Therapeutic Targeting of SARS-CoV-2

Created by: Michael D'Ercole, Onela Esho, Jasmine Leung, Meet Patel, and Amandeep Nagi

Pinto, et al. identify and describe both the binding properties and kinetics of neutralizing antibodies that target the S glycoprotein of SARS-CoV-2. The antibodies were identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003.

Senior PI's:

• David Veesler, PhD., Associate Professor of Biochemistry at the University of Washington School of Medicine.
• Davide Corti, PhD., Senior Vice President of Antibody Research at Vir.

The research published was conducted by Vir’s subsidiary Humabs BioMed SA in Switzerland in collaboration with the Institute for Research in Biomedicine (Pinto et al., 2020).

Vir Biotechnology is a clinical-stage immunology company focused on combining immunologic insights with cutting-edge technologies to treat and prevent serious infectious diseases (Vir Biotechnology, 2021). Vir has assembled four technology platforms that are designed to stimulate and enhance the immune system. Its current development pipeline consists of product candidates designed to target hepatitis B virus, influenza A, SARS-CoV-2, human immunodeficiency virus, and tuberculosis.

Vir, in collaboration with GlaxoSmithKline plc, is conducting research on COVID-19 monoclonal antibodies based on the S309 antibody identified in this study. It’s candidates include VIR-7831 and VIR-7832.

• VIR-7831 - A monoclonal antibody that has demonstrated the ability to neutralize SARS-CoV-2 in vitro. The antibody binds to an epitope on SARS-CoV-2 that is shared with SARS-CoV, indicating that the epitope is highly conserved, which may make it more difficult for escape mutants to develop. VIR-7831 has been engineered to enhance lung bioavailability and have an extended half-life (Vir Biotechnology, 2021).
  • Escape mutants are viruses in which “escape” mutations arise as a result of selection pressure to survive. These mutations allow the viral mutants to survive and replicate. In the case of antibodies, the mutations can be a change in the antigen which can simply prevent the viral mutants from being recognized by the antibodies.

• VIR-7832 - A monoclonal antibody that has demonstrated the ability to neutralize SARS-CoV-2 in vitro. The antibody binds to an epitope on SARS-CoV-2 that is shared with SARS-CoV, indicating that the epitope is highly conserved, which may make it more difficult for escape mutants to develop. VIR-7832 has been engineered to enhance lung bioavailability, have an extended half-life and to potentially function as a therapeutic and/or prophylactic T cell vaccine (Vir Biotechnology, 2021).

Figure 1: Vir Biotechnology's COVID-19 monoclonal antibody candidates (Vir Biotechnology, 2021).

Prior to this study, the therapeutic use of monoclonal antibodies (mAbs) had shown success in treating infectious viral diseases such as Ebola. In addition, several studies had identified potently neutralizing human mAbs from the memory B cells of individuals infected with SARS-CoV or Middle East respiratory syndrome coronavirus (MERS-CoV) (Pinto et al., 2020). However, none of these mAbs neutralized SARS-CoV-2.

Figure 2: Memory B cell screening (Created using BioRender, adapted from Traggiai, 2012).

A 2004 study by Traggiai et al. previously identified 35 neutralizing monoclonal antibodies (mAbs) from the peripheral blood of a patient who had contracted SARS-CoV in 2003 (Traggiai et al., 2004). This new study by Pinto et al. builds upon these findings by identifying mAbs from the same patient with cross-reactivity to SARS-CoV-2. A memory B cell (MBC) screening was first conducted to identify and analyze a set of candidate mAbs. The screening was performed using peripheral blood mononuclear cells isolated from a 2013 blood sample (Pinto et al., 2020).

MBCs are a form of B lymphocyte or white blood cell. Upon exposure to a novel pathogen, B and T cells are activated and elicit a primary immune response. MBCs are later formed from naïve B cells within the germinal centers of secondary lymphoid organs (Gatto & Brink, 2010). MBCs persist after infection in a quiescent state and can elicit a specific secondary immune response, should an infection occur by the same pathogen (Weisel & Shlomchik, 2017). MBC screening first requires the isolation of MBCs from peripheral blood using magnetic and fluorescence-activated cell sorting. The MBCs are then immortalized using Epstein-Barr virus in combination with irradiated mononuclear cells and a CpG oligonucleotide which induces MBC proliferation (Traggiai et al., 2004; Bernasconi et al., 2002). The supernatant of immortalized MBC cultures can then be analyzed for the presence of specific antibodies via immunostaining (Traggiai et al., 2004). Pinto et al. evaluated a panel of 25 mAbs, 19 of which were identified in the screen from 2004, and 6 recombinant IgG-LS antibodies. (Pinto et al., 2020).

