Therapeutic Targeting of SARS-CoV-2

Created by: Bisman Singh, Carter Nattrass, Jessica Xing, Lamisa Syed, Luke Cheon, & Matthew Fernandes

The Kim et al paper presents a novel monoclonal antibody (mAb) called CT-P59 against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. After creating an antibody library from a recovered COVID-19 patient’s peripheral blood mononuclear cells, a high affinity monoclonal antibody, CT-P59, against the RBD was found (Kim et al., 2021). The authors tested CT-P59 neutralizing ability using in vitro plaque assays, and structural analysis. To further prove CT-P59’s therapeutic ability, in vivo challenge studies in animal models were conducted (Kim et al., 2021). Finally, the authors used antibody dependent enhancement (ADE) assays to assess the potential risk of this novel mAb.

Firstly, peripheral Blood Mononuclear Cells (PBMCs) were isolated from the blood of a recovering COVID-19 patient to obtain RBD-binding single chain variable fragments genetic material from the top part of the antibodies formed in the blood while fighting SARS-CoV-2 (Kim et al., 2021). This was done by collecting samples 48 hours after the patient’s symptoms disappeared (Kim et al., 2021). Two consecutive respiratory specimens, each collected after 24 hours, were also confirmed negative for SARS-CoV-2 by PCR before blood sampling (Kim et al., 2021). PBMCs were isolated from blood using Ficoll-Paque and subsequently mRNA was extracted using TRIzol reagent (Kim et al., 2021). Isolated mRNA was then finally converted to cDNA using SuperScript III Reverse Transcriptase (Kim et al., 2021). The cDNA extracted contained all genetic information which produced antibodies when the patient was infected with SARS-CoV-2.

Phage display is a technique by which the foreign DNA fragment encoding polypeptide is fused with the coding gene of phage surface protein and the resulting fusion polypeptide will be displayed on the surface of phage maintaining a relative spatial structure and biological activity (Tennant et al., 2018).

Antibody variable regions (VL and VH) present in the cDNA produced were then amplified using PCR with appropriate primers for phage display (Kim et al., 2021). These regions were amplified as they make up the genetic material for single chain variable fragments (scFvs) of antibodies which will help us to find the perfect antibody that binds to SARS-CoV-2 RBD (Kim et al., 2021). scFvs cDNA was generated by linking VL and VH and then was directly cloned in phagemid vector pComb3xSS cells, for phage display library construction (Kim et al., 2021). ER2738 competent cells were then transformed with scFvs cDNA library and then cultured in SB medium containing VCSM13 (M13) helper phage cells overnight at 37°C (Kim et al., 2021). This helped the scFv cDNA to be incorporated into the M13 bacteriophage cell (Kim et al., 2021).

Figure 1: scFvs Expressed on Phages using Phage Display Technique. Image Source : Phage Display Technology - Creative Biolabs (Updated Version), 2020.

The next day, the phages displaying scFv were harvested for biopanning which is a procedure of selecting binding partners from phage display libraries (Ganten & Ruckpaul, 2006). This helped to screen for SARS-CoV-2 RBD-binding scFv displayed on phage (Kim et al., 2021). Then SARS-CoV-2 was coated on magnetic beads and incubated with the phage library (Kim et al., 2021). The magnetic field helped the SARS-CoV-2 RBD-bound phages to be retained while the other phages got washed away after washing (Kim et al., 2021). After that, SARS-CoV-2 RBD-bound phages were eluted and used to infect fresh ER2738 cells (Kim et al., 2021).

After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by enzyme-linked immunosorbent assay (ELISA) which is an immunological assay used to measure the concentration of an analyte in solution (Thermo Fisher Scientific, n.d.). For this study, the phage-ELISA was performed to check if the scFvs were binding to the RBD of SARS-CoV-2.

Figure 2: Biopanning procedure to screen for scFvs binding to SARS-CoV-2 RBD. Image Source : Phage Display Technology - Creative Biolabs (Updated Version), 2020 .

