Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe aggravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process, however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. We first isolated a MERS-CoV neutralizing antibody that is reactive to the receptor-binding domain (RBD) of spike (S) glycoprotein. To increase stability, we introduced mutations into the complementarity-determining regions (CDRs) of the antibody. In the HCDRs (excluding HCDR3) in this clone, two hydrophobic residues were replaced with Glu, two residues were replaced with Asp, and four residues were replaced with positively charged amino acids. In LCDRs, only two Leu residues were replaced with Val. These modifications successfully generated a clone with significantly greater stability and equivalent reactivity and neutralizing activity following nebulization compared to the original clone. In summary, we generated a MERS-CoV neutralizing human antibody that is reactive to recombinant MERS-CoV S RBD protein for delivery via a pulmonary route by introducing stabilizing mutations into five CDRs.

Stereotypic neutralizing V H antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals

“…None of the 12 clones, including A,B,G-42, reacted against the recombinant RBD proteins from either SARS-CoV or MERS-CoV ( Figure S9). None of the 37 identified MERS-RBD-binding human mAbs (from two patients) were encoded by IGHV3-53/IGHV3-66 and IGHJ6 (Table S4) based on analysis of a prior study (17). Therefore, the presence of these stereotypic-naïve IGH clonotypes in the healthy population, and their light chain plasticity needed to achieve SARS-CoV-2 RBD binding, may be unique to SARS-CoV-2, which might provide a rapid and effective humoral response to the virus among patients who express these clonotypes.…”

“…S9). None of the 37 identified MERS-RBD-binding human mAbs (from two patients) were encoded by IGHV3-53/IGHV3-66 and IGHJ6 (table S4) based on analysis of a prior study (17). Therefore, the presence of these stereotypic naïve IGH clonotypes in the healthy population, and their light chain plasticity needed to achieve SARS-CoV-2 RBD…”

Computational Design of Potent D-Peptide Inhibitors of SARS-CoV-2

“…51 The neutralization assay was performed as previously described. 52,53 Briefly, Vero cells were seeded in 96-well plates (1.5 × 10 4 cells/well) in Opti-PRO SFM (Thermo Scientific, Waltham, MA, USA) supplemented with 4 mM L-glutamine and 1× Antibiotics-Antimycotic (Thermo Scientific, Waltham, MA, USA) and grown for 24 h at 37 °C in a 5% CO 2 environment. Peptides were dissolved in 100% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and diluted in PBS (Welgene, Gyeongsan, Republic of Korea) from the maximum concentration of 100 μM to the lowest concentration of 0.1953 μM.…”

Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population

“…10). In our prior experiment, none of the 37 identified MERS-RBD-binding human monoclonal antibodies, from two patients, were encoded by IGHV3-53/IGHV3-66 and IGHJ6 (Supplementary Table 4) 19 . Therefore, the presence of these stereotypic-naïve IGH clonotypes in the healthy population, and their light chain plasticity to achieve SARS-CoV-2 RBD binding, may be unique to SARS-CoV-2, which might provide a rapid and effective humoral response to the virus among patients who express these clonotypes.…”