408I, and W436R) and three in the RBM (K458R, G476S, and V483A). The core domain mutations are far away from the ab8 epitope, thus these mutations do not affect VH-Fc ab8 binding to RBD. Those three RBM mutations also did not affect ab8 binding although they are close to the ab8 epitope, suggesting these mutations may not affect ab8 neutralizing activity although neutralization of whole virus carrying these mutations is needed to definitely demonstrate this possibility. Interestingly, VH-Fc ab8 effectively inhibited the mouse ACE2-adapted SARS-CoV-2 with a Q498T/P499Y mutation in RBD, indicating that this double mutation also does not affect VH-Fc ab8 binding to RBD. These results suggest that VH-Fc ab8 may be a broadly cross-reactive SARS-CoV-2 neutralizing antibody.
In conclusion, we identified a fully human antibody VH domain that shows strong competition with ACE2 for binding to RBD and potent neutralization of SARS-CoV-2 in vitro and in two animal models. This potent neutralizing activity combined with its specificity and good developability properties warrants its further evaluation for prophylaxis and therapy of SARS-CoV-2 infection. Our elucidation of its unique epitope and mechanism of neutralization could also help in the discovery of more potent inhibitors and vaccines.
Reference & Source information: https://www.cell.com/
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