Researchers have turned naturally generated llama antibodies into potentially effective therapies against SARS-CoV-2

As the fight against COVID-19 continues, scientists have turned to an unlikely source for a potentially effective treatment: tiny antibodies naturally generated by llamas.

While the world has welcomed the news of multiple vaccines against COVID-19, the search for effective treatments for those who contract the virus is ongoing. Now scientists are looking to what might seem to be an unlikely source: the South American llama.

Researchers are using the ultrabright X-rays of the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science User Facility at DOE’s Argonne National Laboratory, to help turn naturally generated llama antibodies into potentially effective therapies against SARS-CoV-2, the virus that causes COVID-19.

Antibodies are the immune system’s natural defense against infection, and when extracted from blood, they can be used to design treatments and vaccines.

“Llamas generate these nanobodies naturally in high yields, and they fit into the pockets on the surface of proteins that larger-size antibodies can’t access.” says Jason McLellan, The University of Texas at Austin.

“We have received more than 50 llama antibodies with several proteins of SARS-CoV-2,” said Andrzej Joachimiak, director of the Structural Biology Center (SBC) at the APS and co-director of the Center for Structural Genomics of Infectious Diseases. (Researchers at the APS do not work with the live virus, but with crystals grown from simulated proteins.)

These antibodies are part of ongoing collaborations with several partners, including researchers at the National Institutes of Health (NIH) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), Joachimiak said, and will be analyzed using the APS to see if they combat the virus’s infectivity.

While it may seem surprising that scientists are turning to llamas, there’s a very good reason for it.

Llamas belong to a group of mammals called camelids, a group that also includes camels and alpacas. Thanks to a quirk of nature, camelids produce a unique type of antibody against disease. These antibodies, often referred to as nanobodies, are about half the size of the antibodies produced by humans. They’re also remarkably stable and easy for scientists to manipulate.

This genetic quirk, which causes camelids such as llamas to produce these smaller antibodies with single protein chains, was discovered by accident in the late 1980s by scientists in Belgium. Since then, scientists have worked with camelid nanobodies to create treatments against several diseases with great success.

Their small size allows them to bind to areas of viral proteins that larger antibodies cannot fit into, blocking those proteins from connecting with cells.

“Llamas generate these nanobodies naturally in high yields, and they fit into the pockets on the surface of proteins that larger-size antibodies can’t access,” said Jason McLellan, an associate professor at The University of Texas at Austin.

McLellan has years of experience working with camelid nanobodies. He and his graduate student Daniel Wrapp, along with Xavier Saelens’ group in Belgium, have isolated nanobodies that have proven effective against respiratory syncytial virus (RSV) and two coronaviruses: severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).

When the genetic sequence of SARS-CoV-2 was released in January of 2020, McLellan, Wrapp and Saelens worked quickly to test whether any of the antibodies that they had previously isolated against the original SARS-CoV (taken from a Belgian llama named Winter) could also bind and neutralize SARS-CoV-2.

They discovered that one of these nanobodies, which they had characterized using the SBC beamlines at the APS, might be effective against SARS-CoV-2. McLellan said this nanobody – called VHH72 – is now under development as a treatment for COVID-19. He and Wrapp received a 2020 Golden Goose Award for this research.

McLellan will tell you that while his results were good, his hopes were a little higher.

“We were seeking one potent antibody that neutralized all coronaviruses,” he said. “We immunized Winter hoping to elicit that one nanobody. And maybe we elicited it, but we didn’t isolate it.”

Isolating these tiny nanobodies is tricky, since the body generates an enormous number of them and only a small fraction is intended to fight a particular virus. That’s exactly the problem that Yi Shi, professor of cell biology at the University of Pittsburgh, is trying to fix.

In a paper published in Science, Shi and his colleagues unveiled a new advanced mass spectroscopy method of analyzing those nanobodies from samples of llama blood. The result, according to Shi and research assistant Yufei Xiang (the paper’s lead author), is a large set of nanobodies that bind well to the SARS-CoV-2 virus.

“This is thousands of times better than the current technology, specifically in its selecting properties,” Shi said. “We want nanobodies that bind tightly to SARS-CoV-2, and with this method we can get a drug-quality nanobody that is up to 10,000 times more potent.”

