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

PAVAN MARÍA FLORENCIA; BOK MARINA; MALITO JUAN PABLO; MARCOPPIDO GISELA ARIANA; FRANCO DIEGO RAFAEL; SCHAMMAS JUAN MANUEL; BAUMEISTER ELSA; AUGUSTE ALBERT J; STONE WILLIAM B; YUAN LIJUAN; WIGDOROVITZ ANDRES; PARREÑO VIVIANA; IBAÑEZ LORENA ITATÍ

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

Keystone Symposia & Digitell, Inc

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. To date, there have been more than 79.9 million confirmed cases of COVID-19 and over 1.75 million deaths reported to the World Health Organization. Despite the recent approval of vaccines in several countries, the development of therapeutic and prophylactic molecules is still essential as a way to mitigate the effects of the disease, especially in cases of vaccine inefficiency or contraindication. The SARS-CoV-2 spike protein (S), containing the receptor-binding domain (RBD), is a potential therapeutic target. Llama-derived single domain antibodies (sdAbs) or nanobodies are small molecules with high stability, solubility and affinity that can be produced at low cost. For this reason, in this work we propose the development of neutralizing sdAbs to block the SARS-CoV-2 infection.In order to obtain sdAbs, a llama was immunized with the pre-fusion and locked S and RBD proteins expressed in the HEK-293T cell line. After immunization high antibody titers against both proteins with neutralizing activity were observed. To generate a sdAb library, total RNA from the llama peripheral blood lymphocytes was extracted and used as template to produce cDNA. sdAbs encoding sequences were amplified by PCR and cloned in the phagemid vector pMECS-GG. More than 80 clones of sdAbs against S and RBD proteins were selected by phage display. From these clones, 52 unique sdAbs were expressed in Escherichia coli WK6 and purified by immobilized metal chelate chromatography (IMAC) followed by size exclusion chromatography (SEC). At least 10 sdAbs were able to block transduction in a pseudovirus neutralization assay as well as infection of Vero cells with the wild-type SARS-CoV-2 virus circulating in Argentina and the United States of America. These sdAbs will be modified so as to generate bivalent antibodies, and their ability to inhibit the SARS-CoV-2 infection in vivo will be assessed in animal models. The strong neutralizing activity of some of these molecules highlights their potential to develop an intranasal treatment for COVID-19, that will not need cold chain. In conclusion, we have developed sdAbs capable of neutralizing the SARS-CoV-2 virus in vitro. These promising results make Argentina one of the few countries in Latin America capable of producing this new type of technology and turning these sdAbs into therapeutic products.