Abstract
The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind to distinct sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies.
Highlights
SARS-CoV-2, the viral causative agent of COVID-1 9, is estimated to have infected some 10% of the world’s population, killing a confirmed ~5 million but likely considerably more
We sought to isolate a large repertoire of highly diverse nanobodies against SARS-CoV-2 spike protein
We built on our existing nanobody generation pipeline (Fridy et al, 2014), further optimizing each step, explicitly designing it to yield hundreds of high-q uality, highly diverse nanobody candidates (Figure 1A)
Summary
SARS-CoV-2, the viral causative agent of COVID-1 9, is estimated to have infected some 10% of the world’s population, killing a confirmed ~5 million but likely considerably more. Nanobodies are derived from the variable domain (VHH) of variant heavy chain-o nly IgGs (HCAb) found in camelids (e.g., llamas, alpacas, and camels) They can bind in modes different from typical antibodies, covering more chemical space and binding with very high affinities (comparable to the very best antibodies) (Jovčevska and Muyldermans, 2020; Muyldermans, 2013). A major advantage of nanobodies is their potential for direct delivery by nebulization deep into the lungs (Wölfel et al, 2020; Nambulli et al, 2021) This route can provide a high local concentration in the airways and lungs to ensure rapid onset of therapeutic effects, while limiting the potential for unwanted systemic effects (Erreni et al, 2020) as exemplified by clinical trials (Van Heeke et al, 2017; Zare et al, 2021). The resulting repertoire provides a plethora of synergistically potent and escape resistant therapeutics
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