Fragment-crystallizable modified COVID-19 antibodies effective in two animal models

A recent article on the bioRxiv* The preprint server identified numerous potent antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in convalescent SARS-CoV-2 patients.

Study: Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models. Photo credit: NIAID

background

The SARS-CoV-2 pandemic continues through the successive virus variants. Various coronavirus disease 2019 (COVID-19) vaccines have been developed, focusing on the original Wuhan strains of SARS-CoV-2. Coincidentally, they were effective against the virus strains subsequently awakened.

The number of SARS-CoV-2 infections decreased in certain countries, possibly due to the effectiveness of vaccination. However, the global COVID-19 pandemic has not yet been contained.

Antiviral therapy is effective during the SARS-CoV-2 replication phase, which occurs in the early stages of infection. Similarly, the use of therapeutic neutralizing antibodies against COVID-19 has been significantly effective. Unfortunately, there are few useful antibodies to fight developing SARS-CoV-2 strains.

About the study

In the present research, scientists developed several monoclonal antibodies from SARS-CoV-2 convalescent patients. Since the beginning of the SARS-CoV-2 epidemic in Japan in March 2020, the authors have collected peripheral blood samples from recovering COVID-19 patients, which were used to generate neutralizing antibodies.

Investigators obtained blood samples from discharged SARS-CoV-2 patients from Keio University Hospital. The cell-based SARS-CoV-2 spike (S) angiotensin converting enzyme 2 (ACE2) inhibition assay was used to assess the neutralizing capacity of sera. The team selected 12 patients with pronounced neutralizing concentrations for antibody generation.

The authors profiled antibodies from patient strains by 1) assessing S-ACE2 inhibition and 2) the association between the binding ability of these antibodies to S-expressing cells and their potential to inhibit ACE2 binding to S-expressing cells to prevent, investigated. To analyze these antibodies in more detail, they also examined their neutralizing potential using a cell fusion experiment. Researchers performed an endpoint microneutralization screen to verify that the selected antibodies can neutralize authentic SARS-CoV-2.

To further identify potential antibodies, scientists assessed affinity for the SARS-CoV-2 receptor-binding domain (RBD) antigen and analyzed epitope overlap. They selected five antibodies and used a pseudovirus containing the S protein of the original SARS-CoV-2 Wuhan sequence and four significant variants to conduct a neutralization experiment before variants of concern (VOCs) emerged. After the appearance of VOCs, they tested the antibodies’ ability to neutralize the original WK-521 virus and its variants, including beta, alpha, gamma, kappa, delta, and Omicron BA.2 and BA.1.

The team performed a cryo-electron microscopy (cryo-EM) study to gain a structural understanding of antibodies and the SARS-CoV-2 S protein. The currently discovered antibodies that are used in the in vivo The study possessed the N297A mutation in the crystallizable (Fc) region of the immunoglobulin G1 (IgG1) fragment to prevent antibody-dependent enhancement (ADE). In addition, the N297A mutation reduces adherence to the Fc receptor. The researchers then studied the effects of these antibodies in two animal models (a cynomolgus macaque model and a hamster model) to see the effects of these antibodies in vivo the settings.

Cryo-EM structure of neutralizing antibodies (A) The structures of RBD and Ab159, Ab188, Ab326, Ab354, Ab445 and Ab496 are shown.  Only the variable domains of antibodies are modeled and drawn as cartoon tubes (individual color) on the RBD surface (grey), and the epitope of each antibody is colored the same as each antibody.  The red area in the central RBD is the binding residue of ACE2 (7A94) (Benton et al., 2020), showing the relationship between the binding sites of the antibodies, which are roughly divided into three groups.  The positions of important amino acids are indicated by black arrows.  (B) Spike residues 400-506 are shown.  The epitopes revealed by cryo-EM are colored red and the residues affected by the mutation described in Figure 3A are shown in squares.Cryo-EM structure of neutralizing antibodies (A) The structures of RBD and Ab159, Ab188, Ab326, Ab354, Ab445 and Ab496 are shown. Only the variable domains of antibodies are modeled and drawn as cartoon tubes (individual color) on the RBD surface (grey), and the epitope of each antibody is colored the same as each antibody. The red area in the central RBD is the binding residue of ACE2 (7A94) (Benton et al., 2020), showing the relationship between the binding sites of the antibodies, which are roughly divided into three groups. The positions of important amino acids are indicated by black arrows. (B) Spike residues 400-506 are shown.