As with McLellan’s research, Shi’s experiment began with a llama, this one named Wally because he resembles (and therefore shares a name with) his black Labrador. The team immunized Wally against SARS-CoV-2, waiting two months for nanobodies to be generated, and then Xiang used their new method to analyze the nanobodies, identify and quantify them. They ended up with 10 million nanobody sequences.

These nanobodies can sit at room temperature for six weeks, and are small enough that they can be aerosolized, meaning therapeutics designed from them can be inhaled directly to the lungs instead of moving through the bloodstream. To confirm the nanobodies’ effectiveness, Cheng Zhang, assistant professor at the University of Pittsburgh, determined structures of the nanobodies bound to the SARS-CoV-2 virus at the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) at the APS.

“With this method we can discover thousands of distinct, ultrahigh-affinity nanobodies for specific antigen binding,” Shi said. “These nanobodies may or may not provide a treatment for COVID-19, but the technology used to isolate them will be important in the future.”

Most recently, a team of scientists led by the University of Bonn in Germany reported newly discovered nanobodies that bind to SARS-CoV-2 and may prevent what is called “mutational escape.”

That’s the ability of a virus to avoid immune responses by mutating, and a treatment that prevents the virus from doing so would guard against reinfection.

This research team combined several nanobodies into molecules that attack different parts of the virus simultaneously, helping to prevent virus mutations from reducing therapeutic effectiveness. These nanobodies were taken from a llama and an alpaca immunized against the SARS-CoV-2 virus, and out of several million candidates they ended up with four molecules that proved to be effective.

Ian Wilson, professor of structural biology at the Scripps Research Institute in California, led the team that conducted X-ray diffraction studies at GM/CA at the APS to determine structures of these molecules bound to the virus.

“From crystal structures determined from data collected at APS and the Stanford Synchrotron Radiation Lightsource (SSRL), we were able to identify the binding sites of the nanobodies on the SARS-CoV-2 receptor binding domain,” Wilson said.

“The X-ray structural information, combined with cryo-electron microscopy data, was used to help design even more potent multivalent antibodies to prevent COVID-19 infection. The X-ray structural work was greatly facilitated by immediate access to the APS.”

Only time (and further tests) will tell whether the various nanobodies will translate into effective treatments against COVID-19. But if they do, we’ll have the lovable llama to thank for it.

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Nbs as new approach for the prevention and treatment of COVID-19
Camel heavy chain antibodies and Nbs

In addition to conventional antibodies (heterotetrameric structure with two heavy and two light chains) produced by all mammals, camelidae members family including Old World species (camels and dromedaries) and New World species (llamas, alpacas, and vicuñas) are able to produce non-conventional antibodies (Hamers-Casterman et al. 1993).

Structures of these IgG are devoid of the complete light chain and the first constant heavy domain CH1 and then named HcAbs. The account of HcAbs in the sera varies between camel species. It can reach 50–80% in Camelus bactrianus and Camelus dromedarius; however, it does not exceed 25% in the serum of the South American camelids (alpacas and llamas) (De Simone et al. 2008; Blanc et al. 2009).

HcAbs were reffered to as IgG2 (IgG2a and IgG2b) and IgG3 IgG-subclasses to distinguish them from conventional antibodies (IgG1). Despite their particular structure, HcAbs antibodies are fully functional and still able to bind antigens with a high affinity through their antigen-binding site known as VHH (Fig. 2; adapted from Smolarek et al. 2012).

Structure and peculiar characteristics of VHHs

Despite it shares general structural features with human variable heavy domain (VH), the camelid heavy chain variable named as VHH, with a molecular weight of 15 kDa, presents an important difference with VH. In fact, there are four major amino acid substitutions observed in the framework region 2 (FR2) that substitute the hydrophobic residues (involved in the VH/VL interaction in conventional antibodies) by more hydrophilic amino acids (Nguyen et al. 2001).

These substitutions compensate the lack of the variable light domain (VL) and confer the higher solubility of VHHs when compared to other single-domain antibodies. Additionally, complementarity determining regions (CDRs) exhibit a long CDR3 loop that increases the antigen- binding loop size in VHHs and enables theme to bind concave epitopes that cannot be recognized by traditional antibodies ((Muyldermans et al. 1994; Vu et al. 1997).