The remaining scFvs after ELISA were cloned into a constant fragment (Fc) fusion vector region of antibody which was then expressed in Chinese hamster ovary cells (CHO) (Kim et al., 2021). For the expression of full-length Immunoglobulin G, synthesized DNAs of heavy and light chain for each mAb were inserted to MarEx vectors using enzymatic digestion (NheI (NEB)/PmeI (NEB) and HpaI (NEB)/ClaI (NEB)) (Kim et al., 2021). Recombinant Anti-SARS-CoV-2 Spike Glycoprotein S1 Antibody (CR3022) (abcam, 2022) was reconstituted with variable sequences for light and heavy chain (Kim et al., 2021). Then, expression by co-transfection was performed in Chinese hamster ovary cells (Kim et al., 2021). scFv-Fc and full-length recombinant IgG were purified using affinity chromatography (Kim et al., 2021).

Plaque reduction neutralization test (PRNT) was conducted to evaluate mAbs neutralizing activity against SARS-CoV-2 virus. PRNT is a method for measuring antibodies that neutralize and prevent virions from infecting cultured cells. This helps to determine the effectiveness of the monoclonal antibody against the virion (Sirivichayakul et al., 2014).

Kim et al. did this by serially diluting mAbs and incubating them at 37 °C for 2 h first, then followed by antibody-virus mixture inoculation into 24-well plate seeded with Vero E6 cells incubated at 37 °C for 1 h and an overlay of 1mL of 0.5% agarose (Kim et al., 2021). The cells were then incubated for 2 to 3 days and fixed with 4% paraformaldehyde and visualized plaques with crystal violet (Kim et al., 2021). Two independent experiments were performed for each mAb and the data were obtained using a dose-response inhibition model and the half-maximal inhibitory concentration (IC50) of each mAb was calculated (Kim et al., 2021).

BLI is an experimental technique that uses optical signals to quantify the kinetics and affinity of protein-protein interactions (CMI, 2022).One molecule is immobilized onto a biosensor and different analytes are used as the flow through (CMI, 2022). After binding to the immobilized molecule and the following wash out steps, the interference patterns are measured in real time and the dissociation constant (KD) is determined to measure the binding affinities (CMI, 2022). Kim et al., (2021) used the Octet QKe system to evaluate the binding specificity and affinity between CT-P59 and ACE2 of SARS-CoV-2 RBDs. Immobilized wild-type and mutant SARS-CoV-2 RBD proteins were saturated with CT-P59, and then flowed with CT-P59 in the presence or absence of ACE2 to determine the competitive characteristics between CT-P59 and ACE2 (Kim et al., 2021). The binding specificity of CT-P59 to SARS-CoV-2 RBD, SARS-CoV S1, HCoV-HKU1 S1, MERS RBD was also measured by loading them onto Anti-Penta-HIS Biosensor, and then CT-P59 flowed (Kim et al., 2021). CT-P59 was loaded onto anti-human IgG Fc Capture Biosensor and flowed to evaluate binding affinity between CT-P59 and SARS-CoV-2 RBD wild type and mutants (Kim et al., 2021).

X-ray crystallography is a method to visualize a protein's structure through purifying a protein crystal and bombarding it with X-rays to obtain diffraction patterns and to synthesize the data using computational programs such as Phenix to solve the structure (Kim et al., 2021). Kim et al. did by mixing the CT-P59 Fab with purified SARS-CoV-2 RBD and preparing CT-P59 Fab/SARS-CoV-2 RBD complex (Kim et al., 2021). This was followed by obtaining diffraction quality crystals by sitting-drop vapor-diffusion method (Kim et al., 2021). The crystals were then cryo-protected, and X-ray diffraction data was collected, and Phaser was used to determine CT-P59 Fab/SARS-CoV-2 RBD complex structure followed by Phenix, and Coot being used for model building and refinement of the complex structure (Kim et al., 2021).

