Special antibodies that llamas make can be directed against SARS-CoV-2 to help find our way out of the pandemic

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Rocky and Marley are used to the company of scientists. Like many other members of their species, the two llamas, living in pastures in rural Massachusetts, have for many years participated in research aimed to harness the wonders of their immune system.

Today, however, the stately creatures are facing an unprecedented level of international attention: scientists hope the special antibodies that llamas make can be directed against SARS-CoV-2 to help find our way out of the pandemic.

Humans, too, make antibodies against SARS-CoV-2, and many groups are working on developing treatments based on them. Llama antibodies, however, come in a simpler design than their human counterparts.

“For reasons that we don’t really understand, these animals make this variant of antibody that just has fantastic properties,” says Michael P. Rout, a structural biologist at Rockefeller.

“It contains the good disease-recognizing parts of a human antibody, packed into a condensed warhead.”

This “warhead” is just one-tenth the size of a normal antibody, and can be cloned out to form miniature antibodies, termed nanobodies. Easy to mass-produce, nanobodies are an attractive source for developing treatments that boost people’s immunity to a particular pathogen.

“We need doses of therapeutics and diagnostic tests for many millions of people,” says biochemist Brian T. Chait. “Nanobodies could become one more weapon in our arsenal against COVID-19, and potentially a widely available one.”

In order to get there, Rout, Chait and their colleagues are now extracting antibodies from llamas and examining their molecular properties to identify those most effective against the virus.

Like all coronavirus research, this project is in its infancy; but if successful, it will allow the scientists to advance these potent antibodies towards the development of both treatments and diagnostic tests.

Their study is one of nearly 20 COVID-19 projects that have been launched by Rockefeller researchers since early March in an effort to better understand the SARS-CoV-2 virus and speed the development of new treatments.

Credit: Rockefeller University

Natural arsenal

In most mammals including humans, a typical antibody consists of two proteins arranged in a sophisticated formation of four protein subunits. Llamas, camels, and other species of the Camelidae family make antibodies consisting of only one protein that, in spite of their simplicity, have been shown to be highly effective.

Researchers hope that llama nanobodies will turn out to have some unique advantages over human antibodies when it comes to fighting SARS-CoV-2.

Their small size may allow them to better access the dense pack of spike proteins that cover the surface of the coronavirus and enable its entry into host cells.

While small, they can also be more stable, and potentially could be nebulized and taken in an inhaler, like asthma medicines, as opposed to an injection. This means therapeutic antibodies will be brought directly to the sites of viral replication in our lungs and airways.

Moreover, it’s possible to combine multiple nanobodies, each targeting a different part of the virus, into one super molecule that hits multiple sites at once. “Because they are so small we can treat them like little molecular Lego and test combinations that the virus can’t wiggle its way out of.”

Marley and Rocky are cared for by Capralogics, a facility that provides antibody production services for research, and for development of diagnostics. There, the team’s collaborators give the llamas an injection of coronavirus proteins, like a vaccination.

The llamas don’t get sick but their immune system starts to produce antibodies against the virus. Antibodies are naturally highly diverse molecules – each individual produces a huge variety, of which only a fraction is adequately effective against the pathogen. “Camelid immune systems are amazing – they have terrific capability of quality control, producing stable and potent antibodies,” Chait says.

“So we let the masters, Rocky and Marley, figure it out for us.” After a few booster shots, blood samples are taken and sent to the researchers. “That’s where the llamas’ role stops,” Rout says. “They then go off and gallop away into their paddock.”

To analyze the sample, Rout and Chait use methods they have previously developed for an efficient nanobody identification and production pipeline: The antibody-producing cells are isolated and their DNA is sequenced.

Simultaneously, researchers examine the binding properties of the antibodies, using mass spectrometry to identify which have the right properties. They then search for the DNA sequence of those select antibodies, using custom software called “Llama Magic,” and prepare them to be expressed in bacteria for mass production – a step that will allow further lab tests to scrutinize the antibodies as candidates for drug development.