A staining assay was conducted using Chinese hamster ovary cells expressing either the SARS-CoV or SARS-CoV-2 S glycoprotein. The spike-transfected cells were stained with the purified mAbs and an Alexa-647-labelled secondary antibody anti-human IgG Fc. Binding was analyzed via flow cytometry and positive binding was identified by comparing staining against mock-transfected cells (Pinto et al., 2020; Traggiai et al., 2004).

Figure 3: Binding of a panel of 10 mAbs to Chinese hamster ovary cells expressing the SARS-CoV-2 (a) or SARS-CoV (b) S glycoprotein (Pinto et al., 2020).

Out of the 25 mAbs tested, 8 mAbs bound to either the SARS-CoV or SARS-CoV-2 S glycoprotein with relatively high potency. These eight mAbs are listed below (Pinto et al., 2020):

• S309
• S303
• S304
• S306
• S310
• S315
• S110
• S109

An indirect enzyme-linked immunosorbent assay (ELISA) was used to evaluate binding of the mAbs to the following coronavirus spike proteins and spike protein domains (Pinto et al., 2020):

• SARS-CoV S^B domain (RBD)
• SARS-CoV-2 S^B domain (RBD)
• HCoV-OC43 S glycoprotein prefusion-stabilized ectodomain trimer
• MERS-CoV S glycoprotein prefusion-stabilized ectodomain trimer
• SARS-CoV S glycoprotein prefusion-stabilized ectodomain trimer
• SARS-CoV-2 S glycoprotein prefusion-stabilized ectodomain trimer

ELISA is a form of labelled immunoassay used to detect and quantify proteins such as antibodies in a biological sample. ELISA is commonly performed using 96 or 384 well microtiter plates, and thus, is an efficient technique for detecting binding between panels of antigens and antibodies (Thermo Fisher Scientific, n.d.).

Figure 4: Indirect ELISA (Created using BioRender).

1. Antigens are immobilized to the surface of wells in a microtiter plate (Thermo Fisher Scientific, n.d.).
2. A solution containing a non-reacting protein such as Bovine serum albumin is added to block areas on the well surface not covered by the antigen (Pinto et al., 2020; Thermo Fisher Scientific, n.d.). This step of ELISA is known as plate or surface blocking and is done to prevent non-specific binding. This increases the sensitivity of the assay and decreases background signals (Thermo Fisher Scientific, n.d.).
3. The antibodies of interest (primary antibodies) are added to the wells and the plate is incubated (Pinto et al., 2020; Thermo Fisher Scientific, n.d.).
4. A buffer is used to wash away any non-bound primary antibodies (Thermo Fisher Scientific, n.d.).
5. The detection of bound primary antibodies can be done using either a direct or indirect approach (Thermo Fisher Scientific, n.d.).
   • Direct detection uses a primary antibody that is conjugated to a detectable enzyme or fluorophore (Thermo Fisher Scientific, n.d.).
   • Indirect detection uses an enzyme or fluorophore-conjugated secondary antibody that binds with specificity to the primary antibody (Thermo Fisher Scientific, n.d.).
6. If indirect detection is used, a secondary wash is performed to remove any non-bound secondary antibodies (Thermo Fisher Scientific, n.d.).
7. A substrate is added, and it is converted to a detectable signal by the enzyme conjugated to the primary or secondary antibody (Thermo Fisher Scientific, n.d.).
8. The optical density of each well is measured using a plate reader. The optical density is proportional to the quantity of primary antibodies bound to antigens (Pinto et al., 2020; Thermo Fisher Scientific, n.d.).

Figure 5: Binding of a panel of 10 mAbs to the S glycoprotein ectodomain trimer or SB domain of SARS-CoV, SARS-CoV-2, MERS-CoV or HCoV-OC43 (Pinto et al., 2020).

While none of the tested mAbs bound to either the MERS-CoV or HCoV-OC43 S glycoprotein, four of the mAbs (S309, S315, S304 and S303) bound to the S^B domain (RBD) of both the SARS-CoV and SARS-CoV-2 S glycoprotein. Interestingly, these results coincide with the classification of these coronavirus strains. While SARS-CoV and SARS-CoV-2 are members of the subgenus Sarbecovirus, MERS-CoV and HCoV-OC43 are not (Pinto et al., 2020).

Figure 6: Biolayer interferometry interference pattern shift (Created using BioRender, adapted from 2Bind molecular interactions, 2020).

