The respiratory tract is the major target for SARS-CoV-2 infection. High respiratory viral load correlates with severe disease in patients. To date, almost all neutralizing monoclonal antibodies (mAbs) that have been tested in clinical trials are the IgG1 isotype and are administered through intravenous (IV) infusion. Importantly, emerging SARS-CoV-2 VOCs are resistant to many potent neutralizing IgG1 mAbs, including those in clinical trials and authorized for emergency use. Therefore, developing new antibody therapies that can overcome these challenges is urgently needed.
IgM and IgA are mucosal antibodies that constitute the first line of defense against mucosal pathogens. Typically, IgM assembles into pentamers and IgA1 into dimers in the presence of the joining chain (J-chain), which facilitates efficient mucosal transcytosis of antibodies. IgM and IgA1 can also be nebulized and reach airway tissues after inhalation. Due to avidity effects, multivalent antibodies can exhibit enhanced neutralization of SARS-CoV-213 and reduce antibody evasion by virus. An IgM pentamer is naturally decavalent owing to the repetitive antigen-binding variable fragments (Fvs). These unique features make IN delivery of IgM neutralizing mAbs appealing for COVID-19 prevention and treatment.
Engineering of neutralizing IgM and IgA1
To develop human IgM and IgA1 neutralizing mAbs, antibody engineering was performed based on the CR3022 mAb15 and five IgG1 mAbs (CoV2-06, 09, 12, 14, 16) previously isolated from a phage-displayed antibody library. These six mAbs recognize different epitopes on the RBD.
The Fv of IgG1 was engineered into human IgM or IgA1 scaffolds for co-expression with the J-chain. The engineered IgM is a pentamer and IgA1 is a dimer. After production, the mAbs of different isotypes correctly assembled and showed >95% purity. IgM and IgA1 bound to RBD more strongly than did IgG1. These results demonstrate successful engineering of IgM and IgA1 mAbs.
Enhanced potency of IgM over IgG
To examine how epitopes affect the potency of engineered IgMs, we also characterized the IgM and IgG1 pairs of CoV2-06 (IgM-06 and IgG-06). IgM-14 showed much stronger ELISA binding to spike (S) than IgG-14.
In kinetic binding, IgM-14 demonstrated faster association to and slower dissociation from S than did IgG-14. In neutralization titrations on Vero and human ACE2-overexpressing A549 (A549-ACE2) cells, IgM-14 dramatically shifted the curves toward higher potency than did IgG-14.
The contrast between CoV2-06 and CoV2-14 supports the conclusion that selection of IgG1 epitope is critical for identifying the most potent neutralizing IgM. To understand the structural mechanism of this observation, antibody docking was performed to simulate the Fv and RBD complex using Rosetta-based protocols. IgM-14 inhibited RBD/ ACE2 interaction more strongly than did IgG-14. In contrast, IgFv-06 has a smaller steric clash with ACE2. IgM-06 also blocked RBD/ACE2 interaction more strongly than did IgG06, however, neither IgM-06 nor IgG-06 achieved full blocking even at the highest concentration tested. Results demonstrate that epitope-dependent steric hindrance is an important mechanism for IgM-14 to exhibit potent neutralization.
Broad coverage of variants by IgM-14
SARS-CoV-2 escapes from antibody neutralization by acquiring mutations in resistance-selection experiments and in natural circulation. Previously has identified neutralization-resistant RBD mutations K444R for IgG-06 and E484A for IgG-141 . To test whether IgM can neutralize these IgG-escape mutants, three SARS-CoV-2 variants were constructed that contain K444R, E484A or K444R+E484A mutations.
IgG-14 effectively neutralized the K444R variant and marginally neutralized the E484A and K444R+E484A variants. Importantly, IgM14 potently neutralized all three variants including the K444R+E484A variant, which is resistant to the cocktail of IgG-06+IgG-14.
IgM-14 is 2,343- and 1,949-fold more potent than IgG-14 in neutralizing the E484A and K444R+E484A variants, respectively. In contrast, IgM-06 only neutralized the E484A variant but not the K444R or K444R+E484A variants.
These data demonstrate that IgM-14 can effectively neutralize IgG-14 escape variants.
To assess the neutralizing activities of IgM-14 and IgG-14 against the recently emerged SARS-CoV-2 VOCs, recombinant viruses were consteucted based on the US-WA1 strain and replaced its full spike gene with that of the B.1.1.7, P.1, and B.1.351 variants. The data demonstrate that IgM-14, but not IgG-14, can tolerate the VOC RBD mutations. IgM-14 is superior to IgG14 in covering viral escape mutations.
IN-delivered IgM-14 targets airway
The feasibility of IgM-14 was evaluated for IN administration by tracking antibody bio-distribution in mice. After a single IN dose, IgM14 was enriched in the nasal cavity and lasted for at least 96h in whole body imaging.
IgM-14 enriched in the nasal cavity and lung at various time points and was still evident in the nasal cavity at 168h. The blood and other organs had minimal antibody exposure. These results indicate that IN-dosed IgM-14 mainly targets the respiratory tract with long-term retention in the nasal cavity and lung. Nasal epithelium is the dominant initial site for SARS-CoV-2 respiratory tract infection, followed by aspiration of virus into the lung. Therefore, IN administration can efficiently load IgM-14 to the airway, which should confer protection against respiratory infection.
In vivo protection of VOCs by IgM-14
The protective efficacy of IgM-14 was evaluated using IN administration with a mouse-adapted virus that contains an N501Y mutation. Three isotypes of CoV2-06 and CoV2-14 at a prophylactic dose of 3.5 mg/kg were tested.
Peak lung viral loads on day 2 post-infection were reduced to undetectable levels in all four mice of the IgG-06 and IgM-06 groups and in three of the four mice of the IgA1-06, IgG-14, IgA1-14 and IgM-14 groups.
Then the focus was on IgM-14 and performed dose range evaluations. Five dose levels (3.5, 1.2, 0.4, 0.13, and 0.044 mg/kg) were tested for prophylactic treatment and three dose levels (3.5, 1.2, and 0.4 mg/kg) were tested for therapeutic treatment.
The data demonstrate that IgM-14 confers protection with an effective dose as low as 0.044mg/kg for prophylactic treatment and 0.4mg/kg for therapeutic treatment. IN administration of IgM-14 confers highly efficacious respiratory protection and that IgM-14 is superior to IgG-14 for protection against the VOCs.
Preclinical pharmacokinetics and safety
To further evaluate the translational potential of IgM-14, an IN pharmacokinetic study in mice was conducted.
A single IN administration of 5mg/kg of IgM-14 resulted in low levels of antibody in the blood (~100ng/ml) that persisted for several hours. In COVID-19 patients, the positive rate of viral RNA is high (93%) in bronchoalveolar lavage samples but very low (<1%) in blood samples. Therefore, IN-administered IgM-14 is more focused on targeting the site of virus replication in the respiratory tract compared to IV-infused IgG1. To further assess the tolerability of IgM-14, a pilot safety study was conducted in rats. Groups of rats were IN-dosed with IgM-14 twice daily for five consecutive days. All animals survived to study termination with no change in body weight.
In summary, they demonstrate engineered IgM as a promising drug modality with potent neutralization, broad coverage of variants, desirable pharmacokinetics and safety profiles, and effective respiratory protection. They envision that engineered, IN-administered IgM can serve as a new therapeutic platform for COVID-19 as well as for other respiratory viral diseases.