Development of neutralizing single domain antibodies against SARS-CoV-2 virus.

PAVAN MF; BOK M; MALITO JP; MARCOPPIDO G; FRANCO D; SCHAMMAS J; BAUMEISTER E; AUGUSTE A; STONE W; YAUN L; WIGDOROVITZ A; PARREÑO V; IBAÑEZ LI

Virtual

Simposio; Antibodies and Vaccines as Drugs for COVID-19; 2021

Virtual Keystone eSymposia

The recent emergence of the disease caused by the SARS-CoV-2 virus,COVID-19, is the third major coronavirus outbreak in the past 20 years. Todate, there have been more than 79.9 million confirmed cases of COVID-19 andover 1.75 million deaths reported to the World Health Organization. In spite ofthe recent approval of vaccines in several countries, the development ofmolecules with therapeutic and prophylactic capacity remains essential as a wayto mitigate the effects of the disease, especially for persons for whomvaccines are ineffective or contraindicated. The SARS-CoV-2 spike protein (S), whichcontains the receptor-binding domain (RBD), is a potential therapeutic target. Singledomain antibodies (sdAbs) or nanobodies are small molecules with highstability, solubility and affinity that can be produced at low cost. For thisreason, in this work we propose the development of neutralizing sdAbs to blockthe SARS-CoV-2 infectioIn order to obtain sdAbs, a llama was immunized with the pre-fusion S and RBD proteinsexpressed in the HEK-293T cell line. The humoral immune response was assessedthroughout the immunization schedule, and high antibody titers against bothproteins were observed. To generate a sdAbs library, total RNA from a llamaperipheral blood lymphocytes was extracted and used as template to produce cDNA.sdAbs encoding sequences were amplified by PCR and cloned in the phagemidvector pMECS-GG. More than 80 clones of sdAbs against S and RBD proteins wereselected by phage display. From these clones, 52 unique sdAbs were expressed inEscherichia coli WK6 and purified by immobilized metal chelatechromatography (IMAC). At least 10 sdAbs were able to block transduction in a pseudovirusneutralization assay as well as infection of Vero cells with the wild-typeSARS-CoV-2 virus circulating in Argentina and the United States of America.These sdAbs will be modified so as to generate bivalent antibodies, and theirability to inhibit the SARS-CoV-2 infection in vivo will be assessed. In conclusion, we have developed sdAbs capable of neutralizing theSARS-CoV-2 virus in vitro. These promising results make Argentina one ofthe few countries in Latin America capable of producing this new type of technologyand turning these sdAbs into therapeutic products.