Biolayer interferometry (BLI) was then used to measure the affinity and avidity of S309, S315, S304 and S303 for the S^B domain (RBD) of SARS-CoV and SARS-CoV-2 (Pinto et al., 2020). BLI is a label-free technique used for analyzing biomolecular interactions such as the binding of antibodies to antigens. BLI is an optical technique that measures a shift in the interference pattern of white light reflected from two surfaces on the tip of a biosensor. The first reflection surface is engineered into the biosensor itself while the second reflection surface is a layer of biomolecules immobilized to the exterior surface of the biosensor (Creative Biolabs, n.d.). For this study, mAbs comprised the second reflection surface (Pinto et al., 2020). The binding of biomolecules to the second reflection surface results in a shift in the interference pattern of reflected light. The size of this shift is proportional to the number of biomolecules bound to the second reflection surface (Creative Biolabs, n.d.). Shifts in the interference pattern are measured in real-time, allowing for the generation of association and dissociation curves (2Bind molecular interactions, 2020).

Figure 7: Generation of real-time association and dissociation curves using biolayer interferometry (Created using BioRender, adapted from 2Bind molecular interactions, 2020).

1. The biosensor is put into a blank buffer (2Bind molecular interactions, 2020).
2. Antibodies are immobilized to the exterior surface of the biosensor (2Bind molecular interactions, 2020).
3. A baseline interference pattern is recorded (2Bind molecular interactions, 2020).
4. The biosensor is put into a solution containing the antigen (S^B domain). Shifts in the interference pattern are measured in real-time and an association curve is generated. The association curve is used to calculate the association rate constant (K_on ) (2Bind molecular interactions, 2020).
5. The biosensor is put into a blank buffer and the bound antigens dissociate. Shifts in the interference pattern are measured in real-time and a dissociation curve is generated. The dissociation curve is used to calculate the dissociation rate constant (K_off) (2Bind molecular interactions, 2020).
6. K_off and K_on are used to calculate the equilibrium dissociation constant (K_D) (2Bind molecular interactions, 2020).

Figure 8: Affinity and avidity of S309, S303, S304 and S315 for the SB domain (RBD) of SARS-CoV-2 (Pinto et al., 2020).

BLI was conducted using different concentrations of the S^B domain (RBD) of the SARS-CoV and SARS-CoV-2 S glycoproteins. All four mAbs bound to the SARS-CoV and SARS-CoV-2 S^B domains (RBDs) with nano- to sub-picomolar affinities. S309 in particular bound to the SARS-CoV-2 S^B domain (RBD) with the greatest affinity (Pinto et al., 2020).

Figure 9: Pseudovirus neutralization assay (Created using BioRender, adapted from Berthold, 2020).

The neutralization potency of S309, S315, S304 and S303 was evaluated using a pseudovirus neutralization assay (Pinto et al., 2020). A pseudovirus is a retrovirus that has been engineered to express the envelope protein of another virus, such as the spike protein of a coronavirus. Pseudoviruses are unable to replicate and therefore offer a safer alternative to working with highly pathogenic natural viruses such as SARS-CoV and SARS-CoV-2 (Berthold, 2020). Pinto et al. used a murine leukemia virus (MLV) pseudotyping system to generate the pseudoviruses SARS-CoV-MLV and SARS-CoV-2-MLV (Pinto et al., 2020). Additionally, the pseudovirus genome contains the gene for luciferase, an enzyme that emits light via bioluminescence. Luciferase is only expressed when the pseudovirus enters a cell, and thus, the intensity of light is inversely proportional to the neutralization potency of the mAbs being tested (Berthold, 2020).

Figure 10: Neutralization of SARS-CoV-MLV and SARS-CoV-2-MLV by S309, S315, S304, S303, S310 and S306 (Pinto et al., 2020).

• S309 potently neutralized SARS-CoV-MLV and SARS-CoV-2 MLV (Pinto et al., 2020).
• S315 and S304 weakly neutralized SARS-CoV-MLV and SARS-CoV-2-MLV (Pinto et al., 2020).
• S303 only neutralized SARS-CoV-MLV (Pinto et al., 2020).

To study the mechanisms of S309-mediated neutralization, the complex between the S309 Fab fragment and a prefusion stabilized ectodomain trimer of SARS-CoV-2 S glycoprotein is characterized using single-particle Cryo-electron microscopy (Pinto et al., 2020).

Cryo-electron microscopy (cryo-EM) is a structural molecular and cellular biology technique that enables structure determination of macromolecular objects and their assemblies (Carroni & Saibil, 2016).

Figure 11: Cryo-electron microscopy Work Flow (Chung & Kim, 2017).