Since they were recognized in the 1990s, camelid antibodies have been studied by various research groups for developing treatments for a range of diseases, from the flu to cancer, with the first of such drugs, caplacizumab, approved last year for treating a blood-clotting disorder.

Now, many camelid researchers around the world have shifted their work to COVID-19, each using slightly different methods, to increase the chances of finding antibodies that will successfully work in people. “Given the enormity of the current crisis, we just need as many people as possible to try as many things as possible,” Rout says.


DISCUSSION

Here, we report the isolation and characterization of two potently neutralizing single-domain antibodies from a llama immunized with prefusion-stabilized MERS-CoV and SARS-CoV-1 spikes. These VHHs bind to the spike RBDs with high affinity and are capable of neutralizing S pseudotyped viruses in vitro. To our knowledge, the isolation and characterization of SARS-CoV-1

S-directed VHHs have not been described before. Several MERS-CoV S-specific VHHs have been described, all of which have been directed against the RBD. Several of these VHHs have also been reported to block DPP4 binding, much like MERS VHH-55 (Stalin Raj et al., 2018; Zhao et al., 2018).

By solving the crystal structures of these newly isolated VHHs in com- plex with their respective viral targets, we provide detailed in- sights into epitope binding and their mechanisms of neutralization.
A number of RBD-directed conventional antibodies that are capable of neutralizing SARS-CoV-1 or MERS-CoV have been described. The epitope of MERS VHH-55 overlaps with the epi- topes of several of these MERS-CoV RBD-directed antibodies including C2, MCA1, m336, JC57-14, D12, 4C2, and MERS-27

(Chen et al., 2017; Li et al., 2015; Wang et al., 2015; 2018; Ying et al., 2015; Yu et al., 2015) (Figure S7A). The epitope of SARS VHH-72 does not significantly overlap with the epitopes of any previously described antibodies other than that of the recently described CR3022, which can also bind to the RBDs of both SARS-CoV-1 and SARS-CoV-2 S (Hwang et al., 2006; Pak et al., 2009; Prabakaran et al., 2006; Walls et al., 2019; Yuan et al., 2020) (Figure S7B). However, unlike SARS VHH-72, CR3022 does not prevent the binding of ACE2 and it lacks neutralizing activity against SARS-CoV-2 (Tian et al., 2020; Yuan et al., 2020).

This discrepancy in function, despite the partially overlapping epitope, is likely due to the different angles of approach that these two antibodies adopt (Figure S7C). Because SARS VHH-72 binds with a nanomolar KD to a portion of the SARS-CoV-1 S RBD that exhibits low sequence variation, as demonstrated by its cross-reactivity with the WIV1-CoV and SARS-CoV-2 RBDs, it may broadly bind S proteins from other SARS-CoV-like viruses. We show that by engineering a bivalent VHH-72-Fc construct, we can compensate for the rela- tively high off-rate constant of the monovalent SARS VHH-72.

This bivalent molecule expresses well in transiently transfected ExpiCHO cells (~300 mg/L) and can neutralize SARS-CoV-2 S pseudoviruses in vitro. Future panning efforts using existing li- braries and SARS-CoV-2 S may yield even more potent neutralizers.
Because of the inherent thermostability and chemostability of VHHs, they have been investigated as potential therapeutics against several diseases. HIV- and influenza-directed VHHs have been reported previously, and there are multiple RSV-

directed VHHs that have been evaluated (Detalle et al., 2015; Iban˜ ez et al., 2011; Koch et al., 2017; Rossey et al., 2017). The possibility of administering these molecules via a nebulized spray is particularly attractive in the case of respiratory patho- gens because the VHHs could theoretically be inhaled directly to the site of infection in an effort to maximize bioavailability and function (Larios Mora et al., 2018).

Because of the current lack of treatments for MERS, SARS, and COVID-19 and the devastating effects associated with pandemic coronavirus out- breaks, both prophylactic and therapeutic interventions are sorely needed. It is our hope that because of their favorable bio- physical properties and their potent neutralization capacity, MERS VHH-55, SARS VHH-72, and VHH-72-Fc may serve as both useful reagents for researchers and as potential therapeutic candidates.



