# Neutralization of SARS-CoV-2 by IgM-14 via engagement of two distinct spike epitopes

## TL;DR

This study explores an engineered IgM antibody, IgM-14, which strongly neutralizes early SARS-CoV-2 variants but loses effectiveness against newer Omicron subvariants, offering insights into improving antibody therapies.

## Contribution

The study reveals a novel mechanism of IgM-14 neutralization through dual epitope engagement on the spike protein, which could guide the design of more effective antibodies.

## Key findings

- IgM-14 neutralizes pre-Omicron variants but is ineffective against Omicron BA.1 and JN.1.
- Two RBD mutations (G476D and F486P) confer resistance to IgM-14.
- IgM-14 binds two distinct spike epitopes, including a secondary site in the 'down' conformation.

## Abstract

Engineered immunoglobulin M (IgM) antibodies typically exhibit superior neutralization potency and avidity compared to their parental IgG counterparts, primarily due to multivalent binding to repeated epitopes on a targeting antigen. In this study, we characterize the neutralization breadth and mechanism of action of IgM-14, a previously reported intranasally deliverable antibody targeting SARS-CoV-2. IgM-14 demonstrates remarkably potent antiviral activity against all pre-Omicron variants but significantly reduced efficacy against Omicron BA.1, and complete loss of activity against the later subvariant JN.1. Resistance selection identified two key mutations in the receptor-binding domain (RBD), G476D and F486P, which disrupt IgM-14 binding and confer strong resistance. Cryo-electron microscopy analysis uncovered two distinct Fab-RBD interfaces: a primary interface overlapping the angiotensin-converting enzyme 2 (ACE2)-binding region, and a unique secondary interface formed only when the RBD adopts the ACE2-inaccessible “down” conformation, involving a neighboring spike protomer. Site-directed mutagenesis and structural modeling revealed a critical role of this secondary site in IgM-14-mediated neutralization. Unlike IgG-14, structural modeling suggested that IgM-14 can simultaneously engage both interfaces in diverse modes, indicating a noncanonical avidity mechanism. Collectively, these findings highlight the structural and functional uniqueness of IgM-14 and offer valuable insights into the rational design of next-generation spike-targeted antibody therapeutics with enhanced breadth and potency.

Antibody therapies are essential for COVID‑19 prevention and treatment. However, many traditional IgG‑based antibodies have become less effective as SARS‑CoV‑2 continues to evolve. Engineered IgM antibodies offer advantages because they can bind to viruses at multiple sites simultaneously, increasing both strength and breadth of protection. In this study, we investigated IgM‑14, an engineered IgM antibody designed for intranasal delivery. We found that IgM‑14 strongly neutralizes early SARS‑CoV‑2 variants but loses effectiveness against Omicron lineages, including complete inactivation against the recent JN.1 variant. We also identified two key spike mutations that enable the virus to escape IgM‑14. To understand the basis of IgM‑14’s activity, we used cryo‑electron microscopy to examine how it binds to the viral spike protein. Surprisingly, the antigen‑binding fragment of IgM‑14 engages two distinct sites on the receptor‑binding domain: a primary site that overlaps that of the ACE2 receptor, and a second site that appears only when the spike is in the “down” position. Structural modeling and mutational analysis reveal that this secondary site contributes to IgM‑14’s unusually strong neutralizing ability. Our work highlights a previously underappreciated mechanism by which engineered IgM antibodies achieve high potency and offer insights for designing next‑generation antibodies that better resist viral evolution.