1. A purified sample is applied to the grid and then vitrified with liquid ethane (Chung & Kim, 2017).
2. Particles embedded in the thin ice will have various random orientations, which are imaged by transmission electron microscopy (TEM) followed by motion correction and contrast transfer function (CTF) determination/correction (Chung & Kim, 2017).
3. Individual particles are selected and aligned for two-dimensional (2D) class average (Chung & Kim, 2017).
4. Three-dimensional (3D) classification and further iterative refinement of 3D reconstruction will finally provide the high-resolution cryo-EM structure (Chung & Kim, 2017).

Figure 12: Cryo-EM structures of the SARS-CoV-2 S glycoprotein in complex with the S309 neutralizing mAb Fab fragment (Pinto et al., 2020).

• Figure 12a: Ribbon diagram of the partially open SARS-CoV-2 S-glycoprotein trimer (one S^B domain is open) bound to three S309 Fabs (Pinto et al., 2020).
• Figure 12b: Ribbon diagrams of the closed SARS-CoV-2 S-glycoprotein trimer bound to three S309 Fabs (Pinto et al., 2020).
• Figure 12c: Ribbon diagrams of the Upper view of the closed SARS-CoV-2 S-glycoprotein trimer bound to three S309 Fabs (Pinto et al., 2020).

• S309 recognizes a proteoglycan epitope on the SARS-CoV-2 S^B, distinct from the receptor-binding motif (Pinto et al., 2020).
• The epitope is accessible in both the open and closed states of the S glycoprotein, which explains the stoichiometric binding of Fab to the trimer of the S glycoprotein (Pinto et al., 2020).

To better understand the interaction of S309 and the proteoglycan epitope of the SARS-CoV-2 S^B, a further close-up view of the Cryo-structure is examined (Pinto et al., 2020).

Figure 13: Close-up view of the S309 epitope (Pinto et al., 2020).

• Figure 13d: Show the contacts formed with the core fucose (labelled with * sign) and the rest of the glycan at proximal glycosylation N343 site (Pinto et al., 2020).
• Figure 13e: Showing the CDRH3 sitting atop the S^B helix that comprises residues 337–344 (Pinto et al., 2020).

• Figure 13d shows the contacts formed between the core fucose on the S309 antibody and the rest of the glycan at position N343 (glycosylation sites on the SARS-CoV-2 S^B domain epitope (Pinto et al., 2020).
• Core fucosylation is an important modification of the N-glycan core structure, which is one of the most common modifications involving oligosaccharides on glycoproteins (Fernández-Quintero et al., 2019).
• It has been known as an important key for the structural stability and function of N-glycoproteins (Fernández-Quintero et al., 2019).

Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding (Polonelli et al., 2008). The S309 paratope is composed of all 6 CDR loops, which bury a surface area of about 1,150 Ų at the interface with S^B through electrostatic interactions and hydrophobic contacts.

• CDRH3: contacts the edge of the S^B 5-stranded β-sheet (residues 356–361), overall accounting for about 50% of the buried surface area and sits atop the S^B helix that comprises residues 337–344; CDRH3 loop is known to play a central role in antigen recognition & binding and has on average the highest counts of contacts with antigens (Hwang et al., 2020).
• CDRL1 & CDRL2: extend the epitope by interacting with the helix that spans residues 440–444 (Pinto et al., 2020).
• CDRH3 & CDRL2: sandwich the glycan of the SARS-CoV-2 S glycoprotein at position N343, through contacts with the core fucose moiety (Pinto et al., 2020).

These interactions between S309 and the glycan bury an average surface of about 300 Ų and ultimately stabilize the N343 oligosaccharide (Pinto et al., 2020).

• Both figure 13d and 13e shows selected residues involved in interactions between S309 and SARS-CoV-2 S glycoprotein (Pinto et al., 2020).
• This helps us to study the structural conservation of the S309 epitope comparing to other zoonotic sarbecoviruses (Pinto et al., 2020).

Figure 14: SARS-CoV-2 S glycoprotein trimer that shows the S309 epitope on one promoter colored as residue conservation (dark blue color) (Pinto et al., 2020).

• Figure 14f: Molecular surface representation of the SARS-CoV-2 S-glycoprotein trimer with S309 epitope on one promoter colored by residue conservation among SARS-CoV-2 and SARS-CoV S glycoprotein. The other two promoters are colored pink and gold.

• The structural data explain the S309 cross-reactivity between SARS-CoV-2 and SARS-CoV, as 17 out of 22 residues of the epitope are strictly conserved (Pinto et al., 2020).

Figure 15: Alignment of the full RBD of multiple sarbecoviruses.Dashed boxes indicate glycosylation sites and orange asterisks indicate the N343 glycosylation site. (Pinto et al., 2020).

• 17 out of 22 residues of the epitope are strictly conserved (Pinto et al., 2020).
• 4 residues are conservatively substituted (Pinto et al., 2020).

