Next-generation sequencing (NGS)-enabled analysis of immune repertoires is commonly used to identify antibodies; however, the technique has yet to be standardized for the identification of miniature nanobodies, which are also known as nanobodies. To help close this gap and facilitate nanobody discovery, Karolinska Institute scientists have developed a new approach that combines next-generation, phage display, and multivariate enrichment.

Using this approach, the scientists identified numerous anti-SARS-CoV-2 nanobodies. The scientists report that in cell cultures and mice, the nanobodies blocked infections in experiments with different SARS-CoV-2 variants.

Detailed findings appeared in Science Advances, in an article titled, “Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralizing SARS-CoV-2 nanobodies.”

“The most potent nanobodies bind to the receptor-binding motif of the receptor-binding domain (RBD), and we identify two exceptionally potent members of this category,” the article’s authors wrote. “Other nanobodies bind to a more conserved epitope on the side of the RBD and are able to potently neutralize the SARS-CoV-2 founder virus, the Beta variant (B.1.351/501Y.V2), and also cross-neutralize the more distantly related SARS-CoV-1.”

The study’s authors, who were led by microbiologist Gerald McInerney, PhD, and computational biologist Ben Murrell, PhD, suggested that their new approach was less laborious than conventional approaches for the isolation and characterization of nanobodies. In addition, the authors asserted that their approach was well suited for “the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.”

Despite the roll-out of vaccines and antivirals, the need for effective therapeutics against severe COVID-19 infection remains high. Nanobodies—which are fragments of antibodies that occur naturally in camelids and can be adapted for humans—are promising therapeutic candidates as they offer several advantages over conventional antibodies. For example, they have favorable biochemical properties and are easy to produce cost-effectively at scale.

“We were able to identify a panel of nanobodies that very effectively neutralized several variants of SARS-CoV-2,” McInerney said. According to first author Leo Hanke, PhD, a postdoctoral researcher who established the nanobody technology in the McInerney group, “These nanobodies represent promising therapeutic candidates against several SARS-CoV-2 variants.”

The researchers are currently applying the same techniques to identify which nanobodies from this set are best able to neutralize Omicron, the now dominating SARS-CoV-2 variant.

“Once established, these libraries can be expanded and mined for nanobodies that neutralize new emerging variants,” added Murrell.

The current study builds on work that was described a few months ago in Nature Communications, in a paper titled, “A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo.” This paper described a single nanobody, Fu2 (named after the alpaca Funny), that significantly reduced the viral load of SARS-CoV-2 in cell cultures and mice. The paper also described how the researchers used electron cryo-microscopy to determine that Fu2 naturally binds to two separate sites on the viral spike, thus inhibiting the virus’s ability to enter the host cell.

Extending this work, the researchers delved deeper into the alpaca’s nanobody repertoire, as described in the Science Advances paper. “Multiplexed NGS of the starting library and of each distinct enrichment step provides us with massively parallel information about the affinity of individual nanobodies against each target,” the paper’s authors detailed. “In contrast to conventional panning and colony picking, this approach relies on enrichment metrics instead of postpanning abundance, which enables the identification of high-affinity nanobodies even when they exist at low abundance in the baseline library, and are not sufficiently enriched to be sampled during traditional colony picking.”