To check for in vivo efficacy, animal models were used to conduct series of viral challenge studies (Kim et al., 2021). All animals were challenged with SARS-CoV-2 via various infection routes (Kim et al., 2021). Twenty-four hours after inoculation, CT-P59 mAbs were administered (Kim et al., 2021). Remdesivir was was administered along with CT-P59 in Ferrets (Kim et al., 2021). Ferrets in the control group were given human IgG isotype and Golden Syrian hamster and Rhesus Monkey were given vehicles as control (Kim et al., 2021). All animals were euthanized to obtain samples certain days after the infection (Kim et al., 2021).

In order to determine novel neutralizing antibodies that target SARS-CoV-2, RBD-binding single-chain variable fragments were isolated using recombinant SARS-CoV-2 RBD as bait for phage display screening (Kim et al., 2021). The neutralization potency of CT-P59, a monoclonal antibody reformatted to IgG, was evaluated via in vitro PRNT against wild type SARS-CoV-2 and SARS-CoV-2 D614G variants (Kim et al., 2021).

Figure 3: Serial twofold-diluted CT-P59 incubated with SARS-CoV-2 wild type and D614G viruses. Upon incubation with VeroE6 cells, the neutralization activity was evaluated by counting plaques.

It was determined that CT-P59 significantly inhibits the viral replication of a Korean SARS-CoV-2 clinical isolate, which contains an S protein genome sequence that is identical to the primary virus (Kim et al., 2021). This was reflected as a low IC50of 8.4 ng/ml and an IC50 of 5.7 ng/ml when examining the D614G variant (Kim et al., 2021). The latter result indicated that CT-P59 reduced the replication of the D614G variant to a similar extent as the wild-type virus (Kim et al., 2021).

Additionally, a competitive binding assay with biolayer interferometry (BLI) later revealed that CT-P59 completely inhibits the binding of RBD-ACE2 (Kim et al., 2021). RBD-binding and ACE2 interference test with RBD mutant proteins further demonstrated that CT-P59 can also bind to the mutants in order to completely inhibit their interaction with ACE2 (Kim et al., 2021).

Figure 4: SARS-CoV-2 RBD was saturated with CT-P59 and flowed over a biosensor surface in the presence or absence of the ACE2 receptor.

The binding specificity of CT-P59 to SARS-CoV, HCoV-HKU1, and MERS-CoV was also evaluated by BLI and the test indicated that CT-P59 can specifically bind to SARS-CoV-2. Surface plasmon resonance analysis also indicated that CT-P59 has a high affinity for SARS-CoV-2 RBD with a KD value of 27 pM (Kim et al., 2021).

Figure 5: Binding affinity of CT-P59 was determined by SPR.

Significantly, these results suggest that SARS-CoV-2 RBD mutations might alter the binding affinity of the virus for ACE2. Specifically, mutations V367F, W436R, and D364Y were observed to increase the virus binding affinity for ACE2 (Kim et al., 2021). This has broader implications for the acceleration of viral spread via genome mutations and the further perpetuation of the pandemic (Kim et al., 2021).

To confirm CT-P59’s ability to neutralize the spike protein, structural analysis with X-ray crystallography was used to visualize CT-P59’s blocking mechanism against the RBD of SARS-CoV-2 (Kim et al., 2021). The protein crystal complex of CT-P59 Fab/SARS-CoV-2 RBD was solved at a resolution of 2.7Å (Kim et al., 2021). CT-P59’s Fab (fragment antigen binding) region was found to bind to the receptor binding motif (RBM) within the receptor binding domain (RBD) in SARS-CoV-2’s spike protein, seen in Figure 6 (Kim et al., 2021).

Figure 6: The X-ray crystallography structure of the solved CT-P59 Fab protein (heavy chains seen in magenta and the light chains as yellow) complexed with the SARS-CoV-2 RBD (green) is visualized. Within the RBD lies the RBM (orange) (Kim et al., 2021).The PDB entry for this complex is 7CM4.

