Researchers at The University of Texas MD Anderson Cancer Center developed fully human bispecific antibodies that bind to two different epitopes of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and neutralize a wide range of variants, including the Omicron variant, in a recent study published on the bioRxiv* preprint server.
Background
SARS-CoV-2 is an enveloped, single-stranded ribonucleic acid (RNA) virus that enters mammalian host cells via interactions between the RBD in the S1 subunit of the spike protein and the mammalian cell’s angiotensin-converting enzyme-2 (ACE-2) receptor. The mammalian cell’s transmembrane serine protease 2 (TMPRSS2) aids viral entrance by cleaving the spike protein.
The accumulation of mutations in SARS-CoV-2 has resulted in the creation of several variations, including Alpha, Beta, Delta, and Omicron, with Omicron and its subvariants containing the most unique mutations. These alterations in the spike protein area have allowed the Omicron subvariants to avoid neutralizing antibodies elicited by coronavirus disease 2019 (COVID-19) vaccinations as well as monoclonal antibodies that have previously been employed to treat infections caused by previous variants.
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Although monoclonal antibody combinations remain somewhat successful against Omicron subvariants, bispecific monoclonal antibodies that bind to two separate epitopes on the virus simultaneously present a more time and cost-effective approach that should be investigated further.
Concerning the research
The researchers used single B cell cloning and humanized transgenic mice to create monoclonal antibodies capable of binding and neutralizing several SARS-CoV-2 variants. Female ATX-GK mice were inoculated subcutaneously with spike protein dissolved in phosphate-buffered saline and an adjuvant, and one booster dose was given per week for three weeks. An additional booster dosage was injected intraperitoneally three days following the third booster dose, and the mice were euthanized four days after the last booster dose. To obtain antibodies, bone marrow, spleen, blood, and draining lymph nodes were collected.
Using a single B-cell closure method, memory B cells from alloy humanized transgenic mice were used to amplify the varied heavy and light chains. To create the bispecific monoclonal antibodies, two antibody clones that identify separate spike protein epitopes were chosen. Micro-scale transfection with a single expression vector produced in Chinese hamster ovary cells was employed for the high-throughput manufacture of monoclonal antibodies.
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The antibodies were purified from the culture supernatant using fast protein liquid chromatography, and the concentration of the monoclonal antibodies was measured using a NanoDrop. In addition, horseradish peroxidase (HRP)-conjugated anti-monoclonal immunoglobulin G (mIgG) titers of SARS-CoV-2-specific antibodies were detected, and the bio-layer interferometry (BLI) assay was utilized to screen for anti-SARS-CoV-2 monoclonal antibodies. Additionally, ACE-2-blockage assays and pseudovirus experiments were performed to see if the monoclonal antibodies could attach to the viral and block ACE-2 receptor binding.
Results
According to the findings, the two selected monoclonal antibodies — SARS-CoV2-83 and SARS-CoV2-81 — had moderate to strong affinities for numerous SARS-CoV-2 variations, including the Omicron form. The two monoclonal antibodies, however, were unable to attach to one of the Delta variant’s mutations (L452R). Bispecific antibodies created by pairing the heavy or light chains of SARS-CoV2-81 with the single chain variable segment of SARS-CoV2-83 bind to all SARS-CoV-2 variations tested, including the Delta variant L452R mutant.
Additionally, the bispecific antibodies bound to the RBD of the Omicron variation with greater avidity than the two monoclonal antibodies did independently. In the in vitro assay, one of the bispecific antibodies (Bs-A) was also able to neutralize the Omicron pseudovirus. These findings suggested that bispecific antibodies were more avid while binding and had stronger neutralizing capacity than the two parent monoclonal antibodies.
Conclusions
Overall, the findings showed that the fully human, bispecific monoclonal antibodies created using two unique monoclonal antibodies, SARS-CoV2-83 and SARS-CoV2-81, bind and neutralize several SARS-CoV-2 variations, including the Omicron variant. SARS-CoV2-81 avidity was also shown to be comparable to GSK’s currently used Sotrovimab, indicating that it could be a promising monoclonal antibody candidate for clinical application.
While bispecific antibodies are effective against multiple SARS-CoV-2 variants, the authors believe that the rapid emergence of new variants calls into question the efficacy of these antibody therapies, emphasizing the ongoing need for the development of broadly neutralizing bispecific antibodies that target different epitopes.