• The 1 residue left is semi-conservatively substituted (Pinto et al., 2020).

The oligosaccharide at position N343 is also conserved in both viruses and corresponds to SARS-CoV N330, for which core-fucosylated glycopeptides were previously detected by mass spectrometry that would allow for similar interactions with the S309 Fab (Pinto et al., 2020).

• S309 recognizes a proteoglycan epitope on the SARS-CoV-2 S^B domain which explains the binding of S309 Fab to the S glycoprotein trimer (Pinto et al., 2020).
• Core flucosylation was observed in the structural model, which is important for the structural stability and the proper functioning of N-glycoprotein - in this case, N343 in S glycoprotein (Pinto et al., 2020).
• 6 CDR loops was observed in the S309 epitope and CDRH3 was found to be sitting atop of the S^B helix that comprises residues 337–344, which is important for antigen recognition and binding (Pinto et al., 2020).
• Residues of the epitope is strictly conserved (Pinto et al., 2020).
• The degree of conservation is consistent with the moderate rates of evolution of SARS-CoV-2 (Pinto et al., 2020).
• Collectively, structural data suggest that S309 could neutralize potentially all SARS-CoV-2 isolates known to be circulating to date, and possibly many other zoonotic sarbecoviruses (Pinto et al., 2020).

Figure 16: Ribbon diagrams of S309 and ACE2 bound to SARS-CoV-2 SB. Generated using the SARS-CoV-2 S glycoprotein–S309 cryo-EM structure, and a crystal structure of SARS-CoV-2 SB bound to ACE2 (Pinto et al., 2020).

Following the characterization of the complex between the S309 Fab fragment and a prefusion stabilized ectodomain trimer of SARS-CoV-2 S glycoprotein, the study focuses on analyzing the specific mechanisms of S309-mediated neutralization. Figure 16 shows a composite model generated using the cryo-EM structure of S309 bound to SARS-CoV-2 S glycoprotein (Pinto et al., 2020), and a previously published crystal structure of SARS-CoV-2 S^B bound to ACE2 (Lan et al., 2020), indicates that the Fab fragment engages an epitope distinct from the receptor-binding motif (ACE2), and the physical structure of Fab fragment does not interfere or clash with ACE2 (Pinto et al., 2020).

Figure 17: Results from biolayer interferometry showing the competition of S309 or S230 mAbs with ACE2 to bind to SARS-CoV SB (A) and SARS-CoV-2 SB (B)(Pinto et al., 2020).

To further examine the context of this finding, the researchers then performed biolayer interferometry analysis of S309 Fab to the SARS-CoV-2 S^B domain or the ectodomain trimer of S glycoprotein. The results, as seen in figure 17, show association curves of the competition of S309 or S230 mAbs with ACE2 to bind to SARS-CoV S^B (Figure 17a) and SARS-CoV-2 S^B (Figure 17b) (Pinto et al., 2020). In both graphs, the x-axis follows the duration of the experiment, and the y-axis plots the shifts in the interference pattern (in nm) representing the association of mAb-complexed or free S^B to solid-phase ACE2 (Shah et al., 2014). The vertical dashed line indicates the start of these associations. The researchers indicate each mAb was tested in at least two experiments with similar results (Pinto et al., 2020).

It is evident although free SARS-CoV and free SARS-CoV-2 S^B domains are able to bind to the solid phase-phase ACE2, the association between S309 complexed S^B domain and ACE2 biolayer is more significant for both experiments. The association between S309 and S^B domain does not interfere with ACE2 association, essentially confirming the absence of competition between S309 and ACE2 for binding to the SARS-CoV-2 S glycoprotein. In figure 17a, it is also evident S309 outcompetes S230 antibody, and the graph serves as an example of the expected shift in the association curve in the presence of competitive binding (Pinto et al., 2020).

Figure 18: Transfection steps in the S309 Fab and S309 IgG mediated neutralization comparison assay (Created using BioRender).

To further investigate the mechanism of S309-mediated neutralization, the researchers analyzed the neutralization of SARS-CoV–MLV by S309 recombinant ®IgG1, in comparison with S309 Fab mediated neutralization. The use of fundamental tissue culturing and transfection techniques were used, as summarized in figure 18, to analyze and compare the neutralizing capacity of S309 Fab with S309 rIgG1. The MLV-based SARS-CoV S-glycoprotein-pseudotyped viruses were essentially prepared by co-transfecting HEK293T cells with a SARS-CoV or SARS-CoV-2 S-glycoprotien-encoding-plasmid, and the MLV transfer vector encoding a luciferase reporter (Pinto et al., 2020).

