A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.

Generation and utility of a single-chain fragment variable monoclonal antibody platform against a baculovirus expressed recombinant receptor binding domain of SARS-CoV-2 spike protein.

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.

A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants.

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19.

Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment.

The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.

Rational development of a human antibody cocktail that deploys multiple functions to confer Pan-SARS-CoVs protection.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein.

A phage library displaying 1010 variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD with the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.

Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

Implementing a method for engineering multivalency to substantially enhance binding of clinical trial anti-SARS-CoV-2 antibodies to wildtype spike and variants of concern proteins.

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.

A rapid, point-of-care red blood cell agglutination assay detecting antibodies against SARS-CoV-2.

The COVID-19 pandemic has caused significant morbidity and mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess past infection, evaluate responses to vaccines in development, and determine individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which have not scaled to meet the demand of hundreds of millions of antibody tests so far. Herein, we present an alternative method of antibody testing that depends on one protein reagent being added to patient serum/plasma or whole blood with direct, visual readout. Two novel fusion proteins, RBD-2E8 and B6-CH1-RBD, were designed to bind red blood cells (RBCs) via a single-chain variable fragment (scFv), thereby displaying the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Mixing mammalian-derived RBD-2E8 and B6-CH1-RBD with convalescent COVID-19 patient serum and RBCs led to visible hemagglutination, indicating the presence of antibodies against SARS-CoV-2 RBD. B6-CH1-RBD made in bacteria was not as effective in inducing agglutination, indicating better recognition of RBD epitopes from mammalian cells. Given that our hemagglutination test uses methods routinely used in hospital clinical labs across the world for blood typing, we anticipate the test can be rapidly deployed at minimal cost. We anticipate our hemagglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.

Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library.

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.

Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins.

Coronavirus disease 2019 (COVID-19) is a newly emerged human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a global pandemic, development of a cheap, rapid, accurate, and easy-to-use diagnostic test is necessary if we are to mount an immediate response to this emerging threat. Here, we report the development of a specific lateral flow immunoassay (LFIA)-based biosensor for COVID-19. We used phage display technology to generate four SARS-CoV-2 nucleocapsid protein (NP)-specific single-chain variable fragment-crystallizable fragment (scFv-Fc) fusion antibodies. The scFv-Fc antibodies bind specifically and with high affinity to the SARS-CoV-2 NP antigen, but not to NPs of other coronaviruses. Using these scFv-Fc antibodies, we screened three diagnostic antibody pairs for use on a cellulose nanobead (CNB)-based LFIA platform. The detection limits of the best scFv-Fc antibody pair, 12H1 as the capture probe and 12H8 as the CNB-conjugated detection probe, were 2 ng antigen protein and 2.5 × 104 pfu cultured virus. This LFIA platform detected only SARS-CoV-2 NP, not NPs from MERS-CoV, SARS-CoV, or influenza H1N1. Thus, we have successfully developed a SARS-CoV-2 NP-specific rapid diagnostic test, which is expected to be a simple and rapid diagnostic test for COVID-19.

Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2.

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.

An approach towards development of monoclonal IgY antibodies against SARS CoV-2 spike protein (S) using phage display method: A review.

The present state of diagnostic and therapeutic developmental race for vaccines against the SARS CoV-2 (nCOVID-19) focuses on prevention and control of this global pandemic which also represents a critical challenge to the global health community. Although development of novel vaccines can prevent the SARS CoV-2 infections, it is still impeded by several other factors and therefore novel approaches towards treatment and management of this disease is the urgent need. Passive immunotherapy plays a vital role as a possible alternative to meet this challenge and among various antibody sources, chicken egg yolk antibodies (IgY) can be used as an alternative to mammalian antibodies which have been previously studied against SARS CoV outbreak in China. In this review, we discuss the strategies for the use of chicken egg yolk (IgY) antibodies in the development of rapid diagnosis and immunotherapy against SARS CoV-2. Also, IgY antibodies have previously been used against various respiratory bacterial and viral infections in humans and animals. Compared to mammalian antibodies (IgG), chicken egg yolk antibodies (IgY) have greater binding affinity to specific antigens, ease of extraction and lower production costs, hence possessing remarkable pathogen-neutralizing activity of pathogens in respiratory and lungs. We provide an overall importance for the use of monoclonal chicken egg yolk antibodies (IgY) using phage display method describing their potential passive immunotherapeutic application for the treatment and prevention of SARS CoV-2 infection which is simple, fast and safe way of approach for treating patients effectively.