The Fab region is found on the N-terminal of the light and heavy chain of the antibody, and it facilitates specific binding to an antigen on the target protein (Nelson, 2010). Past structural analysis of human ACE2 complexed with the SARS-CoV-2 spike protein revealed that the RBM is a key functional motif that directly binds the viral spike protein to hACE2 and the surrounding region of the RBM promotes the RBD’s stability (Yi, 2020).

The association angle between CT-P59 and the RBD was unique when compared to the other 12 mAbs and their interactions with the SARS-CoV-2 spike protein (Kim et al., 2021). This was confirmed by solving the protein structure of CT-P59 in complex with the RBD and compared to other neutralizing antibodies, seen in Figure 7. (Kim et al., 2021).

Figure 7: Twelve monoclonal antibodies and CT-P59 are listed with their respective epitope-binding residues on the SARS-CoV-2 RBD (Kim et al., 2021). The RBD residues are listed at the top of the image and the red residues represent the ACE2 binding residues on the RBD (Kim et al., 2021).

CT-P59’s heavy and light chains also buried solvent-accessible regions on the spike protein’s RBD with a surface area of 825 Å2 and 113 Å2, respectively (Kim et al., 2021). This was calculated using PISA (Protein Interfaces, Surfaces and Assemblies), a macromolecule assembly calculator which generates an estimate of the size of protein-protein interactions (Krissinel, 2015). The CT-P59’s heavy chain interaction is similar to the surface area that is buried when ACE2 binds to the RBD (864 Å2) (Lan et al., 2020).

Three complementarity-determining regions (CDR) in CT-P59 were all found to interact with SARS-CoV-2’s RBD, shown in Figure. 8 (Kim et al., 2021). CDRs are structured loops which form the antigen binding site in antibodies (Gabrielli et al., 2009). Antibodies contain six CDRs in total: three in the light chain (L1, L2 and L3) and three in the heavy chain (H1, H2 and H3) (Gabrielli et al., 2009). In the Kim et al study, interactions between CT-P59 and the RBD were primarily found in the heavy chain, and with some interactions in the light chain (Kim et al., 2021). Sixteen residues from CT-P59’s heavy chain interacted with 19 of the RBD’s residues (Kim et al., 2021). CT-P59’s CDR H3 component formed a beta hairpin structure which contributed to strong binding by forming eight hydrogen bonds and hydrophobic interactions with the center of the ACE2-binding site within the RBD (Kim et al., 2021). On the other hand, there were fewer interactions between the light chain (mostly CDR L1 and CDR L2) and the RBD with three residues interacting with four SARS-CoV-2 residues (Kim et al., 2021).

Figure 8: A zoomed in view of the CDR H1-H3 interactions (magenta) with the RBD of the spike protein (gray). The CDR H1-H3 structures are shown as a stick model while the SARS-CoV-2 spike protein is represented as a gray translucent surface model structure (Kim et al., 2021). Hydrogen-bonds are represented as blue dashed lines.

The Kim et al team’s structural analysis further elucidated that CT-P59 blocked substantial areas on the ACE2 binding regions on the S protein, after aligning the CT-P59 Fab-RBD complex against the ACE2-RBD complex, as seen in Figure 9 (Kim et al., 2021). CT-P59 also potentially changed the conformation of a certain region in the B5 and B6 loop region of the RBD (residues 473- 488), where the majority of the ACE2 contacting residues exist (Kim et al., 2021; Lan et al., 2020). The heavy chain fully overlaps with the ACE2 binding regions on the RBD, and the light chain only partially overlaps with the ACE2 binding sites (Kim et al., 2021). Out of the 21 RBD residues that interact with ACE2, 12 residues were bound by CT-P59 at a distance cut-off of 4.5A (Kim et al., 2021). On a structural basis, CT-P59 overlaps with most of the SARS-CoV-2 RBD surface area that would have bound to ACE2 which indicates that it is a good fit to neutralize the viral protein and prevent binding in permissive human cells with the cognate ACE2 receptors (Kim et al., 2021).

