PROJECT SUMMARY/ABSTRACT
The outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly
efficacious therapeutics are urgently needed. Camelid VHH antibodies or nanobodies (Nbs) are small, highly
stable, easily bioengineered, and can be rapidly and economically manufactured from microbes. They are
highly robust and are flexible for administration, including possible delivery by nebulization. Together these
unique properties of Nbs make their uses against respiratory viruses such as SARS-CoV-2 especially
appealing.
We recently developed a disruptive proteomic technology for large-scale identification of multi-epitope, drug-
quality Nbs (Xiang et. al, Cell Systems. 2021). Using this technology, we identified > 8,000 high-affinity Nbs for
the SARS-CoV-2 spike (S) receptor-binding domain (RBD) including Nbs that target highly neutralizing
epitopes with sub-pM affinities and can neutralize SARS-CoV-2 at sub-ng/ml concentrations, which are
unprecedented for antiviral antibody fragments. Structural proteomics revealed multiple distinct epitopes and
potential neutralization mechanisms. Bioengineering of multi-epitope and multivalent constructs improved the
potency to below 0.1 ng/ml (Xiang, et. al, Science. 2020). Most recently, we have demonstrated the high
preclinical efficacy of an ultrapotent and stable trimeric Nb construct (PiN-21) for inhalation treatment of SARS-
CoV-2 infection in a sensitive COVID-19 model (Nambulli, et. al, Science Advances. 2021). Intranasal delivery
of PiN-21 at 0.6 mg/kg substantially reduces viral burdens in both airways. Critically, aerosol delivery of PiN-21
at 0.2 mg/kg decreases lung viral titers by 6-logs, minimizing lung pathology post-infection and preventing viral
pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a
convenient and cost-effective option to mitigate the evolving pandemic and future events.
In the revision, we aim to identify and characterize highly potent Nbs that are highly resistant to the variants of
concern (VOCs) of SARS-CoV-2, investigate the neutralization mechanisms by structural approaches, and
develop ultrapotent Nb constructs into safe and effective therapeutics. Our central hypothesis is that Nbs can
be bioengineered into multivalent and ultrapotent forms to resist the mutational escape and the variants of
concerns (VOCs) of SARS-CoV-2.
Completion of our proposed studies will lead to cost-effective and convenient COVID-19 therapeutic
candidates for translation into clinical trials. High-resolution structural studies will provide critical insights into
how Nbs uniquely target the virus for high-affinity binding and neutralization. Critically, this project will serve as
the testbed of our multidisciplinary platform to develop potent therapeutic and diagnostic reagents for future
pandemics caused by coronaviruses or other pathogens.