The stability of extended CDR3 loops is maintained by a disulfide bond between CDR1 and CDR3 or between FR2 and CDR3. These particular features increase the stability and the solubility of VHH even under denaturing conditions or high temperatures (Van der Linden et al. 1999; Dumoulin et al. 2002; Conrath et al. 2005; Kunz et al. 2018). Moreover, VHHs are easily engineered with high yields and low-cost production using various expression systems (Liu and Huang 2018; De Marco 2020).

The most important characteristics of VHH fragments include negligible immunogenicity in the human body, rapid penetration into the tissue, a nanomolar affinity for their target and flexible formatting (multimerization). More importantly, the high stability of VHHs under harsh conditions (in the presence of proteases, chaotropic agents as well as at extreme pHs) facilitate their administration by inhaled delivery for the treatment of respiratory diseases. These distinctive properties provide VHHs numerous advantages compared to conventional antibodies and their recombinant fragments and make them a powerful tool for immunotherapy as well as immunodiagnostics immunoassay development.

Production process of VHHs

Over three decades ago, the phage display technology has been shown to be an effective and efficient platform to develop and produce therapeutic antibodies. Numerous recombinant antibodies with desired functional properties are selected from immune, naïve, or synthetic libraries via phage display technology. This fast methodology enables the selection of Nbs with a reasonable specificity and affinity by successive rounds of bio-panning (Silacci et al. 2005).

Nbs are successfully expressed in a variety of expression systems including prokaryotic, eukaryotic and plant hosts. Nbs are characterized by large-scale production, solubility and stability compared to conventional antibody fragments (antigen-binding fragments (Fab) or single-chain variable fragments (scFv)) that can aggregate due to their low solubility (Van der Linden et al. 1999).

Other strategies, such as ribosome display and yeast surface display can be used for the selection of specific VHHs. The mono-domain format of VHH offers significant advantages in cost of production and engineering compared to conventional antibodies.

Camel nanobodies: promising therapeutic tools to combat the emerging pandemic virus
Neutralizing VHH against viral zoonosis
Due to their peculiar properties, rapid progress has been made regarding the production of VHH domains for therapeutic and diagnostic applications (Wesolowski et al. 2009; Khodabakhsh et al. 2018; Lafaye and Li 2018; Sanaei et al. 2019; Chames and Rothbauer 2020). It has been demonstrated that these Nbs can be easily engineered without loss of functionality. Currently, several Nbs produced by Ablynx (now Sanofi), are in different clinical trials and with Caplacizumab, the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) approved the first sdAb-based medicine for adults in November 2018 and in February 2019, respectively (Morrison 2019; Jovčevska and Muyldermans 2020).

Viral neutralizing sdAb has been described as valuable biomolecules, with high potentials, able to perturb different steps in the viral life cycle with many examples of viral neutralizing Nbs (De Vlieger et al. 2019; Sroga et al. 2020). These Nbs have been generated from an immune or synthetic phage display library against a wide range of target classes. Table 1 shows Nbs selected for the prevention and treatment of infectious viral diseases including that of SARS-CoV-2.

Table 1

Nanobodies-based therapeutic tools for viral infection diseases including emerging corona viruses