Results

Researchers discovered 494 antibodies from COVID-19 recoveries, most of which showed identical SARS-CoV-2 neutralizing ability to clinically used antibodies in the neutralization assessment. Originally, antigen-specific memory B cells and antigen-nonspecific plasma cells were used to produce antibodies. Nonetheless, the former contained superior antibodies, underscoring the importance of selecting B cells by antigen. The data from the authentic endpoint virus neutralization assay confirmed the results of the cell-based S-ACE2 inhibition and cell fusion assays used to screen for neutralizing antibodies.

Cryo-EM and cell-based mutant S-ACE2 inhibition experiments identified the epitopes on the S protein as antibodies were selected by competition with ACE2, classifying antibody binding to S as class 1/2. The N297 insertion on IgG1-Fc was one of the features of the antibodies discovered in this study. This mutation nearly abolished adhesion to Fc receptors. In fact, it stopped the Fc-facilitated uptake of the virus into Raji cells.

The selected antibodies were comparable or better than imdevimab, a COVID-19 therapeutic, in neutralization tests against the Wuhan strain and VOCs using authentic viruses and pseudoviruses. Regarding the in vivo activity of these antibodies, they showed potential for therapeutic application in macaque and hamster models. At doses of approximately 5 to 7 mg/kg, the current antibodies demonstrated therapeutic efficacy in hamsters and macaques without causing an increase in virus uptake via ADE.

Conclusions

Overall, in the current study, the authors generated many antibodies from the B cells of convalescent COVID-19 patients infected with the SARS-CoV-2 D614G mutant or the Wuhan strain. In addition, they identified numerous neutralizing antibodies with strong neutralizing properties against SARS-CoV-2 variant strains.

These Fc-modified neutralizing antibodies from SARS-CoV-2 recoveries had neutralizing properties comparable to clinical COVID-19 antibodies. The effectiveness of these antibodies has been illustrated by infection research using macaque and hamster models in vivo and authentic virus and pseudovirus neutralization assays in vitro. These results indicated that the currently discovered antibodies had sufficient antiviral activity to serve as treatment options for COVID-19.

*Important NOTE

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be relied upon as conclusive, guide clinical practice/health behavior, or be treated as established information.

Magazine reference:

  • Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models; Masaru Takeshita, Hidehiro Fukuyama, Katsuhiko Kamada, Takehisa Matsumoto, Chieko Makino-Okamura, Tomomi Uchikubo-Kamo, Yuri Tomabechi, Kazuharu Hanada, Saya Moriyama, Yoshimasa Takahashi, Hirohito Ishigaki, Misako Nakayama, Cong Thanh Nguyen, Yoshinori Kitagawa, Yasushi Itoh, Masaki Imai, Tadashi Maemura, Yuri Furusawa, Hiroshi Ueki, Kiyoko Iwatsuki-Horimoto, Mutsumi Ito, Seiya Yamayoshi, Yoshihiro Kawaoka, Mikako Shirouzu, Makoto Ishii, Hideyuki Saya, Yasushi Kondo, Yuko Kaneko, Katsuya Suzuki, Koichi Fukunaga, Tsutomu Takeuchi. bioRxiv Preprint 2022. DOI: https://doi.org/10.1101/2022.06.21.496751, https://www.biorxiv.org/content/10.1101/2022.06.21.496751v1

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