Development of high affinity monobodies recognizing SARS-CoV-2 antigen

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a threat to global public health. Prompt patient identification and quarantine is the most effective way to control its rapid transmission, which can be facilitated by early detection of viral antigens. Here we present a platform to develop and optimize the fibronectin-based affinity-enhanced antibody mimetics (monobodies) for recognizing viral antigens. Specifically, we developed monobodies targeting SARS-CoV-2 nucleocapsid (N) protein. We showed that two monobodies, NN2 and NC2, bind to N protein’s N- and C-terminal domains respectively with a Kd in nM range.The specificity of the recognition was confirmed with co-immunoprecipitation and immunofluorescence assays. Furthermore, we demonstrated that one round of in vitro maturation using mRNA display can improve the binding affinity of monobodies. Machine learning algorithms were integrated with deep sequencing data for selecting candidates that improve the detection sensitivity of N. Using this pair of monobodies, we have developed an enzyme-linked immunosorbent assay (ELISA) for viral detection. We were able to detect recombinant N at 4 pg/ml and detect N in viral culture supernatant, with no cross-reactivity with other CoV. Integrating high-dense mutagenesis, mRNA display, deep sequencing and machine learning, this platform can be applied through iterations to identify and optimize monobodies against emerging viral antigens, potentiating point-of-care detection of communicable diseases in a cost-and time-sensitive manner.Authors Yushen Du, Tian-hao Zhang, Xiangzhi Meng, Yuan Shi, and Menglong Hu contributed equally to this work.

Intracellular Degradation of SARS-CoV-2 N-Protein Caused by Modular Nanotransporters Containing Anti-N-Protein Monobody and a Sequence That Recruits the Keap1 E3 Ligase

“…The target protein was the nucleocapsid protein (N-protein) of the SARS-CoV-2 virus. The binding of the developed MNTs to this protein was carried out by another antibody mimetic, monobody NC2 [ 35 ]. According to the literature data, this monobody is characterized by the highest affinity for the N-protein, when compared to similar monobodies (equilibrium dissociation constant of the NC2 complex with the N-protein equals 6.7 nM) [ 35 ].…”

“…The binding of the developed MNTs to this protein was carried out by another antibody mimetic, monobody NC2 [ 35 ]. According to the literature data, this monobody is characterized by the highest affinity for the N-protein, when compared to similar monobodies (equilibrium dissociation constant of the NC2 complex with the N-protein equals 6.7 nM) [ 35 ]. The proposed MNTs can not only bind to the N-protein but also provide its degradation due to the presence of an amino acid sequence recruiting the Keap1 E3 ligase.…”

“…The NC2 monobody within MNT 1 and the free monobody possess different affinities for the N-protein. Indeed, according to the literature, the equilibrium dissociation constant of the NC2 monobody complex with the N-protein of the SARS-CoV-2 virus is 6.7 nM [ 35 ], whereas within the MNT 1, its constant is 47.2 ± 2.5 nM. If MNT 1 is subjected to cleavage by cathepsin B, the resulting dissociation constant hits 17.2 ± 4.4 nM.…”