Virus	Target	Nanobodies	Source	References
MERS-CoV	RBD	Monomeric VHH: NbMS10	Llama immune VHH library (phage display)	Zhao et al. (2018)
MERS-CoV	RBD	VHH and camel/human chimeric (HcAbs:HCAb-83)	Dromedary camel immune VHH library (phage display)	Raj et al. (2018)
MERS-CoV	RBD	Mono-Nb, dimeric Nb (Di-Nb) and Trimeric Nb (Tri-Nb)	Llama immune VHH library (phage display)	He et al. (2019)
SARS-CoV-2	RBD	Monomeric VHH: NIH-CoVnb-112	Llama immune VHH library (phage display)	Esparza and Brody (2020)
SARS-CoV-2	RBD	Multivalent Nb: Nb213 and Nb203	Llama immune VHH library (phage display)	Xiang et al. (2020)
SARS-CoV-2	RBD	Monomeric VHH	Alpaca immune VHH library (phage display)	Nieto et al. (2020)
SARS-CoV-2	RBD	Monomeric VHH: NM1226, NM1228 and NM1230	Alpaca immune VHH library (phage display)	Wagner et al. (2020)
SARS-CoV-2	RBD	Trimeric Nb: mNb6-tri	Llama synthetic VHH library (yeast display)	Schoof et al. (2020)
SARS-CoV-2	RBD	Monomeric VHH: H11-D4 and H11-H4; chimeric fusions:H11-H4-Fc and H11-D4-Fc	Naïve llama VHH library	Huo et al. (2020)
SARS-CoV-2	RBD	Monomeric VHH: Ty1	Alpaca immune VHH library (phage display)	Hanke et al. (2020)
SARS-CoV-2	RBD	Monovalent Nb: Nb11-59	Camel immune VHH library (phage display)	Gai et al. (2020)
SARS-CoV-2	RBD	Monomeric VHH: SR31	Synthetic sdAb phage display library	Yao et al. (2020)
SARS-CoV-2	RBD	Monovalent VHH: 2F2, 3F11 and 5F8Fc-fused sdAbs	Synthetic sdAb phage display library	Chi et al. (2020)
SARS-CoV-2	RBD	Sybody Sb23	Three sybody libraries (concave, loop and convex)	Custódio et al. (2020)
SARS-CoV-2	RBD	Sybody MR3	Three sybody libraries (concave, loop and convex)	Li et al. (2020a)
SARS-CoV-2	RBD	Sybodies	Three large combinatorial libraries, using ribosome and phage display	Walter et al. (2020)
SARS-CoV-2	Recombinant SARS-CoV-2 S protein	Bispecific VHH-Fc antibody, Tri-specifc VHH-Fc antibody	Naïve and synthetic llama VHH library	Dong et al. (2020a, b, c)
SARS-CoV-2	RBD, S1 protein	Human single-domain antibodies n3130	Naïve antibody libraries	Wu et al. (2020)
SARS-CoV-2, MERS-CoV, SARS-CoV-1	prefusion-stabilized coronavirus spikes	Bivalent VHH:VHH-72 VHH-55, VHH-72-Fc	Llama immune VHH library (phage display)	Wrapp et al. (2020a)
Influenza A and B viruses	Hemaglutinins	Multivalent VHH: MD3606	Llama immune VHH library	Laursen et al. (2018)
H1N1	Hemaglutinin	bivalent VHH: R1a-B6	Alpaca immune VHH library (phage display)	Hufton et al. (2014)
Respiratory syncytial virus	Fusion (F) protein	Trivalent nanobody: ALX-0171	Llama immune VHH library (phage display)	Van Heeke et al. (2016), Detalle et al. (2015), Wilken et al. (2017)
Hepatitis B virus	Capsid protein: HBcAg	VHH intrabodies	Llama immune VHH library (phage display)	Serruys et al. (2010)
HIV	gp120	Monovalent VHH: A12, C8, and D17,	Llama immune VHH library (phage display)	Forsman et al. (2008)
HIV	gp140	Monovalent VHH: 2E7	Llama immune VHH library (phage display)	Strokappe et al. (2012)
Influenza A virus	Nucleoprotein (NP)	Monovalent VHH:NP-VHHs	Alpaca immune VHH library (phage display)	Ashour et al. (2015)
Influenza A virus	Native M2 ion channel protein	Monovalent VHH: M2-7A	Synthetic Camel single-domain antibody (VHH) libraries	Wei et al. (2011)
H5N1	Hemaglutinin	Trivalent VHH	Llama immune VHH library (phage display)	Hultberg et al. (2011)
H5N1	Influenza virus neuraminidase (NA) Neuraminidase	Bivalent VHH: N1-VHHb, N1-VHH-Fc	Alpaca immune VHH library (phage display)	Cardoso et al. (2014)
Poliovirus type 1	Capsid	Monovalent VHH: PVSS21E	Dromedary immune VHH library (phage display)	Strauss et al. (2016)
Norovirus	VLPs	Monomerci: Nano-26 and Nano-85	Alpaca immune VHH library (phage display)	Koromyslova et al. (2017)
Rotavirus	VP6 inner capsid protein	Monovalent VHH	Llama immune VHH library (phage display)	Van der Vaart et al. (2006)
Chikungunya virus (CHIKV)	CHIKV virus-like particles contained the capsid, E1 and E2 proteins	CC3 VHH	Llama immune VHH library (phage display)	Liu et al. (2019)
Ebola virus	Recombinant EBOV GP and EBOV VLPs	sdAbs	Llama immune VHH library (phage display)	Liu et al. (2017)