Figure 9: The structural comparison between the CT-P59 Fab binding sites (magenta) on the SARS-CoV-2 RBD (gray surface structure) is visualized against the ACE2 binding sites (cyan) on the RBD (Kim et al., 2021).

To determine the therapeutic efficacy of CT-P59, Kim et al. (2021) conducted a series of virus challenge studies in ferrets, golden Syrian hamsters, and rhesus monkeys (macaques). These three animal models were chosen to be evaluated in combination to reflect the clinical symptoms in COVID-19 patients, as no single animal model accurately represents the COVID-19 clinical symptoms (Kim et al., 2021). Each animal model was set up to compare the therapeutic efficacy of CT-P59 to a control group and remdesivir by varying the doses administered (Kim et al., 2021). Remdesivir was specifically chosen as it is a US FDA approved drug and widely used in treating hospitalized COVID-19 patients (Kim et al., 2021). For the ferret model, the researchers employed an IgG isotype control by administering human IgG isotype (Kim et al., 2021). Isotype controls are a type of negative control that are highly similar structurally to the primary antibody tested but bind to an antigen irrelevant to the experiment (“Isotype control antibodies,” n.d.). The isotype control shares the same host species, class and subclass of antibody, and is used in the same experimental conditions and procedure (“Isotype control antibodies,” n.d.). The non-specific binding effect of the primary antibody can be determined by visualizing and comparing the primary antibody to the isotype control (“Isotype control antibodies,” n.d.; Kim et al., 2021). The IgG isotype was chosen as CT-P59 is a reformatted IgG antibody (Kim et al., 2021). For the other two animal models, the researchers treated the animals with vehicles (Kim et al., 2021). Vehicle-treated control is another type of negative control that only administers the vehicle, or the delivery substance (Johnson et al., 2002; Kim et al., 2021). This excludes the experimental substance to determine the effect of the administered vehicle (Johnson et al., 2002; Kim et al., 2021). Unlike the other two models, the control group of the ferret model was administered with human IgG, to replicate IgG-mediated viral clearance in humans to compare the efficacy of CT-P59 and remdesivir (Kim et al., 2021). The samples from the animal studies were obtained by euthanizing animals at specific days post infection (dpi) (Kim et al., 2021).

In the ferret model, the virus was administered via intranasally and intratracheal routes, and CT-P59 and the isotype control was administered 1-day post-infection (Kim et al., 2021). 30 mg/kg of human IgG isotype was administered and CT-P59 was administered in 3 mg/kg and 30 mg/kg doses (Kim et al., 2021). The results from the upper respiratory tract showed that CT-P59 administration resulted in a better therapeutic efficacy compared to control and remdesivir (Kim et al., 2021). At 2 dpi, Remdesivir and 3 mg/kg showed a similar level of viral titer and viral RNA, but 30 mg/kg showed a decrease in both compared to remdesivir (Kim et al., 2021). By 4 dpi, both 3 mg/kg and 30 mg/kg showed a reduction in viral RNA compared to remdesivir, but remdesivir showed a lower viral titer than 3 mg/kg (Kim et al., 2021). Lower respiratory tracts showed similar results with high dosage of CT-P59, showing attenuated viral titer by 3 dpi and viral titer undetectable by 7 dpi (Kim et al., 2021). In the ferret study, the therapeutic effect of lower doses of CT-P59 was not significant and in some cases performing worse than remdesivir (Kim et al., 2021). However, at higher doses, the difference in therapeutic effect is significant, as results showed that high dose of CT-P59 can reduce viral titer to pre-infection level and significantly reduce the viral RNA by 6 dpi (Kim et al., 2021). This contrasts with the delayed clearance of SARS-CoV-2 in remdesivir-administered ferrets (Kim et al., 2021).

Figure 10: (a) Viral titer and (b) viral RNA copy number in upper and lower respiratory tracts of ferrets (adapted from Kim et al., 2021).

