Researchers in China, The Netherlands, France, and Switzerland have identified synthetic nanobodies or “sybodies” that exhibit potent neutralizing activity against severe acute respiratory syndrome coronavirus (SARS-CoV-2) – the agent responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic.
Structural and biophysical studies revealed that the sybodies stopped the receptor-binding domain (RBD) of the viral spike protein interacting with the human host cell receptor angiotensin-converting enzyme 2 (ACE2).
As recently reported in the journal Nature Communications, the sybody MR3 exhibited the highest binding affinity for the spike RBD and the most potent neutralizing activity against SARS-CoV-2 pseudoviruses.
The team also showed that prophylactic administration of this potent MR3 construct reduced viral load and protected against lung damage in hamsters infected with the virus.
“Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak,” writes Dianfan Li from the Chinese Academy of Sciences in Shanghai and colleagues.
More about the nanobodies as potential therapeutics
The initial stage of the SARS-CoV-2 infection process is mediated by the viral spike protein when its RBD binds to the human host cell receptor ACE2.
Therefore, the spike RBD has been a primary target for the development of neutralizing antibodies that prevent this ACE2 binding.
Llama-derived single-domain antibodies are nanobodies of around 15 kilodaltons that offer advantages over conventional antibodies in terms of their bioavailability, ease of production, and amenability to protein engineering.
Furthermore, “as single-chain antibodies, nanobody libraries are less complex to construct and screen, enabling in vitro selection of high-affinity binders in a relatively short time, typically 2–4 weeks,” writes Li and colleagues.
Over recent weeks, several independent research groups have reported the generation of nanobodies that neutralize SARS-CoV-2.
“However, the in vivo efficacy of such nanobodies remains to be investigated,” says the team.
What did the researchers do?
Li and colleagues selected 99 (sybodies) against the spike RBD by in vitro selection using three high-diversity nanobody libraries. About half of the sybodies exhibited neutralizing activity against SARS-CoV-2.
“The in vitro platform was efficient in generating neutralizing sybodies; the selection process took two weeks,” says the team.
Structural and biochemical studies showed that three of the sybodies – SR4, MR17, and MR3 – all neutralized SARS-CoV-2 by blocking the RBD–ACE2 interaction.
All three sybodies formed high-affinity complexes with the spike RBD, with MR3 exhibiting the greatest affinity (KD=1.0nM).
Neutralization assays also revealed that MR3 exhibited the most potent neutralizing activity against SARS-CoV-2 pseudoviruses.
Increasing potency by increasing valency
Since increasing valency is a common technique used to enhance the potency of nanobodies, the researchers engineered three types of divalent sybodies, including the biparatopic fusion of two different sybodies and the Fc-fusion and tandem fusion of the same sybody.
Compared with the monovalent MR3, the divalent MR3-MR3 increased the potency of this sybody by more than 40-fold.
Investigating potency of the sybodies in vivo
To investigate in vivo protection against SARS-CoV-2, the team conducted an experiment using hamsters, which are known to develop severe symptoms following infection with the virus.
Since nanobodies have very short serum half-lives owing to their minute size, the team fused MR3-MR3 to the N-terminus of an albumin-binding domain (ABD), which is known to extend the circulating half-life of its fusion partners.
In addition to MR3-MR3-ABD, Fc-MR3 was also used, along with the non-neutralizing sybodies Sb92-Sb44-ABD and Fc-Sb2 for the ABD and Fc-fusion, respectively, as controls.
Compared with uninfected hamsters, challenge with SARS-CoV-2 live virus caused severe weight loss – of around 20% – by day four following infection.
The team reports that intraperitoneal administration of MR3-MR3-ABD six hours prior to infection reduced weight loss among these hamsters by around 50%.
Furthermore, although the Fc-MR3 hamsters lost weight during the first 2 days following infection, they regained weights that almost reached those of the animals in the noninfected group. The non-neutralizing sybody controls did not show any protection against weight loss among the animals.
What about viral load and lung damage?
The viral RNA load in the lungs was reduced by approximately 7-fold among the hamsters that received MR3-MR3-ABD, compared with those that received Sb92-Sb44-ABD.
Consistent with the weight loss results, the administration of Fc-MR3 reduced the viral RNA load by a dramatic 6 Log10, falling to the detection limit.
Finally, histopathologic analysis confirmed that MR3-MR3-ABD provided modest protection against lung damage, while Fc-MR3 provided almost full protection.
A preliminary basis for nanobody therapeutics to fight COVID-19
The team says the results form a preliminary basis for the development of nanobody therapeutics for COVID-19.
“Using the most potent construct, we have demonstrated that nanobodies can provide prophylactic protection of hamsters from SARS-CoV-2 infection,” says Li and colleagues.
“Our results should encourage the development of nanobody therapeutics to fight COVID-19 or future viral outbreaks,” they conclude.
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