Figure 18 summarizes the neutralization assay conducted for this experiment. VeroE6 cell line was used, where the cells were transfected with human ACE2 and cultured in antibiotic (1% penicillin–streptomycin) supplemented media to ensure successful transfection occurred. Then, the prepared concentrated pseudovirus with or without antibodies was added, and the cells were further incubated in antibiotic supplemented media. The plates were then measured for luciferase signals (Pinto et al., 2020). In a pseudovirus luciferase assay, the inhibition of viral entry into cells by the antibody is correlated to the decreased levels of luciferase signals in the cells (Nie et al., 2020). The researchers indicate that measurements were done in duplicate and relative luciferase units were converted to percent neutralization, as evident in figure 19 (Pinto et al., 2020).

Figure 19: Neutralization of SARS-CoV–MLV by S309 recombinant (r)IgG1 or S309 Fab, plotted in nM (Pinto et al., 2020).

As evident by the results in figure 19, both experiments yielded comparable IC50 values (3.8nM for IgG and 3.5 nM for Fab), which indicates that the potencies for IgG and Fab are similar. However, it is also evident that S309 IgG-mediated neutralization reached 100%, whereas S309 Fab mediated neutralization plateaued at around 80%, which indicates that there may be one or more IgG-specific bivalent mechanisms that may possibly contribute to the ability of S309 to fully neutralize pseudovirions (Pinto et al., 2020). Some of these mechanisms may include (Pinto et al., 2020):

• S-glycoprotein trimer cross-linking
• Steric hindrance
• Aggregation of virions

Figure 20: Steps in antibody-dependent cell cytotoxicity (ADCC) mediated by natural killer cells (Created using BioRender).

To further analyze the S309 mediated neutralization mechanism, the researchers shifted focus to investigate Fc-dependent effector mechanisms. Antibody-dependent cell cytotoxicity (ADCC) mediated by natural killer cells, is a mechanism that can contribute to viral control in individuals infected with the virus (Pinto et al., 2020). After an antibody selectively coats the target cell, effector immune cells, like natural killer (NK) cells, recognize and bind to the Fc fragment of the antibody, activating the effector cells to lyse the target eventually (Parekh et al., 2012). This mechanism has been adopted to create in-vitro cell-killing assays to ascertain the efficacy of therapeutic monoclonal antibodies (InvivoGen, n.d.). Figure 20 summarizes the core principles behind the ADCC assay protocols conducted in this study. As evident in the figure, cross-linking of CD16 Fc receptors (Fc gamma RIIIa) on the effector cell, bound to the antibody Fc fragment, triggers downstream signaling leading to degranulation into lytic synapse, which results in apoptosis of target cell (Weiner et al., 2010).

Figure 21: Comparison between antibody-dependent cellular cytotoxicity mediated by natural killer cells, and antibody-dependent cellular phagocytosis (Created using BioRender, adapted from Weiner et al., 2010).

However, the researchers explored another Fc-dependent effector mechanism, one which relies on macrophages as the effector cells. Similar to antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis (ADCP) relies on effector cells to interact with the Fc fragment of the bound antibody. ADCP, mediated by macrophages or dendritic cells, can contribute to viral control by clearing viruses and infected cells, and by stimulating a T cell response through the presentation of viral antigens (Pinto et al., 2020). This mechanism is summarized in figure 21, which shows that CD32a Fc receptors (Fc gamma RIIa) on effector cells like phagocytes (peripheral blood mononuclear cells used in ADCP assay for this study), engage with the Fc fragment to trigger a signaling cascade leading to the engulfment of the target cell (InvivoGen, n.d.).

Figure 22: Mechanism for LDH release assay, highlighting the colorimetric method for cytotoxicity detection (Created using BioRender, adapted from AG Scientific, n.d.).

This rate of apoptosis of ADCC was measured using LDH release assay, which is summarized in figure 22 (AG Science, n.d.). The LDH release assay is a cell death assay used to assess the level of plasma membrane damage in a cell population. Lactate dehydrogenase (LDH) is a stable enzyme, present in all cell types, which is rapidly released into the cell culture medium upon damage of the plasma membrane (Lobner et al., 2000). And through the electron transfer chain of reactions presented in figure 22, the production of Formazan (a dye used as a colorimetric indicator of enzymatic activity) can be directly measured (AG Science, n.d.). Formazan production is converted to ADCC percentage on the y-axis of the resulting curve, as seen in figure 23a. ADCP assays were performed using expiCHO target cells transiently transfected with SARS-CoV-2 S glycoprotein and were tagged with a fluorescent label. Antibody-mediated phagocytosis was determined by flow cytometry. The results, as seen in figure 23b, present the y-axis as ADCP percentage (Pinto et al., 2020).

