Broadly neutralizing SARS-CoV-2 peptidic knobs

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Abstract Targeted therapeutic agents range in size from very small organic molecules (100’s of Da) to protein-based molecules like monoclonal antibodies (~150,000 Da). Small disulfide bonded peptides have evolved in many species, including plants and animals, to have ideal pharmacological properties including high affinity target binding, stability to proteases, heat and other stresses. Such peptides include “cyclotides” or “knottins” which can inhibit enzymes, ion channels, and GPCRs with high potency and are often the major active component of venoms of many predator organisms. We have uncovered convergent evolution between ultralong third complementary determining regions (CDR H3s) in the heavy chain of an unusual class of cow antibodies and cyclotide/knottin peptides. We can produce these “knob” peptides in microbial systems and they retain the binding and potency properties of the parent antibody. These tiny peptide-based molecules are small (~4-6 kDa), highly stable, and can bind targets at subnanomolar KD. We have already developed a panel of virus neutralizing knob peptides against SARS-CoV-2 which bind unique epitopes, and some of which maintain high affinity binding to various SARS-CoV-2 variants, including the recent ‘delta’ and ‘omicron’ strains. The high stability, potency, and straightforward manufacturing path enables multiple routes of administration, potentially including inhaled or intranasal delivery, which could be very important prophylactic or treatment in the current or future coronavirus pandemic. Our goals in this project are to further develop the technology to identify peptidic knob domains, expand our panel of knobs against SARS-CoV-2 variants and other coronaviruses like MERS-CoV and SARS-CoV- 1, understand the structural basis of their binding, and validate their activity in vitro and in vivo. The knobs identified here can potentially be used as monotherapy or combination therapy in the current or a new coronavirus pandemic, and will be a valuable new therapeutic class to add to the arsenal against coronavirus disease.