For example, sdAb specific for influenza viruses has been successfully isolated through selections against the nucleoprotein and M2 ion channel protein of Influenza A, the neuraminidase and trimeric spike protein and hemagglutinin of H5N1 and H1N1 (Wei et al. 2011; Hultberg et al. 2011; Cardoso et al. 2014; Hufton et al. 2014; Ashour et al. 2015; Laursen et al. 2018). An sdAb-specific viral protein (PV1) of poliovirus (PV) was identified from the immunised phage display library. The mechanism by which these VHHs reduce viral loads is by blocking the ligand–receptor interactions (Strauss et al. 2016). Human norovirus is classified among the leading cause of gastroenteritis worldwide. Several Nbs against virus-like particles (VLPs) of norovirus have shown promise for disease treatment (Koromyslova and Hansman 2017).

Ebola virus (EBOV) is extremely virulent and causes fatal hemorrhagic fever in  ~ 50% of the cases. The interaction between viral envelope glycoprotein GP and host cell receptors plays a critical role in pathogenicity of this virus. An immune sdAb phage display library derived from a llama immunized with killed EBOV and recombinant GP was constructed and SdAbs specific for Ebola GP were selected and evaluated for their affinity and thermal stability (Liu et al. 2017). Also, monovalent, bivalent and trivalent VHHs targeting the glycoproteins, gp120 and gp41 of VIH have been generated to disrupt virus entry into cluster of differentiation 4 (CD4)+ T cells (Forsman et al. 2008; Strokappe et al. 2012; Weiss and Verrips 2019). Chikungunya virus (CHIKV) is a member of Togaviridae family that causes severe joint pain which is associated with fever, rash and headache. Five anti-CHIKV sdAbs have been generated in immunized llamas. These viral neutralizing sdAbs have been successfully isolated through selections against the CHIKV VLPs (Liu et al. 2019).

Nbs developed against the core antigen of hepatitis B (HBcAg) have an effect on the viral life cycle in HBV-transfected hepatocytes (Serruys et al. 2010). Moreover, VHHs directed against rotavirus serotype G3 reduce morbidity of rotavirus-induced diarrhea in vivo (Van der Vaart et al. 2006). In another example, it has been shown by Terryn et al. (2016) that multi-merization of VHH domains significantly improves protection of mice from lethal rabies infection.

Nbs have been described as inhaled bio-therapeutics for lung diseases. Human respiratory syncytial virus (RSV) belongs to the Pneumoviridae family and represents the most important cause of lower respiratory tract infections in young infants. Successful phase I/IIa clinical trials were completed for an anti-RSV VHH domain called ALX-0171 (Detalle et al. 2015; Van Heeke et al. 2017; Wilken and McPherson 2018). Neutralizing activities of ALX-0171 were compared to Palivizumab, a marketed neutralizing monoclonal antibody, and it was demonstrated that ALX-0171 inhibits virus replication in 87% of viruses tested versus 18% observed with Palivizumab administration. In addition, to develop lung-targeting drugs, Nbs targeting pulmonary surfactant protein A (SPA) have been isolated. The authors showed fast accumulation of selected Nbs in the lungs (Wang et al. 2015).

