Special llama antibodies “nanobodies” could be a powerful weapon against COVID-19


Today in Science, researchers at the University of Pittsburgh School of Medicine describe a new method to extract tiny but extremely powerful SARS-CoV-2 antibody fragments from llamas, which could be fashioned into inhalable therapeutics with the potential to prevent and treat COVID-19.

These special llama antibodies, called “nanobodies,” are much smaller than human antibodies and many times more effective at neutralizing the SARS-CoV-2 virus. They’re also much more stable.

“Nature is our best inventor,” said senior author Yi Shi, Ph.D., assistant professor of cell biology at Pitt.

“The technology we developed surveys SARS-CoV-2 neutralizing nanobodies at an unprecedented scale, which allowed us to quickly discover thousands of nanobodies with unrivaled affinity and specificity.”

To generate these nanobodies, Shi turned to a black llama named Wally – who resembles and therefore shares his moniker with Shi’s black Labrador.

Shi and colleagues immunized the llama with a piece of the SARS-CoV-2 spike protein and, after about two months, the animal’s immune system produced mature nanobodies against the virus.

Using a mass spectrometry-based technique that Shi has been perfecting for the past three years, lead author Yufei Xiang, a research assistant in Shi’s lab, identified the nanobodies in Wally’s blood that bind to SARS-CoV-2 most strongly.

Dr. Yi Shi, senior author on a paper published today in Science, explains how “nanobodies” could be a powerful new weapon against the COVID-19 pandemic. Credit: UPMC

Then, with the help of Pitt’s Center for Vaccine Research (CVR), the scientists exposed their nanobodies to live SARS-CoV-2 virus and found that just a fraction of a nanogram could neutralize enough virus to spare a million cells from being infected.

These nanobodies represent some of the most effective therapeutic antibody candidates for SARS-CoV-2, hundreds to thousands of times more effective than other llama nanobodies discovered through the same phage display methods used for decades to fish for human monoclonal antibodies.

Shi’s nanobodies can sit at room temperature for six weeks and tolerate being fashioned into an inhalable mist to deliver antiviral therapy directly into the lungs where they’re most needed.

Since SARS-CoV-2 is a respiratory virus, the nanobodies could find and latch onto it in the respiratory system, before it even has a chance to do damage.

In contrast, traditional SARS-CoV-2 antibodies require an IV, which dilutes the product throughout the body, necessitating a much larger dose and costing patients and insurers around $100,000 per treatment course.

“Nanobodies could potentially cost much less,” said Shi. “They’re ideal for addressing the urgency and magnitude of the current crisis.”

In collaboration with Cheng Zhang, Ph.D., at Pitt, and Dina Schneidman-Duhovny, Ph.D., at the Hebrew University of Jerusalem, the team found that their nanobodies use a variety of mechanisms to block SARS-CoV-2 infection.

This makes nanobodies ripe for bioengineering. For instance, nanobodies that bind to different regions on the SARS-CoV-2 virus can be linked together, like a Swiss army knife, in case one part of the virus mutates and becomes drug-resistant.

“As a virologist, it’s incredible to see how harnessing the quirkiness of llama antibody generation can be translated into the creation of a potent nanoweapon against clinical isolates of SARS-CoV-2,” said study coauthor and CVR Director Paul Duprex, Ph.D.

Coronaviruses are positive sense, single-stranded RNA viruses. There are seven types of coronaviruses known to infect humans, including the recent 2019 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).[1, 2] Patients infected with these viruses develop respiratory symptoms of various severity and outcomes. Since the beginning of the century, there have been three major world-wide health crises caused by coronaviruses: the 2003 SARS-CoV-1 outbreak, the 2012 MERS-CoV outbreak, and the 2019 SARS-CoV-2 outbreak.[3] To date, hundreds of thousands of people have succumbed to the virus during these outbreaks.

The SARS-CoV-2 virus gains entry to human cells via the angiotensin converting enzyme 2 (ACE2) receptor by the SARS-CoV-2 receptor binding domain (RBD) of the spike protein on the viral surface.[4-8] This
RBD-ACE2 interaction provides a clear therapeutic target for binding and prevention of infection (Figure 1. S).

