In a recent study posted to the bioRxiv* preprint server, researchers proposed and constructed receptor-blocking conserved non-neutralizing antibodies (ReconnAbs) effective towards the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) treatment.
Out of the seven monoclonal antibodies (mAbs) authorized for the coronavirus disease 2019 (COVID-19) treatment, all but sotrovimab were not therapeutically effective against the omicron variant infection. Hence, there is a pressing need to develop therapeutic agents efficient in mitigating the evolution of SARS-CoV-2 and its variants of concern (VOC).
In the present study, the researchers hypothesized and developed broad-spectrum SARS-CoV-2 inhibitors, ReconnAbs, by modifying the existing non-neutralizing antibodies targeted towards the highly conserved epitopes of the SARS-CoV-2 spike (S) protein combined with a receptor-blocking component. The ReconnAbs are broadly neutralizing since they target the conserved epitopes in SARS-CoV-2.
Non-neutralizing, cross-reactive SARS-CoV-2 antibodies were profiled via two techniques; 1) analysis of a collection of SARS-CoV-2 antibodies phylogenetic tree and, 2) excluding those likely to bind with the receptor-binding domains (RBD).
The affinity of the non-RBD antibodies towards SARS-CoV-1 spike protein was determined to be similar to the development of the mAb, sotrovimab. Further, the team evaluated and developed the ReconnAb’s neutralizing capacity of SARS-CoV-2 variants of concern (VOCs) and a bispecific ReconnAb capable of neutralizing all SARS-CoV-2 VOCs.
The results indicated that from the 696 antibody sequences that bound to the S protein outside of the RBD of COVID-19 convalescent donors, the researchers developed phylogenetic trees for antibody light chain (LC) and heavy chain (HC) using amino acid sequences of full-length V-genes, one allele of each germline V-gene, and the CDR3 region.
Further, the team constructed non-RBD-binding antibodies by clustering 48 non-RBD-binding sequences within the HC phylogenetic tree, and these sequences also showed diversity in the LC phylogenetic tree.
The 48 non-RBD-binding antibodies showed 82% staining with the SARS-CoV-2 S and 21% staining with the SARS-CoV-1 S when tested using the SARS-CoV-1 spike as a surrogate for epitope conservation.
Further, 10 antibody sequences that target highly conserved regions of the SARS-CoV-2 S protein were identified using fluorescence-activated cell sorting (FACS) and SARS-CoV-1 spike protein as bait. Of the 10 antibodies, seven were strong binders of SARS-CoV-1, and one antibody, COV2-2449, also binds to OC43 and MERS S proteins, confirmed via biolayer interferometry (BLI).
Among the seven antibodies, five were unique for SARS-CoV-2 in a binding competition assay between SARS-CoV-1 and SARS-CoV-2 S using BLI. The five non-neutralizing, cross-reactive antibodies were converted into ReconnAbs by fusion to the angiotensin-converting enzyme 2 (ACE2) ectodomain, which acts as the receptor-blocking element in the ReconnAb design. The SARS-CoV-2 neutralizing capacity of ReconnAbs depends on both its binding and inhibitory components. Moreover, the SARS-CoV-2 neutralization capacity of ReconnAbs will be lost if the linker joining the inhibitory and binding components is cleaved at the TEV protease site.
Additionally, a bifunctional ReconnAb, CV10-2449-ACE2-CrossMAb, developed by linking ACE2 with a bispecific antibody targeting two non-overlapping conserved epitopes, CV10 and COV2-2449, demonstrated neutralizing activity against all SARS-CoV-2 VOCs, including Omicron, at sub-nanomolar concentrations.
The study findings indicated that ReconnAbs developed by converting cross-reactive, non-neutralizing antibodies, potentially act as broad-spectrum antiviral agents against SARS-CoV-2 and other emerging pandemic diseases due to their conserved targets and modular nature.
The usage of the ACE2 domain as the inhibitory component supported the proof-of-concept of the ReconnAb design since the therapeutics containing ACE2 are known to elicit autoimmunity in humans. RBD-directed mAbs, ACE2 domains with enhanced RBD-binding activity or aptamers can be used instead of the ACE2 module.
A limitation of the present study is that the library of SARS-CoV-2 non-RBD-binding antibodies was profiled from the early COVID-19 pandemic sequences, thus does not account for the cross-reactive, non-neutralizing vaccine-derived antibodies.
Developing future ReconnAb designs considering the below-mentioned recommendations will increase their therapeutic applications not just for SARS-CoV-2 but also viruses like influenza, human immunodeficiency virus-1 (HIV-1), or other human coronaviruses:
Firstly, by assessing elements like fusion partners, modifications to the Fc domains, linkage locations, and length. Secondly, by targeting the interaction of dipeptidyl peptidase 4 (DPP4), which is a receptor for other coronaviruses, as well as accounting for a wide SARS-CoV-2 non-RBD library of antibodies. Finally, therapeutic applications can also be increased by focusing on neutralizing antibodies that bind to conserved epitopes, as well as evaluating existing antibody libraries for highly conserved, non-neutralizing binders.
Overall, the study emphasizes that the rapid administration of customized ReconnAbs in a pandemic setting helps ease the initial impact of a new pathogen and buys more time for other mitigation measures or therapeutics to be devised.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.