In a recent study posted to the bioRxiv* preprint server, researchers developed bispecific-antibodies (bsAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sub-lineages.

Study: Function and Cryo-EM structures of broadly potent bispecific antibodies against multiple SARS-CoV-2 Omicron sublineages. Image Credit: Naeblys/Shutterstock

Background

SARS-CoV-2 evolution is considered one of the numerous concerns of the continuing coronavirus disease 2019 (COVID-19) pandemic. Many lineages have emerged; some have grow to be dominant while others have diminished. SARS-CoV-2 Omicron was first reported in November 2021 in South Africa, which subsequently became the predominant variant in lots of countries. The mutations in Omicron’s spike protein have reduced the efficacy of SARS-CoV-2 vaccines and therapeutic monoclonal antibodies (mAbs).

Several sub-lineages have emerged from the unique Omicron lineage; these include BA.1, BA.2, BA.3, and BA.4/5. Although several vaccines have been developed, therapeutic mAbs are critical for COVID-19 treatment, particularly for hospitalized patients. Notably, just a few mAbs retained activity against all Omicron sub-lineages.

Thus, it is crucial to proceed developing countermeasures like newer therapeutic antibodies.

The study and findings

In the present study, researchers developed and characterised the bsAbs against the SARS-CoV-2 Omicron using 4 previously developed mAbs. Unlike antibody cocktails, bsAbs could goal two antigens. Five bsAbs were developed via the knob-into-hole (KIH) bispecific CrossMab technology using fragment antigen-binding (Fab) arms from 4 mAbs – PC.03, MB.02, and MB.08, and clone 13A. The resultant bsAbs were CoV2-0203, CoV2-0208, CoV2-0213, CoV2-0813, and CoV2-0803.

Enzyme-linked immunosorbent assay (ELISA) was performed with 4 recombinant RBD proteins from SARS-CoV-2 WA-1, Delta, Omicron BA.1, and BA.2 variants. Sotrovimab (S309), a clinical mAb, was also included within the assessments. Only CoV2-0813 and CoV2-0213 showed significantly lower half-maximal effective concentrations (EC50) to 4 RBD proteins, while the others had lower EC50 values against one or two RBD proteins. Furthermore, CoV2-0213 and CoV2-0813 competed with angiotensin-converting enzyme 2 (ACE2) for RBD.

In pseudovirus neutralization assays, S309 retained neutralizing activity against each BA.1 and BA.1.1, albeit it declined 30-fold against BA.2. In contrast, CoV2-0213 exhibited broad neutralization activity and neutralized BA.1, BA.1.1, and BA.2. CoV2-0213 was 78-fold stronger against BA.2 than S309. Moreover, CoV2-0203 and CoV2-0208 exhibited Omicron-specific neutralizing activity.

CoV2-0203 was potent against the three sub-lineages, albeit 1.5-fold weaker against BA.1 and BA.1.1 than CoV2-0213. CoV2-0203’s activity against BA.2 was comparable to that of S309. The team estimated CoV2-0213’s binding affinity with RBD proteins from three Omicron sub-variants using biolayer interferometry (BLI). BLI revealed a high affinity of CoV2-0213 for BA.1 and BA.2 RBDs. 

Further, cryo-electron microscopy (EM) structures of CoV2-0213 with considered one of its Fab arms (MB.02) complexed with multiple spike variants were resolved. Two conformations of the spike trimer were identified, one with a one-RBD-up state and the opposite with two RBD-up conformation. The spike trimer was certain to a few Fabs, one per RBD in either conformation. MB.02 binding caused flexibility of spike RBD, particularly within the up state.

The cross-reactivity of CoV2-0213 against SARS-CoV-2 and other human CoVs was evaluated by ELISA using six RBD proteins from BA.2, BA.2.12.1. BA.3, BA.4/5, SARS-CoV-1 and middle east respiratory syndrome (MERS)-CoV. Results indicated broad binding activity against Omicron RBDs, with moderate reactivity to SARS-CoV-1 and MERS-CoV RBDs. Further, BLI was performed to measure the binding affinity of CoV2-0213 towards RBD proteins from BA.2.12.1, BA.3, and BA.4/5.

BLI revealed a high binding affinity of CoV2-0213 against the three sub-variants. The opposite Fab arm of CoV2-0213 (clone 13A) primarily engages with the suitable ridge of RBD and won’t cause a steric clash with MB.02. The cryo-EM structure of CoV2-0213 certain to spike from the BA.5 variant was resolved. Just one conformation within the one-RBD-up state was detected.

Further, a subset of cryo-EM particles was identified with density for one Fab each for 2 RBDs within the down conformation and two Fabs on the identical RBD within the up conformation. This meant that three MB.02 and one clone 13A Fabs were certain to the identical spike trimer, possibly from three CoV2-0213 antibodies, with considered one of them having each arms certain to the trimer.

Conclusions

In summary, the authors created five bsAbs that neutralize a bunch of SARS-CoV-2 Omicron sub-variants. Of the five, CoV2-0213 showed high breadth and potency against multiple SARS-CoV-2 variants, including the key sub-lineages of Omicron. Further, cryo-EM revealed that each arms of CoV2-0213 could directly and concurrently engage with the identical spike trimer. The authors reported that CoV2-0213 was primarily derived from humans and might be subject to translational testing.

*Necessary notice

bioRxiv publishes preliminary scientific reports that are usually not peer-reviewed and, due to this fact, mustn’t be thought to be conclusive, guide clinical practice/health-related behavior, or treated as established information.