Nbs against different coronavirus species have been isolated and characterized to block interaction between virus and host cell. As example, Abs targeting RBD of MERS-CoV were identified and selected from the immunized dromedary and it was shown that camel/human chimeric HcAbs bind to their target with picomolar affinity (Raj et al. 2018). In addition, Zhao and colleagues panned an immune VHH library derived from llama immunized with a recombinant MERS-CoV RBD protein, and isolated monomeric VHH (Mono-Nb, NbMS10) (Zhao et al. 2018). He et al. (2019) generated two oligomeric Nbs, dimeric and trimeric Nbs, from an immunized library directed against the RBD of MERS-CoV. According to a multiple recent studies, VHHs exhibited high neutralization potency SARS-CoV-2. Humanized sdAbs-binding SARS-CoV-2 RBD proteins have been discovered in a synthetic sdAb phage display library (Chi et al. 2020).

The inhibition efficiency on SARS-CoV-2 pp and affinity kinetics was tested in vitro. These Nbs, named 2F2, 3F11 and 5F8 could be very advantageous to find new specific drugs to prevent SARS-CoV-2 infection by inhibiting membrane fusion between RBDs of the viral Spike and their host cell receptors and then blocks the entry of SARS-CoV-2 into cells (Fig. 3; adapted from Esparza et al. 2020). Another illustration of a VHH against SARS-CoV-2 is VHHs against prefusion-stabilized MERS-CoV and SARS-CoV-1 spikes of Betacoronaviruses.

These neutralizing sdAbs were isolated from immunized llamas and it was demonstrated that engineered bivalent Nbs exhibits cross-reactivity against SARS-CoV-1 RBD and SARS-CoV-2 RBD and able to neutralize SARS-CoV-2 S pseudoviruses with high affinity (Wrapp et al. 2020a). Using a combiation of two llama VHH libraries, humanized VHH has been constructed (Dong et al. 2020a, b). In their studies, Dong and collaborators demonstrated that multi-specific antibodies showed better affinity and avidity than individual monoclonal VHH-Fcs. More importantly, these multi-specific antibodies showed more potent neutralization activity than a combination of monoclonal antibodies. Hanke et al. (2020) reported the isolation of a monomeric Nb (Ty1) from immunised alpaca.

The highly specificity and high-affinity binding of Ty1 to the RBD have been confirmed. Hanke and colleagues, suggest that the generation of homodimeric or trimeric formats is likely to further increase its neutralization activity. More recently, Schoof et al. (2020) have successfully isolated a panel of Nbs, from synthetic Nbs library, that bind to multiple epitopes on Spike. These Nbs were divided into two classes. Class I bound directly to the RBD and competed with the ACE2 receptor from the surface of human cells. While class II recognized another binding region leading to modification of structural conformation of the RBD so that it cannot recognize ACE2 receptor. Structural analyses have clearly shown that the binding domain of class II Nbs occurred on a protected area on the spike protein well away from the RBD. Two class I Nbs designated Nb6 and Nb11 bound to both open and closed conformations of Spike.

To enhance the reactivity, Nb6 has been considered to make dimers and trimers. The novel structures inhibited more strongly the trimeric spike S protein by binding to more than one RBD on its surface. To assess the binding efficacy the measurement of IC50 revealed that Nb6 had an IC50 of 2 micromolar, while the Nb6-trimeric form (mNb6-tri) was 1.2 nM.

In this assay, the trimeric form showed two-thousand-fold improvement in efficacy. This result was confirmed in a test of Vero cell infection with real SARS-CoV-2 coronavirus where mNb6-tri was able to prevent viral attack with an IC50 of 160 picomolar, which is truly impressive. Moreover, a mutation generate on mNb6-tri reached femtomolar affinity for SARS-CoV-2. Even if at the prophylactic level, Sputnik vaccine and mRNA vaccine have been shown to be effective. The therapeutic mNb6-tri is very interesting with its high interaction with RBD as well as its stability after aerosolization, lyophilization and heat treatment (Schroof el al. 2020). It is easy and not expensive to obtain this Nb in large number by cultivation on yeast.

The study of its efficacy on patient’s infected with SARS-CoV-2 needs to be monitored. In a similar study, the identification and characterization of two other high-affinity Nbs (H11-D4 and H11-H4) have been reported (Huo et al. 2020). Both H11-D4 and H11-H4 Nbs blocked RBD binding to ACE2. Furthermore, in the same study, a bivalent Human Fc-Nb fusion (homodimeric chimeric protein) showed neutralizing activity against SARS-CoV-2 and additive neutralization with the SARS-CoV-1/2 antibody CR3022. In fact, it has been demonstrated that the CR3022 and these Nbs recognized non-overlapping epitopes on RBD. Interestingly, such additive combinations are a well-known strategy to reduce mutational escape.

