Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers.

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 × 10-14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.

Identification of an alpha-1 antitrypsin variant with enhanced specificity for factor XIa by phage display, bacterial expression, and combinatorial mutagenesis.

Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2'-P3' was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3', PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis.