Generation and characterization of a multi-functional panel of monoclonal antibodies for SARS-CoV-2 research and treatment

Abstract

The Coronavirus disease 2019 (COVID19) pandemic caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is an ongoing threat to global public health. To this end, intense efforts are underway to develop reagents to aid in diagnostics, enhance preventative measures, and provide therapeutics for managing COVID-19. The recent emergence of SARS-CoV-2 Omicron variants with enhanced transmissibility, altered antigenicity, and significant escape of existing monoclonal antibodies and vaccines underlines the importance of the continued development of such agents. The SARS-CoV-2 spike protein and its receptor binding domain (RBD) are critical to viral attachment and host cell entry and are primary targets for antibodies elicited from both vaccination and natural infection. In this study, mice were immunized with two synthetic peptides (Pep 1 and Pep 2) within the RBD of the original Wuhan SARS-CoV-2, as well as the whole RBD as a recombinant protein (rRBD). Hybridomas were generated and a panel of three monoclonal antibodies, mAb CU-P1-1 against Pep 1, mAb CU-P2-20 against Pep 2, and mAb CU-28-24 against rRBD, were generated and further characterized. These mAbs were shown by ELISA to be specific for each immunogen/antigen. Monoclonal antibody CU-P1-1 has limited applicability other than in ELISA approaches and basic immunoblotting. Monoclonal antibody CU-P2-20 is shown to be favorable for ELISA, immunoblotting, and immunohistochemistry (IHC), however, not live virus neutralization. In contrast, mAb CU-28-24 is most effective at live virus neutralization as well as ELISA and IHC. Moreover, mAb CU-28-24 was active against rRBD proteins from Omicron variants B.2 and B.4/B5 as determined by ELISA, suggesting this mAb may neutralize live virus of these variants. Each of the immunoglobulin genes has been sequenced using Next Generation Sequencing, which allows the expression of respective recombinant proteins, thereby eliminating the need for long-term hybridoma maintenance. The synthetic peptide sequences, hybridomas, and related mAbs are now protected by Intellectual Property agreements with the Clemson University Research Foundation and are Patent Pending based on their unique amino acids within the complementary determining regions (CDRs).