In a recent study, three potent Nbs (Nb21, Nb20 and Nb89) with picomolar affinity have been isolated from a Llama immune VHH library (Xiang et al. 2020). Multivalent Nbs have been constructed to enhance the antiviral activities. Results showed that up to ~ 30-fold improvement of inhibitory activity was observed with an IC50 of 1.3 picomolar and 4.1 picomolar for the homotrimeric constructs of Nb213 and Nb203, respectively.

Importantly, multivalent constructs keep excellent physicochemical properties after lyophilization and aerosolization make them suitable for inhalation administration. Nieto et al. (2020) reported a rapid selection of monomeric Nb with sub-nanomolar affinity. Nevertheless, the neutralization activity of this Nb against SARS-CoV-2 pseudo-virus needs to be achieved. In another study, Nbs targeting different spike antigens (RBD, S1 domain or homo-trimeric spike) with high neutralizing potency have been successfully isolated from immunised alpaca VHH library (Wagner et al. 2020).

Using these Nbs (NM1226, NM1228 and NM1230), a competitive multiplex binding assay called “NeutrobodyPlex” has been developed. Authors demonstrated that NeutrobodyPlex approach using RBD-specific Nbs was more efficacious than conventional antibodies which showed cross-reactive signals. Furthermore, the test has been validated by analyzing serum samples collected from 18 patients and 4 healthy donors in comparison to standard assays. It can determine whether the examined people carry neutralizing antibodies preventing re-infection. Interestingly, this novel diagnostic test opens the door to surveil the emergence of neutralizing antibodies in infected patients. Thus, it might be useful during vaccination campaigns in the future.

Nbs library derived from immunised camel has been constructed (Gai et al. 2020). It has been shown that seven Nbs represented good binding capacity to RBD including eight SARS-CoV2-RBD mutants. Among these candidates, Nb11-59 exhibited the best neutralizing activities with a good stability. According to these results, Nb11-59 might be novel therapeutic molecule, as an inhaled drug, for COVID-19 treatment. In a study, 63 sybodies, against the SARS-CoV-2 RBD, were generated from three large combinatorial libraries, using ribosome and phage display.

Described flycode technology provides new opportunities for passive immunization to protect people against SARS-CoV-2 escape mutants (Walter et al. 2020). In addition, other studies reported the rapid isolation and characterization of potent synthetic Nbs which neutralize SARS-CoV-2 pseudo-viruses with high affinity (sybodies MR3 and Sb23) (Custodio et al. 2020; Li et al. 2020a). The last one (Sb23) can also bind the RBD in both “up” and “down” conformation (Custodio et al. 2020). Also, Yao et al. (2020) identified a new synthetic Nb. Selected sybody (named SR31) displayed poor neutralization activity against SARS-CoV-2 pseudo-virus. However, when fusioned with two other sybodies, increased binding affinities and neutralizing activities were observed. According to this result, SR31 cannot be used alone. Nevertheless, it may be combined with monoclonal antibodies or other antibody fragments to improve affinity and potency.

In another study, several Nbs that bind to the SARS-CoV-2 RBD have been isolated (Esparza and Brody 2020). Among those, the lead therapeutic candidate named NIH-CoVnb-112 showed high affinity in monomeric form and blocked interaction between ACE2 and several variant forms of the spike protein. Furthermore, Wu et al. (2020) selected fully human single-domain antibodies against five types of epitopes on SARS-CoV-2 RBD, using phage-displayed VHH library by grafting naïve CDRs into FR regions. The use of these Nbs may represent a novel approach to battle against COVID-19 given that human showed immunogenicity towards other antibody fragments (Wu et al. 2020).