Figure 1. Structure of SARS-CoV-2 spike protein, with receptor binding domain in contact with the human ACE2 protein on the surface of a lung epithelial cell. A major therapeutic goal is to develop inhibitory agents that disrupt the interaction between the spike protein and the human ACE2 protein. (Figure generated using BioRender)

Many of the antibodies considered for diagnostic, research, and therapeutic applications have been conventional immunoglobulins (IgG). The use of IgGs as therapeutics, while successful in many diseases, is known to have potential pitfalls due to the risk of receptor-mediated immunological reactions.[9] Treatment or prophylaxis of a pulmonary virus can be delivered via aerosolization and inhalation; thus, the size and biophysical characteristics are paramount considerations.

The camelid family, which includes llamas, produce an additional subclass of IgGs which possess a single heavy-chain variable domain.[10-12] This heavy-chain variable domain has demonstrated the ability to function as an independent antigen-binding domain with similar affinity as a conventional IgG. These heavy chain variable domains can be expressed as a single domain, known as a VHH or nanobody, with a molecular weight 10% of the full IgG. Nanobodies generally display superior solubility, solution stability, temperature stability, strong penetration into tissues, are easily manipulated with recombinant molecular biology methods, and possess robust environmental resilience to conditions detrimental to conventional IgGs.[13-15] In addition, nanobodies are weakly immunogenic which reduces the likelihood of adverse effects compared to other single domain antibody such as those derived from sharks or synthetic platforms. Therefore, nanobodies that bind to the SARS-CoV-2 RBD and block the ACE2 interaction are an attractive therapeutic for prevention and treatment of viral infection.[16]

Using standard methods to immunize llama, B-cell nanobody DNA sequences were isolated and a phage display library with over 108 clones was created (Figure 2.A). From this phage library, 13 unique lead candidate nanobodies that bind to the SARS-CoV-2 spike protein RBD were isolated, several of which block the spike protein-ACE2 interaction with high potency.

Figure 2. Isolation of nanobodies binding SARS-CoV-2 spike protein. A. An adult llama was immunized 5 times over 28 days with purified, recombinant SARS-Cov-2 spike protein. On day 35 after first immunization (7 days after last immunization), llama blood was obtained through a central line, B-cells were isolated, the single heavy-chain variable domains (nanobodies) of the llama antibodies were amplified and cloned to construct a recombinant DNA library containing more than 10^8 clones. The library of clones was expressed in a phage display format, in which each phage expresses between 1-5 nanobody copies on its surface and also contains the DNA sequence encoding that nanobody. Immunopanning was performed (see Figure 3.) to isolate candidate nanobodies for expression and validation studies. B. Reagents required for characterization and validation include recombinant human ACE2, recombinant SARS-Cov-2 receptor binding domain (RBD), and recombinant SARS-Cov-2 Spike protein (S1). Single bands on protein-stained SDS-PAGE gel indicate purity and appropriate size. C-D. Validation that recombinant SARS-Cov-2 RBD and SARS- Cov-2 Spike S1 bind with high affinity to recombinant human ACE2. This high affinity, saturable binding indicates that all 3 recombinant proteins are appropriately folded in vitro. (Figure elements generated using BioRender)

To isolate the candidate nanbodies, a novel screening strategy was designed and executed to specifically select for nanobodies that not only bound to the SARS-Cov-2 spike RBD, but also interfered with the interaction with the human ACE2 protein. The purity of in vitro binding of commercially available recombinant spike protein RBD and human ACE2 protein (Figure 2.B-D) that were used for the screening strategy was validated. In this screening strategy (Figure 3.), recombinant human ACE2 protein was immobilized in tubes. Then, biotinylated- RBD was incubated with the nanobody phage library and allowed to interact with the immobilized ACE2.
Biotinylated-RBD with no associated nanobodies, or with nanobody associations that did not block the ACE2 binding domain, bound the immobilized ACE2. Biotinylated-RBD with associated nanobodies that blocked the ACE2 binding domain remained in solution and were recovered using streptavidin-coated magnetic particles that bind to the biotin. Nanobodies that did not bind to RBD were removed during washing of the magnetic beads. This method allowed for specific enrichment of functional nanobodies that bind to the RBD and compete for the RBD-ACE2 binding surface.

