The COVID-19 pandemic, caused by SARS-CoV-2, necessitates the development of effective vaccines and therapeutics. Convalescent plasma, containing neutralizing antibodies, has shown some efficacy in treating severe COVID-19. However, scalability and variability limit its widespread use. Monoclonal antibodies (mAbs) offer a more consistent and scalable therapeutic alternative. SARS-CoV-2, like SARS-CoV-1, utilizes its spike (S) protein to bind to the human angiotensin-converting enzyme 2 (ACE2) receptor for entry into host cells. The S1 subunit of the S protein, specifically the receptor-binding domain (RBD), is crucial for this interaction. Emerging variants, such as the D614G variant, exhibit increased infectivity. Therefore, targeting the RBD with neutralizing antibodies is a promising strategy, particularly those that avoid antibody-dependent enhancement (ADE). This research focuses on identifying and characterizing a potent neutralizing mAb targeting the RBD of SARS-CoV-2, assessing its efficacy in various in vitro and in vivo models.

Previous research has highlighted the importance of targeting the RBD of the SARS-CoV-2 spike protein for effective neutralization. Studies have shown that convalescent plasma containing neutralizing antibodies can reduce viral replication and improve clinical outcomes in COVID-19 patients. However, the inconsistent antibody levels and scalability issues associated with convalescent plasma necessitate the development of monoclonal antibodies. Several studies have characterized the structural basis of RBD interactions with ACE2, and others have identified neutralizing antibodies targeting different epitopes on the RBD. The D614G mutation, prevalent in many circulating SARS-CoV-2 strains, has been shown to enhance viral infectivity and transmission. This review of the literature underscores the need for a potent and broadly neutralizing antibody that can effectively combat both the wild-type virus and emerging variants, without inducing ADE.

The study involved several key steps: 1. **Antibody Library Construction and Biopanning:** A phage display library was constructed from peripheral blood mononuclear cells (PBMCs) of a convalescent COVID-19 patient. Biopanning was performed against the SARS-CoV-2 RBD to select for RBD-binding antibodies. 2. **Antibody Production and Characterization:** Selected single-chain variable fragments (scFvs) were further cloned and expressed in mammalian cells to produce full-length IgG antibodies. The binding affinity (Kd) of the lead antibody, CT-P59, was determined using surface plasmon resonance (SPR) and biolayer interferometry (BLI). 3. **Neutralization Assays:** In vitro neutralization assays were performed to evaluate the ability of CT-P59 to neutralize SARS-CoV-2 isolates, including the D614G variant. A plaque reduction neutralization test (PRNT) was used to determine the neutralization potency (IC50). 4. **Structural Analysis:** X-ray crystallography was employed to determine the three-dimensional structure of the CT-P59 Fab fragment in complex with the SARS-CoV-2 RBD. This analysis revealed the precise interaction interface and potential mechanisms of neutralization. 5. **In vivo Efficacy Studies:** The therapeutic efficacy of CT-P59 was evaluated in three animal models: ferrets, golden Syrian hamsters, and rhesus macaques. Animals were challenged with SARS-CoV-2, and CT-P59 was administered therapeutically. Viral loads in various tissues (nasal wash, lungs, etc.), clinical symptoms, and lung pathology were assessed. A comparison of the therapeutic efficacy of CT-P59 was conducted against Remdesivir, a US FDA-approved drug for COVID-19. 6. **Antibody-Dependent Enhancement (ADE) Assessment:** In vitro assays were conducted to evaluate the potential for CT-P59 to induce ADE using Fc receptor (FcR)-bearing cells and permissive cells.

The study identified CT-P59, a potent neutralizing monoclonal antibody against SARS-CoV-2. Key findings include: * **Potent Neutralization:** CT-P59 effectively neutralized both wild-type and D614G SARS-CoV-2 variants in vitro, exhibiting a high binding affinity (Kd of 27.9 pM). * **Unique Binding Mechanism:** The crystal structure of the CT-P59/RBD complex revealed a distinct binding mode compared to previously reported neutralizing antibodies. CT-P59's binding overlaps significantly with the ACE2 binding site, effectively blocking viral entry. * **In vivo Efficacy:** Therapeutic administration of CT-P59 in ferret, hamster, and rhesus monkey models significantly reduced viral loads in the upper and lower respiratory tracts, accompanied by an alleviation of clinical symptoms and improved lung pathology. CT-P59 demonstrated superior efficacy to Remdesivir in the ferret model, with faster viral clearance. * **No Antibody-Dependent Enhancement (ADE):** In vitro studies showed no evidence of ADE mediated by CT-P59, indicating a safe therapeutic profile.

The findings demonstrate the efficacy of CT-P59 as a potential therapeutic agent for COVID-19. The potent neutralization activity against both wild-type and D614G variants highlights its broad applicability. The unique binding mode of CT-P59 suggests that it may overcome the limitations of antibodies with similar binding modes. The substantial reduction in viral load and amelioration of clinical symptoms in multiple animal models further supports its therapeutic potential. The absence of ADE is crucial for the safety and efficacy of a therapeutic antibody. Future research should focus on clinical trials to assess CT-P59's efficacy and safety in humans, including investigation of possible combination therapies and assessment of its effectiveness against other emerging SARS-CoV-2 variants.

This study successfully identified and characterized CT-P59, a highly potent neutralizing antibody against SARS-CoV-2. Its unique binding mode, in vivo efficacy, and lack of ADE suggest significant therapeutic potential for COVID-19. Further clinical investigation is warranted to confirm its efficacy and safety in human patients.

The study was conducted in animal models, which may not fully reflect human disease pathogenesis. The sample size in some animal studies was relatively small. Long-term effects of CT-P59 treatment were not assessed. The study focused on a single convalescent patient's immune response, limiting the generalizability of the findings.