Nanobodies as modulators of inflammation

Immune responses play a key role during SARS-CoV-2 virus infection. The latest reports suggest that acute respiratory distress syndrome (ARDS) is the common immunopathological event for this infectious disease (Shi et al. 2020; Wen et al. 2020). One of the main mechanisms for ARDS is the uncontrolled systemic inflammation, named as cytokine storm, resulting from the release of large amounts of pro-inflammatory cytokines (interferons: IFN-α and IFN-γ; interleukins: IL-1β, IL-2, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-18 and IL-33; tumour necrosis factor: TNF-α; transforming growth factor β: TGFβ; etc.) and chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9, CXCL10, etc.) by immune effector cells upon viral infection (Fig. 1) (Ong et al. 2020; Li et al. 2020a, b; de la Rica et al. 2020). Given the pivotal role of these proteins during inflammation, specific inhibitors of their activities might be useful as new tools to modulate immune functions in COVID-19 patients (Shi et al. 2020).

The generation of Nbs directed against chemokines, cytokines, and ecto-enzymes can be tailored to modulate inflammation responses and then beneficial for the recovery of COVID-19 patients. Nbs that modulate immune function have been successfully generated in immunized camelids. Several reports have been published in raising sdAbs directed against cytokine by phage display technology (Nosenko et al. 2017). For example, TNFα is an important cytokine implicated in a number of chronic inflammatory disorders. TNFα-blocking sdAbs have been successfully isolated from a llama immunized with human and mouse TNFα which are more effective at neutralizing TNFα than the conventional TNFα-blocking antibodies Infliximab and Adalimumab (Coppieters et al. 2006). Another Nb that binds to human IL-6-R and IL23 was generated for treatment of rheumatoid arthritis (Tillib et al. 2015; Desmyter et al. 2017).

Koch-Nolte et al. (2007) selected a novel Nb from an immunized phage display library directed against the T cell ecto-enzyme, ART2.2, which plays a key role in inflammatory settings and induces T cell death. Researchers demonstrate that these Nbs block effectively the enzymatic activity of ART2.2 in vivo.

Another study presented an interesting example of generating an ion-channel blocking Nb. The P2X7 ion channel is expressed by both monocytes and T cells. This ion channel responsible for the release of IL-1β during inflammation represents a potential therapeutic target in inflammatory diseases. It has been shown that sdAbs recognizing P2X7 were isolated from immunized llama effectively blocked ATP-induced the release of IL-1β with sub-nanomolar affinity (Danquah et al. 2016).

CXCL10 expression level increases in several diseases including SARS-CoV-2 infection. The group of Sadeghian-Rizi et al. (2019) reported the isolation of anti-CXCL10 polyclonal HcAbs for the development of a specific Nbs that specifically target CXCL10 for in vivo therapeutic applications (Sadeghian-Rizi et al. 2019). Several VHHs targeting other chemokine receptors including CXCR4, CXCR7 and ChemR23 have been described (Jahnichen et al. 2010; Maussang et al. 2013; Peyrassol et al. 2016). Similarly, VHHs Blockade CCL2, CCL5, CXCL11 and CXCL12 have been selected by Blanchetot et al. (2013). The selected Nbs showed preventing chemokine receptor activation-induced immune cells migration in vitro.

The adaptive immune response in SARS-CoV had been extensively investigated. It was reported that CD4 + T cells promoted the proliferation of neutralizing antibodies, whereas CD8 + T cells were responsible for the destruction of viral-infected cells. Increasing evidences have been reported that insufficient T cell responses could play a decisive role in clearance of SARS-CoV (Rajaei and Dabbagh 2020; Vellingiri et al. 2020).

In this context, targeted delivery of antigens to antigen presenting cells (APCs) improve immune responses by enhancing antibody production, activation of CD4+ T cells and elicitation of CD8+ T cell responses. MHC-II products, integrins (CD11b) and scavenger receptors (CD36) are abundantly expressed on APCs. Duarte et al. (2016) showed that VHHs specific for these molecules enhanced immune responses in distinct dendritic cells (DCs) populations. So, VHH can be used to deliver proteins or peptides to APCs to trigger humoral immunity and to track inflammation to treat or prevent SARS-CoV-2 infection.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820838/

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More information: Yufei Xiang et al, Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2, Science (2020). DOI: 10.1126/science.abe4747