Figure 23: The curves related to figure 23. a, ADCC for one donor who is homozygous for high-affinity variant Fc gamma RIIIa 158V (VV). b, ADCP for one donor who is heterozygous for Fc gamma RIIIa 158V (FV) (Pinto et al., 2020).

Figure 23a shows efficient S309 and S306 mediated ADCC of SARS-CoV-2 S-glycoprotein-transfected cells, whereas other monoclonal antibodies show limited or no activity. The researchers hypothesize that these findings might be related to distinct binding orientations and positions of the antibody Fc fragment relative to the Fc gamma RIIIa receptors (Pinto et al., 2020).

Previous studies have shown that the presence of valine (V) versus phenylalanine (F) at position 158 of Fc gamma RIIIa improves the affinity for IgG and is associated with a higher therapeutic response. Increased CD16 expression on natural killer (NK) cells from donors with the VV or VF versus FF genotype has been observed in several studies (Congy-Jolivet et al., 2008). As a result, ADCC was observed only using natural killer (effector) cells that express the high-affinity Fc gamma RIIIa variant (V158) and not the low-affinity variant (F158) (Pinto et al., 2020). These results may suggest that S309 Fc engineering could potentially enhance the activation of natural killer cells with the low-affinity Fc gamma RIIIa variant (F158).

Similar to the ADCC results, S309 and S306 showed the strongest ADCP response (Figure 23b). To summarize, these results collectively show that, additionally to potent in vitro neutralization, S309 may leverage additional protective mechanisms in vivo (Pinto et al., 2020).

Figure 24: In-tandem assay is one of three epitope binning assay formats: the antigen is bound to the biosensor and the saturated mAb (mAb1) and competing mAb (mAb2) are added respectively for competitive binding assessment (Takkar et al., 2020).

The researchers discovered some of epitopes of SARS-CoV and SARS-CoV-2 S^B domain targeted by the panel of mAbs available. To identify these S^B domain epitopes, they first looked at structural and escape mutant analysis data available from other studies. For instance, a study done in 2010 by Rockx et al. on SARS-CoV S^B domain escape mutants shows that many of the available mAbs work by targeting different epitopes. This is due to amino acid changes that alter epitopes of SARS-CoV spike protein. Therefore, the array of mAbs targeting the spike protein were shown to have different neutralization profiles, as they bind to different epitopes (Rockx et al., 2010).

With the findings of this study, Pinto et al. sought to discover the neutralization profiles for the binding of mAbs to both the SARS-CoV and SARS-CoV-2 S^B domain. To do so, they used the Octet system BLI for epitope binning of the mAbs to the S^B domain (Pinto et al., 2020). This technique is used to carry out cross competition assays where they assess the competitive binding of mAbs to specific epitopes of a protein of interest. In each bin there are two mAbs competing with each other for binding to an epitope on the S^B domain. If both antibodies recognize the same epitope, one antibody will prevent the other from binding. In contrast, antibodies that recognize distinct epitopes will have no effect on one another and will both bind to the S^B domain (Takkar et al., 2020).

Although there are different formats available for epitope binning assays, Pinto et al. used an in-tandem assay (Takkar et al., 2020) where the S^B domain of each SARS-CoV and SARS-CoV-2 were HIS-tagged and bound to anti-Penta-HIS biosensors. Kinetics buffer was later added to allow for the competitive binding of the mAbs in each bin (Pinto et al., 2020).

Figure 25: BLI epitope binning: heat mapping competition between mAb pairs on SARS-CoV S protein where each colour corresponds to a distinct binding site (Pinto et al., 2020).

Results

The BLI analysis identified 4 distinct epitopes of the S^B domain of SARS-CoV which serve as targets for neutralization. These receptor binding domains were identified based on the mAbs that bound to them. The sites are listed below and are colour-coordinated through heat mapping in figure 25 (Pinto et al., 2020):

• site I: targeted by S110, S230 and S227
• site II: targeted by S315 (bridges to site III via S124)
• site III: targeted by S124 (bridges to site II via S124)
• site IV: targeted by S309, S109 and S303

Figure 26: BLI epitope binning: heat mapping competition between cross-neutralizing mAb pairs on SARS-CoV-2 S protein (Pinto et al., 2020).

The same experiment was conducted with only the mAbs capable of cross-neutralization of the SARS-CoV-2 S^B domain. The results of figure 26 show the following neutralization profiles (Pinto et al., 2020):

• site IV: targeted by S309 and S303
• site II & III: targeted by S304 and S315

Figure 27: Pseudovirus assay for neutralization potency of S309, (c) S315 and (d) S304 on SARS-CoV-2-MLV S protein (Pinto et al., 2020).

