In a recent study published in the Nature Microbiology Journal, researchers generated six human monoclonal antibodies (mAbs) that prevented infection by all human angiotensin-converting enzyme 2 (ACE2) binding sarbecoviruses tested, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, Delta and Omicron.
They targeted the hACE2 epitope that binds to the SARS-CoV-2 spike (S) glycoprotein rather than targeting the S protein, which all previous therapeutic mAbs for SARS-CoV-2 targeted.
Study: Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies. Image Credit: paulista/Shutterstock.com
Background
The emergence of new variants of SARS-CoV-2, especially Omicron sublineages, made all therapeutic mAbs targeting SARS-CoV-2 S obsolete.
Any new S-targeting mAb therapy will also probably have limited utility because SARS-CoV-2 will continue to adapt to human antibodies. Ideally, mAbs developed in anticipation of future pandemics caused by sarbecoviruses should be resilient to mutations that arise in them.
About the study
In the present study, researchers developed hACE2-binding mAbs that blocked infection by pseudotypes of all tested sarbecoviruses at potencies matching SARS-CoV-2 S targeting therapeutic mAbs. The binding affinity of these mAbs to hACE2 was in the nanomolar to picomolar range.
To develop these mAbs, researchers used the KP and Av AlivaMab mouse strains that generate a human Kappa (κ) light chain and Kappa (κ) and Lambda (λ) light chains carrying antibodies, respectively.
They immunized these mice with monomeric and dimeric recombinant hACE2 extracellular domains. Fusion to the fraction, crystallizable (Fc) portion of human immunoglobulin G1 (IgG1) rendered them dimeric.
Further, the team generated hybridomas from mice using sera that inhibited SARS-CoV-2 pseudotyped viruses. They used enzyme-linked immunosorbent assay (ELISA) to screen hybridoma supernatants for hACE2-binding mAbs.
Furthermore, the researchers tested the ability of the six most potent mAbs to inhibit Wuhan-hu-1 S pseudotyped infection in Huh-7.5 target cells.
The team purified chimeric mAbs from the hybridoma culture supernatants and used a SARS-CoV-2 pseudotype assay to reconfirm their antiviral activity. They also sequenced the human Fab variable regions, VH and VL.
The team cloned VH and VL domains from the six most potent chimeric human-mouse mAbs into a human IgG1 expression vector to generate fully human anti-hACE2 mAbs.
They used single-particle cryo-electron microscopy (cryo-EM) to delineate the structural basis for broad neutralization of anti-hACE2 mAbs.
Specifically, they determined the structure of soluble hACE2 bound to the antigen-binding fragment (Fab) of 05B04, one of the most potent mAbs unaffected by naturally occurring human ACE2 variations.
Finally, the researchers tested these hACE2 mAbs in an animal model and determined their pharmacokinetic behavior.
Results
The researchers identified 82 hybridomas expressing hACE2-binding mAbs, of which they selected ten based on their potency in inhibiting pseudotyped virus infection of Huh-7.5 cells.
These ten mAbs were 1C9H1, 4A12A4, 05B04, 2C12H3, 2F6A6, 2G7A1, 05D06, 05E10, 05G01 and 05H02. Four of the five mAbs from the KP AlivaMab mice, viz., 05B04, 05E10, 05G01, and 05D06, shared identical complementarity-determining regions (CDRs). Conversely, AV AlivaMab mice-derived mAbs were diverse.
While allosteric inhibition of hACE2 activity by the mAbs was theoretically feasible, such inhibition did not occur.
Also, the anti-hACE2 mAbs did not affect hACE2 internalization or recycling, suggesting that the anti-hACE2 mAbs would unlikely undergo accelerated target-dependent clearance from the circulation during in vivo use.
These two findings confirmed that these mAbs would not have harmful side effects based on their target specificity.
In addition, the anti-hACE2 mAbs showed favorable pharmacokinetics and no ill effects on the hACE2 knock-in mice. When used prophylactically in hACE2 knock-in mice, these mAbs conferred near-sterilizing protection against lung SARS-CoV-2 infection.
Moreover, they presented a high genetic barrier to the acquisition of resistance by SARS-CoV-2.
The six anti-hACE2 mAbs also inhibited infection by pseudotyped SARS-CoV-2 variants, Delta, and Omicron, with similar potency, i.e., half maximal inhibitory concentration (IC50) values ranging between 8.2 ng ml−1 and 197 ng ml−1.
A cryo-EM structure of the 05B04-hACE2 complex at 3.3 Å resolution revealed a 05B04 Fab bound to the N-terminal helices of hACE2.
05B04-mediated inhibition of ACE2-binding sarbecoviruses through molecular mimicry of SARS-CoV-2 receptor-binding domain (RBD) interactions, providing high binding affinity to hACE2 despite the smaller binding footprint on hACE2.
None of the four most potent mAbs affected hACE2 enzymatic activity or induced the internalization of hACE2 localized on the host cell surface. Thus, based on their target specificity, these mAbs shall not have deleterious side effects.
Though these anti-ACE2 antibodies could effectively inhibit sarbecovirus infection in humans, the fact that the antibodies target a host receptor molecule rather than the SARS-CoV-2 S protein will necessitate their testing in terms of safety, efficacy, and pharmacological behavior in primate models before human clinical trials.
Conclusions
SARS-CoV-2 might evolve and start using receptors other than ACE2, creating another genetic hurdle to overcome for researchers working on the development of SARS-CoV-2 therapeutics.
However, the human anti-hACE2 mAbs engineered in this study showed exceptional breadth and potency in inhibiting infection by hACE2-utilizing sarbecoviruses.
Thus, they represent a long-term, ‘resistance-proof’ prophylaxis and treatment for SARS-CoV-2, even for future outbreaks of SARS-like coronaviruses.
In addition, these mAbs might prove particularly useful for susceptible patients like those with immunodeficiency and in which vaccine-induced protective immunity is unattainable or difficult to attain.
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