In the Syrian hamster study, the virus was only administered via the intratracheal route (Kim et al., 2021). The test group was administered CT-P59 in doses of 15, 30, 60, and 90 mg/kg at 1 dpi (Kim et al., 2021). Figure 4e shows that all doses of CT-P59 showed significant reduction in viral RNA in the lower respiratory tract at 5 dpi and figure 4b shows that viral titer was undetectable in 30, 60, and 90 mg/kg groups while 15 mg/kg showed significant reduction compared to vehicle control-treated group at 3 dpi in the same location (Kim et al., 2021). Although remdesivir was not administered in Syrian hamsters, the results may indicate that CT-P59 completely inhibits SARS-CoV-2 replication in the lower respiratory tract (Kim et al., 2021).

Figure 11: (a) Viral titer and (b) viral RNA copy number in the lower respiratory tracts of golden Syrian hamsters (adapted from Kim et al., 2021).

In the Rhesus monkey study, the virus was administered via a combination of intranasal, intratracheal, ocular, and oral routes (Kim et al., 2021). This animal study used a vehicle control-treated group and CT-P59 was administered in 45 mg/kg and 90 mg/kg at 1 dpi (Kim et al., 2021). The rhesus monkeys did not show the clinical symptoms of SARS-CoV-2 such as fever, weight loss, and respiratory difficulty in both vehicle control-treated and treatment groups (Kim et al., 2021). In the upper respiratory tract, the virus titer of the vehicle control-treated group peaked at 2 dpi and slowly declined until 6 dpi when it became undetectable (Kim et al., 2021). In contrast, the CT-P59 treatment group showed a significant increase in viral titer clearance, shown by viral titer undetected at 2 dpi (Kim et al., 2021). At 6 dpi, viral RNA remained detected in the middle and lower respiratory tracts (figure 4c) but infectious SARS-CoV-2 virus particles were not detected (figure 4f) (Kim et al., 2021).

Figure 12: Viral titer from (a) nose swab and (b) throat swab in upper respiratory tracts of Rhesus monkeys. (c) Viral titer and (d) viral copy number of samples from upper, middle and lower lobes of left and right lungs at 6 dpi (adapted from Kim et al., 2021).

Antibody-dependent enhancement (ADE) occurs through two main mechanisms: enhanced infection or enhanced immune activation (Lee et al., 2020). Both mechanisms begin with non-neutralizing antibodies or antibodies at sub-neutralizing levels that bind to the virus (Lee et al., 2020). This antibody-virus complex can then be internalized by immune cells that have Fc receptors (FcRs) on their surface, increasing viral entry and replication (Lee et al., 2020; Iwasaki & Yang, 2020). The antibody-virus complex can also cause enhanced immune activation where excessive Fc-mediated effector functions, such as secretion of pro-inflammatory cytokines and immune cell recruitment, lead to increased inflammation and immunopathology (Lee et al., 2020; Iwasaki & Yang, 2020). FcR-independent ADE has also been shown to occur in certain viruses such as HIV-1 through complement mediated mechanisms (Xu et al., 2021). ADE via enhanced immune activation is most common in respiratory pathogens and can be examined in vivo by measuring disease severity (Lee et al., 2020). ADE via increased infection is commonly measured using in vitro assays detecting the antibody-dependent infection of FcR-expressing phagocytic cells, such as monocytes or macrophages (Lee et al., 2020).

In the study by Kim et al. (2021), they performed an ADE assay using two FcR-bearing cells (Raji cells and U937 cells) and a permissive cell (VeroE6, that is highly susceptible to infection). Equal volumes of SARS-CoV-2 (BetaCoV/Korea/KCDC03/2020) were first mixed with serially diluted antibodies (Kim et al., 2021). These antibody-virus mixtures were then used to infect the cells previously mentioned. 24 hours post-infection the virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody, and the data was presented as a graph (Kim et al., 2021).