Figure 5. Neutralizing Mechanisms of MERS VHH-55 and SARS VHH-72
(A) The MERS-CoV spike (PDB ID: 5W9H) is shown as a transparent molecular surface, with each monomer colored either white, gray, or tan. Each monomer is bound by MERS VHH-55, shown as blue ribbons. The clash between MERS VHH-55 bound to the white monomer and the neighboring tan RBD is highlighted by the red ellipse.
(B)  The SARS-CoV-1 spike (PDB ID: 5X58) is shown as a transparent molecular surface, with each protomer colored either white, gray, or pink. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle.
(C)  The SARS-CoV-2 spike (PDB ID: 6VXX) is shown as a transparent molecular surface, with each protomer colored either white, gray, or green. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. The SARS-CoV-2 trimer appears smaller than SARS-CoV-1 S because of the absence of flexible NTD-distal loops, which could not be built during cryo-EM analysis.
(D)  CoV VHHs prevent MERS-CoV RBD, SARS-CoV-1 RBD, and SARS-CoV-2 RBD-SD1 from interacting with their receptors. The results of the BLI-based receptor-blocking experiment are shown. The legend lists the immobilized RBDs and the VHHs or receptors that correspond to each curve.
Figure 6. SARS VHH-72 Bivalency Permits SARS-CoV-2 Pseudovirus Neutralization
(A and B) SARS-CoV-1 S (A) and SARS-CoV-2 S (B) VSV pseudoviruses were used to evaluate the neutralization capacity of SARS VHH-72 and SARS VHH-6. MERS VHH-55 and PBS were included as negative controls. Luciferase activity is reported (n = 3 ±SEM) in counts per second (c.p.s.). NI, cells were not infected. (C and D) Binding of monovalent and bivalent VHHs was tested by ELISA against SARS-CoV-1 S (C) and SARS-CoV-2 RBD-SD1 (D). VHH-72-Fc refers to SARS VHH-72 fused to a human IgG1 Fc domain by a GS(GGGGS)2 linker. VHH-72-Fc (S) is the same Fc fusion with a GS, rather than a GS(GGGGS)2, linker. GBP is an irrelevant GFP-binding protein. VHH-72-VHH-72 refers to the tail-to-head construct with two SARS VHH-72 proteins connected by a (GGGGS)3 linker. VHH-23- VHH-23 refers to the two irrelevant VHHs linked via the same (GGGGS)3 linker.
(E and F) SARS-CoV-1 S (E) and SARS-CoV-2 S (F) pseudoviruses were used to evaluate the neutralization capacity of bivalent VHH-72-Fc. GBP and PBS were included as negative controls. NI, cells were not infected.
Figure 7. VHH-72-Fc Neutralizes SARS-CoV-2 S Pseudoviruses
(A) BLI sensorgram measuring apparent binding affinity of VHH-72-Fc to immobilized SARS-CoV-2 RBD-Fc. Binding curves are colored black, buffer-only blanks are colored gray, and the fit of the data to a 1:1 binding curve is colored red.
(B) Time course analysis of VHH-72-Fc expression in ExpiCHO cells. Cell culture supernatants of transiently transfected ExpiCHO cells were removed on days 3–7 after transfection (or until cell viability dropped below 75%), as indicated. Two control mAbs were included for comparison, along with the indicated amounts of purified GBP-Fc as a loading control.
(C) SARS-CoV-2 S pseudotyped VSV neutralization assay. Monolayers of Vero E6 cells were infected with pseudoviruses that had been pre-incubated with the mixtures indicated by the legend. The VHH-72-Fc used in this assay was purified after expression in ExpiCHO cells (n = 4). VHH-23-Fc is an irrelevant control VHH-Fc (n = 3). NI, cells were not infected. Luciferase activity is reported in counts per second (c.p.s.) ± SEM.

More information: Peter C Fridy et al. A robust pipeline for rapid production of versatile nanobody repertoires, Nature Methods (2014). DOI: 10.1038/nmeth.3170

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