Figure 3. Selection strategy for isolation of nanobody candidates which bind to the RBD:ACE2 interaction surface. Using a phage display library from an adult llama immunized with full length S1 spike protein, nanobodies were isolated which block the interaction between RBD and ACE2. A. In a standard radioimmunoassay tube, ACE2 was immobilized and the surface blocked with non-specific protein. Biotinylated-RBD, was incubated with the nanobody phage library and then added to the immuno-tube and allowed to interact with the immobilized ACE2. Biotinylated-RBD with no associated nanobodies, or with nanobody associations which do not block the ACE2 binding domain, bound the immobilized ACE2. B. Biotinylated-RBD with associated nanobodies that blocked the ACE2 binding domain remained in solution and were recovered using streptavidin-coated magnetic particles that bind to the biotin. C. Nanobodies which did not bind to RBD were removed during washing of the magnetic beads. This method allowed for specific enrichment of nanobodies which both bind to the RBD and compete for the RBD-ACE2 binding surface. (Figure generated using BioRender)

Using the novel screening strategy, hundreds of phage were isolated, which when sequenced, revealed 13 unique full length nanobody DNA sequences, termed NIH-CoVnb-101 through NIH-CoVnb-113 (Figure 4.). These sequences were distinct from the previously published sequences of VHH72[17] and Ty1[18] that also bind SARS-Cov-2 spike protein. The CDR3 domains responsible for much of the specific binding of nanobodies to their targets, in NIH-CoVnb-112 and the other new nanobodies were longer than those in VHH72 and Ty1. These findings indicated that novel nanobody DNA sequences were isolated.

Figure 4. Protein sequences for novel nanobodies that bind to the SARS-Cov-2 spike protein receptor binding domain. Single letter amino acid codes. Clustal Omega algorithm used for alignment. Blue highlights indicate sequence diversity with NIH-CoVnb- 112 set as the reference sequence for comparison. For reference, two previously published nanobody sequences (VHH72 from Wrapp et al., and Ty1 from Hanke et al.) have clearly distinct sequences. Both VHH72 and Ty1 have shorter CDR3 domains (represented in NIH-CoVnb-112 by amino acids 99-120).

Representatives from each of these unique nanobody sequences were produced in bacteria, purified, and tested for binding to SARS-Cov-2 spike RBD (Figure 5). All of the nanobodies bound to recombinant SARS-Cov-2 spike RBD with high affinity. The strongest binding nanobody was NIH-CoVnb-112 (Figure 5E) with an affinity of 4.9 nM. NIH-CoVnb-112 had both a fast on rate (1.3e5/M/sec.) and a slow off rate (6.54e-4/sec.), with kinetics compatible with 1:1 binding. These results indicate that the novel nanobodies bind to the
SARS-Cov-2 spike RBD with very high affinity. The long CDR3 in NIH-CoVnb-112 and the other new nanobodies may in part underlie their extraordinarily high affinity, though this is clearly not the only factor in that the nanobody with the longest CDR3, NIH-CoVnb-110, is not one of the top 5 highest affinity nanobodies.

Figure 5. Affinity binding curves of isolated anti-SARS-CoV-2 RBD nanobodies. Using Biolayer Interferometry on a BioForte Octet Red96 system, association and dissociation rates were determined by immobilizing biotinylated-RBD onto streptavidin coated optical sensors (A-E). The RBD-bound sensors are incubated in specific concentrations of purified candidate nanobodies for a period of time to allow association. The sensors are then moved to nanobody-free solution and allowed to dissociate over a period of time. Curve fitting using a 1:1 interaction model allows for the affinity constant (KD) to be measured for each nanobody as detailed in (F).

In an important validation of the in vitro efficacy of the candidate nanobodies, the nanobodies were able to interfere with SARS-Cov-2 spike RBD binding to human ACE2 protein (
Figure 6. – left panel). Specifically, a competitive inhibition assay was designed and implemented, in which recombinant RBD was coated onto Enzyme Linked Immuno-Sorbent Assay (ELISA) plates and soluble ACE2 binding was assessed. Without any interference, soluble ACE2 binding was indicated by high colorimetric absorbance. At increasing concentrations, each of the new nanobodies, showed progressively less ACE2 binding. For the most potent anti-SARS-CoV-2 RBD nanobody, NIH-CoVnb-112, the concentration at which 50% of the ACE2 binding was blocked (half maximal effective concentration; termed EC50) was found to be
0.02 micrograms/mL, equivalent to 1.11 nM. The rank order of ACE2 competition EC50 matched the rank order of RBD binding affinities for the novel nanobodies assessed. As an additional confirmation, the experiment was repeated using a commercially available SARS-Cov-2 spike RBD- human ACE2 protein competitive inhibition assay (GenScript). The results were very similar to those obtained using the initial assay (Figure 6 – right panel). These results indicate that the novel nanobodies block SARS-Cov-2 spike protein RBD binding to ACE2, an essential human receptor responsible for viral infection.