SARS-CoV-2 was mixed with a wide range of antibodies (CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle)). The virus-antibody complexes were then used to infect a permissive cell (VeroE6) and two FcR-bearing cells: Raji cells (expressing FcγRII) and U937 cells (expressing FcγRI & II). OD values are directly correlated to virus titer and therefore levels of viral infection. VeroE6 cells were used to measure any FcR-independent ADE, while the FcR-bearing cells were used to measure the more common FcR-dependent ADE via enhanced infection (Kim et al., 2021). There was an overall increase in viral infection in VeroE6 cells (Figure xa) compared to the FcR-bearing cells (figure x b and c). This is expected because the VeroE6 cells are highly susceptible to SARS-CoV-2 infection (Kim et al., 2021). Figure xa showed that high antibody concentration of CT-P59 neutralized the virus and did not cause any increase in viral infection via FcR-independent ADE, over the other two antibodies. Furthermore, both figure x b and c, showed that Fc receptor-bearing cells had no increase in viral infection due to ADE when compared to the other antibodies. Therefore, the ADE assay indicated no CT-P59-mediated increase in viral infections for both FcR-bearing and permissive cells.

Figure 14: in vitro ADE assay with authentic SARS-CoV-2 infecting both permissive and Fc receptor (FcR)-bearing cells (Kim et al., 2021).

Through the results collected from in vitro and in vivo studies, CT-P59 targeted the RBD of SARS-CoV-2 (Kim et al., 2021). In the BLI competition assay and X-ray crystallography, they observed that the binding of CT-P59 was effective at sterically hindering the binding of ACE2 to the S protein’s RBD (Kim et al., 2021). This was not only seen with the wild-type strain of SARS-CoV-2 but also with clinical isolates such as the D614G variant (Kim et al., 2021). In addition to D416G; V367F, W436R, and D364Y are variants which also increase the affinity of the RBD for ACE2; however, CT-P59 was able to inhibit the variant’s interaction with ACE2 (Kim et al., 2021). Through the X-ray crystallography data, CT-P59 does not bind to these amino acid residues meaning the mutations do not interfere with the ability for CT-P59 to neutralize these variants (Kim et al., 2021). This means CT-P59 can potentially avoid binding to areas that are prone to antigenic shifts (Kim et al., 2021).

CT-P59 orients in a certain position when binding to the RBD to block the interaction of SARS-CoV-2 to ACE2 (Kim et al., 2021). In addition to CT-P59, there are other groups of neutralizing antibodies which inhibit this interaction to ACE2 (Kim et al., 2021). The immunoglobulin heavy-chain variable region genes (IGHV) 3 germline are one of these groups of antibodies, they include CB616, B3817, CV3018, CC12.119, CC 12.319, C10522, COVA2-0423, and REGN1093314 (Kim et al., 2021). In contrast, another group of antibodies with the IGHV4-38-2 gene consists of the P2B-2F6 antibody (Kim et al., 2021). This group of antibodies differ in their orientation by 90° in comparison to IGHV3 (Kim et al., 2021). With CT-P59, it binds to the RBD at an angle in between the angles bound by these two groups and has the IGHV2-70 gene (Kim et al., 2021). From the structural alignment of CT-P59 to the RBD, CT-P59 binds to the “up” conformation of the RBD, which is the conformation the RBD is able to bind to ACE2 (Kim et al., 2021). However, the “down” conformation (which is the conformation that doesn’t bind to ACE2) hinders CT-P59 binding due to the collision with the RBD’s Asn343 glycosylated site (Kim et al., 2021).