Once they discovered the different antigen binding sites for the cross-neutralizing mAbs, Pinto et al. further tested how these mAbs work alone or when used in combination. They decided to test S309 due to its ability to recognize site IV of both SARS-CoV and SARS-CoV-2 S^B domain. It was first evaluated for its ability to neutralize the protein alone and was then used in combination with each site-II specific S315 and site-II/III specific S304. This was done to see the impact that combining mAbs would have on the overall neutralization potency. These chosen mAbs underwent a pseudovirus neutralization assay with the use of SARS-CoV-2-MLV (Pinto et al., 2020).

Results

The SARS-CoV-2-MLV pseudovirus neutralization assays tested for the neutralization potency of S309, S304 and S315 on their own, as well as combining S309 with either S315 in Fig. 27c or S304 in Fig. 27d. The results showed that S309 alone has a relatively high neutralization potency, while those of S304 and S315 alone are relatively weak. However, combining S309 with either S304 or S315 enhanced the neutralization potency compared to S309 used alone. These findings conclude that the use of mAb cocktails can target more sites of SARS-CoV-2 S^B domain and thus can enhance the neutralization effect (Pinto et al., 2020).

• The study design was simple and effectively showed the cross-neutralization ability of the S309 antibody
• Data provided in the figures was relatively easy to interpret
• Inclusion of the extended figures provided additional information to support the study

• The study would have benefited from using an in vivo model to test the effects of the cross-neutralizing antibodies (e.g. through treating a SARS-CoV-2 infected mouse)
• The study could have focused more on some of the IgG-specific bivalent mechanisms that may possibly contribute to the ability of S309 to fully neutralize pseudovirions, and conducted more in vitro tests to investigate them further

Figure 28: REGN-COV2: an antibody cocktail of casirivimab and imdevimab administered intravenously for COVID-19 disease treatment (Industry Week, 2020).

The findings of this study support the effectiveness of S309 antibody in neutralizing both SARS-CoV and SARS-CoV-2 S protein. They also showed that combining S309 with other non-competing mAbs has a synergistic effect and enhances their neutralization potency (Pinto et al., 2020). This is further supported by research findings from other studies previously done on SARS-CoV, where the use of mAb cocktails has proven to effectively neutralize the virus (Meulen et al., 2006).

One area of concern in the design of antibody treatments is the chance of escape mutations on the S1 or S2 domains of the SARS-CoV-2 S protein. Previous studies done on SARS-CoV have shown that a single amino acid change in the S protein is enough to evade mAb neutralization. However, the use of mAb cocktails with more than one epitope being targeted could offer a potential solution to targeting escape mutant viruses (Meulen et al., 2006).

This concept has shown promising results from a clinical standpoint. As of late November 2020, the FDA has issued emergency use authorization (EUA) of casirivimab and imdevimab for COVID-19 treatment. These are monoclonal antibodies similar to those of the Pinto et al. study and work by binding to the S protein of SARS-COV-2, neutralizing the virus to prevent its entry into the host cell. This mAb cocktail is manufactured by Regeneron pharmaceuticals, an American biotechnology company, and is sold under the name REGN-COV2. Based on clinical trials, it is believed that these antibodies work best to prevent symptoms in mild or moderate cases COVID-19. However, issues may arise when these are used on patients with severe disease or requiring ventilation, as they may exacerbate the symptoms of the disease (FDA, 2020).

Monoclonal antibody treatments are a popular treatment method for COVID-19 due to their extensive testing and promising clinical trial results. As such, many different countries are researching and approving their own antibody treatments for COVID-19 treatment. For instance, the government of Canada has approved of the use of bamlanivimab, an antibody that also targets the S protein of SARS-CoV-2 for neutralization of the virus. This treatment is co-developed by Eli Lilly and AbCellera Biologics, a biotechnology company located in Vancouver. Currently, this antibody has led to reduced hospital visits in high risk patients, however it is continuously being evaluated for its safety and efficacy (Government of Canada, 2020).

In conclusion, the study by Pinto et al., identified neutralizing antibodies from an individual which was previously infected with SARS-CoV that can effectively target and neutralizes SARS-CoV-2. In addition to it’s ability to neutralizes the S protein of SARS-CoV-2, the mAb S309 was found to have broad neutralizing activity against multiple sarbecoviruses. Furthermore, S309 can recruit effector mechanisms such as ADCC and ADCP and shows increased effectiveness when used in combination with weakly neutralizing mAbs such as S304 or S315. As S309 shows promise as an effective countermeasure to the COVID-19 pandemic caused by SARS-CoV-2, Fc variants of S309 have already been developed by Vir and are currently in clinical trials.

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