Figure 6. Competitive inhibition of ACE2 and RBD binding using anti-SARS-CoV-2 RBD nanobodies. Left: RBD coated ELISA plates were blocked with non-specific protein and incubated with dilutions of each candidate anti-SARS-CoV-2 RBD nanobody.
Biotinylated-ACE2 was added to each well and allowed to bind to unoccupied RBD. The ELISA was then developed using a standard streptavidin-HRP and tetramethylbenzidine reaction. Unoccupied RBD allows for a positive reaction signal which is suppressed in the presence of bound competitive nanobody. NIH-CoVnb-112 produces the greatest inhibition of ACE2 binding with an EC50 of 0.02 micrograms/mL (1.11 nM). Right: Comparable findings using the commercially available Genscript SARS-CoV-2 neutralization surrogate assay.

There have been many variants of the spike protein RBD described recently that increase the binding affinity to the human ACE2 receptor.[19] Several of these variants including N354D D364Y, V367F, and W436R have been reported to have up to 100 fold higher affinity for ACE2 than the prototype RBD in vitro. NIH-CoVnb-112 blocked interaction between human ACE2 and three of these variants with similar EC50 compared to its blocking effects on the prototype sequence spike protein RBD (Figure 7, left panel). Binding affinity of NIH-CoVnb-112 to the variants was also similar to that of the prototype sequence (Figure 7, right panel). Thus, NIH-CoVnb-112 may provide robust blocking of SARS-Cov-2 spike protein RBD binding to ACE2.

Figure 7. Interaction of NIH-CoVnb-112 with SARS-Cov-2 Spike Protein RBD variants. Left: RBD “wild type” and 3 variant forms of the RBD coated ELISA plates were blocked with non-specific protein and incubated with dilutions of each the lead candidate anti-SARS-CoV-2 RBD nanobody NIH-CoVnb-112. Biotinylated-ACE2 was added to each well and allowed to bind to unoccupied RBD. The ELISA was then developed using a standard streptavidin-HRP and tetramethylbenzidine reaction. Unoccupied RBD allows for a positive reaction signal which is suppressed in the presence of bound competitive nanobody. NIH-CoVnb-112 produces the inhibition of ACE2 binding to each of the variants with a similar EC50 of 0.02 micrograms/mL (1.11 nM). Right: Binding of NIH- CoVnb-112 to RBD “wild type” and 3 variant forms of the RBD had similar affinity, with half maximal binding at approximately 0.01 micrograms/mL.

Finally, we assessed whether NIH-CoVnb-112 binds to the same spike protein RBD epitope as the previously reported nanobody VHH72 [17]. We reasoned that if both nanobodies bind to the same epitope, their signals would occlude each other when applied at saturating concentrations on bilayer interferometry. In contrast, if

they bind to different epitopes, their signals would be additive on bilayer interferometry. We found that when NIH-CoVnb-112 was applied at 500 nM (>100 x greater than the KD) for long enough to reach steady state, and then VHH72 was applied, the signals were clearly additive (Figure 8). This result clearly indicates that NIH- CoVnb-112 binds to a SARS-CoV-2 spike protein RBD epitope that is distinct from that of VHH72. This result is consistent with the reported findings that VHH72 recognizes a non-ACE2 binding motif on the spike protein [17], whereas NIH-CoVnb-112 has RBD-ACE2 interaction disruption potency that is quantitatively similar to its affinity- a result that is most consistent with NIH-CoVnb-112 binding directly to the ACE2 interaction domain.

Figure 8. NIH-CoVnb-112 binds to SARS-CoV-2 RBD at a distinct epitope from that bound by VHH72. To determine if NIH- CoVnb-112 and VHH72 have competing epitopes the following association Octet experiment was performed: Biotinylated SARS- CoV-2 RBD was bound to streptavidin BLI sensors in blocking buffer. Following baseline stability, the sensor was transferred into a well containing 500nM NIH-CoVnb-112 and allowed to associate. The sensor was then transferred to the adjacent well containing 500nM VHH72 and allowed to associate. If VHH72 had an overlapping epitope blocked by NIH-CoVnb-112 there would be minimal to no increase in response. Based on the observed increase in response, it can be inferred that the two nanobodies have non-competing epitopes on the SARS-CoV-2 RBD.


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More information: “Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2,” Sciencescience.sciencemag.org/cgi/doi … 1126/science.abe4747


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