To evaluate the therapeutic effects of CT-P59 against SARS-CoV-2, animal models such as ferrets, Syrian hamsters, and rhesus monkeys were chosen (Kim et al., 2021). From the in vivo studies, CT-P59 proved to be effective at decreasing the viral load and alleviating common COVID-19 symptoms (Kim et al., 2021). When compared to remdesivir, CT-P59-treated ferrets showed more attenuated viral loads in upper respiratory tracts from 2 dpi (Kim et al., 2021). This comparison between remdesivir and CT-P59 treated ferrets in addition to other animal studies shows that CT-P59 has higher therapeutic efficacy (Kim et al., 2021). From the TCID50 assay, CT-P59 was shown to neutralize SARS-CoV2 and inhibit the replication (Kim et al., 2021). To further improve the effectiveness at clearing the virus, CT-P59 may be combined with drugs such as remdesivir as a combination therapy for patients (Kim et al., 2021). In addition to evaluating the therapeutic effectiveness, the safety for the use of CT-P59 was evaluated (Kim et al., 2021). In vitro and animal studies showed no signs of ADE with no worsening infections with the administration of CT-P59 (Kim et al., 2021). CT-P59 is designed to target the RBD of the spike protein in order to neutralize it; RBD-based vaccines have been previously shown to cause little or no ADE (Kim et al., 2021). Thus, the use of CT-P59 is both safe and effective at treating SARS-CoV2 infections by binding to the RBD (Kim et al., 2021).

• Comprehensive research design that included in vitro and in vivo testing of the novel neutralizing antibody (Kim et al., 2021)
• Kim et al. illustrated the effectiveness of CT-P59 as a novel neutralizing antibody by showing its ability to block the ACE2 receptor and lowering clinical symptoms in animals (Kim et al., 2021)

• SARS-CoV-2 can avoid immune surveillance in it’s ”down” confirmation, thus a neutralizing antibody that is able to detect this conformation would be beneficial in addition to CT-P59’s recognition of the “up” conformation of the spike protein (Kim et al., 2021)
• Could have used human lung epithelial cells in the ADE experiment to better represent potential ADE effects at the primary site of SARS-CoV-2 infection in humans (Essaidi-Laziosi et al., 2021)
• Specific antibodies used not explained for ADE section
• In vivo animal models did not present with clinical symptoms that people with severe COVID-19 experience so, they had to use more than one animal model as a result (Kim et al., 2021)

As the effectiveness of CT-P59 has been demonstrated through this study, the next steps would be the application of CT-P59 as a potential therapeutic. This is observed through its use in clinical trials as well as the use in further research studies to test for its broadly neutralizing capabilities (Kim et al., 2021). At the time of publishing the paper, CT-P59 was evaluated for the safety of its use through a phase I clinical trial as well as a phase II clinical trial in South Korea (Kim et al., 2021). Currently, CT-P59 has been observed to be in phase II and III clinical trials to verify the effectiveness of its use as a therapy for treating patients with COVID-19 as well as the safety of its use (NIH, n.d.). However, no data on the results from the trial has been made available (NIH, n.d.). In addition to the clinical trials, CT-P59 can also be examined for its broadly neutralizing capabilities. As seen in the study, CT-P59 was able to inhibit multiple mutants of SARS-CoV2 such as D416G, V367F, W436R, and D364Y from their binding to the host’s ACE2 receptor (Kim et al., 2021). The mutations at these positions did not interfere with the binding to CT-P59 (Kim et al., 2021). From these results, CT-P59 could be studied for its binding to the RBD of other variants of SARS-CoV2 to test its ability as a broadly neutralizing mAb against SARS-CoV2.

The Kim et al study (2021) evaluated the therapeutic potential of a novel neutralizing antibody, CT-P59, through comprehensive in vitro and in vivo experiments. The key findings are as follows:

• CT-P59 is able to competitively inhibit the binding of RBD-ACE2 (Kim et al., 2021).
• The protein structure of CT-P59 Fab blocked substantial areas of the ACE2 binding sites within the SARS-CoV-2 RBD (Kim et al., 2021).
• Virus titration and quantification showed CT-P59 completely inhibits viral replication, reducing viral RNA copy number and viral titer faster than remdesivir (Kim et al., 2021).
• ADE assay showed no evidence of CT-P59-mediated increase in viral infections via ADE (Kim et al., 2021).

Overall, the Kim et al paper outlines the functional characteristics of a novel neutralizing antibody against SARS-CoV-2 that has great therapeutic potential (Kim et al., 2021).

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