Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library

Abstract

Since it was first reported in Wuhan, China, in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic outbreak resulting in a tremendous global threat due to its unprecedented rapid spread and an absence of a prophylactic vaccine or therapeutic drugs treating the virus. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key player in the viral entry into cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein, and the RBD has therefore been crucial as a drug target. In this study, we used phage display to develop human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2. A human synthetic Fab phage display library was panned against the RBD of the SARS-CoV-2 spike protein (SARS-2 RBD), yielding ten unique Fabs with moderate apparent affinities (EC50 = 19–663 nM) for the SARS-2 RBD. All of the Fabs showed no cross-reactivity to the MERS-CoV spike protein, while three Fabs cross-reacted with the SARS-CoV spike protein. Five Fabs showed neutralizing activities in in vitro assays based on the Fabs’ activities antagonizing the interaction between the SARS-2 RBD and ACE2. Reformatting the five Fabs into immunoglobulin Gs (IgGs) greatly increased their apparent affinities (KD = 0.08–1.0 nM), presumably due to the effects of avidity, without compromising their non-aggregating properties and thermal stability. Furthermore, two of the mAbs (D12 and C2) significantly showed neutralizing activities on pseudo-typed and authentic SARS-CoV-2. Given their desirable properties and neutralizing activities, we anticipate that these human anti-SARS-CoV-2 mAbs would be suitable reagents to be further developed as antibody therapeutics to treat COVID-19, as well as for diagnostics and research tools.

Discovery of Antivirals Using Phage Display

“…Phage display has been one of the most powerful drug discovery technologies to lead to the development of FDA-approved peptide and antibody drugs for various diseases (e.g., hereditary angioedema, immune thrombocytopenic purpura, rheumatoid arthritis, and uveitis) [33,45,206] The success of phage display-derived drug molecules as well as the growing interest in peptides/antibodies in the biopharmaceutical market make phage display technology a popular approach for antiviral discovery. In fact, since the beginning of the latest coronavirus outbreak, several research groups have screened phage libraries to develop neutralizing recombinant antibodies against SARS-CoV-2 [83,191,[207][208][209][210][211][212].…”

Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein

“…A previous study has shown the ability of the related SARS-CoV-1 ORF3a N terminal targeting antibodies, derived from convalescent patients' plasma, to induce the destruction of ORF3a-expressing cells by activation of the complement pathway [60]. In addition, Akerstrom et al (2006) have demonstrated that rabbit antisera specific for the N terminus of SARS-CoV-1 ORF3a (representing amino acids [15][16][17][18][19][20][21][22][23][24][25][26][27][28] resulting in a neutralising effect 48 h post infection in virus infected Vero E6 cells [61]. There was no corresponding neutralizing effect using antisera that recognized the C terminal region of SARS-CoV-1 ORF3a in two separate studies [61,62] after 2-4 days of infection.…”

“…Phage display technology provides a robust and well-validated antibody discovery platform and has been used successfully for the isolation of several neutralising SARS-CoV-2 anti-RBD antibodies [19][20][21][22][23][24]. Anti-viral, antibody-based therapy has well-recognized advantages including high specificity and affinity, long serum half-life and desirable effector functions [25].…”

Passive Immunotherapy Against SARS-CoV-2: From Plasma-Based Therapy to Single Potent Antibodies in the Race to Stay Ahead of the Variants

“…These are often termed “immune libraries”; (ii) libraries constructed from B cells derived from “naïve” donors, i.e., subjects who have not been specifically vaccinated, infected, or diseased in a manner that would skew the antibody repertoire. In this case, the retrieved antibodies are usually relatively low affinity and often need to be affinity matured to improve their chances of being therapeutically relevant; and (iii) synthetic human antibody libraries [ 266 – 268 ], which are made to resemble natural antibodies by sequence and/or structure, usually by modelling hundreds of antibodies for which sequence and X-ray crystallographic structure information is available [ 268 ]. In this third case, synthetic libraries can also be made to mimic unusual antibodies such as the pool of anti-viral VH1-69 germline antibodies that rely on CDR-H2 contacts as part of their binding capacity [ 270 – 272 ], or anti-viral antibodies with long CDR-H3s [ 262 ], another “phenotype” of antibody associated with some anti-viral neutralizing antibodies [ 274 – 276 ].…”