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Developing Nanobodies and Antibodies Against SARS-CoV-2

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Developing Nanobodies and Antibodies Against SARS-CoV-2

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The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to spread across the globe. The number of documented cases has exceeded  205 million, resulting in more than 4.3 million deaths. The virus causes respiratory difficulty as well as multi-organ damage, through a multi-faceted pathological process, involving immune system hyperactivity, microvascular damage and metabolic disturbances. The virus infects the host by binding its spike protein to the angiotensin-converting enzyme 2 (ACE2) cell surface receptor, which facilitates entry into host cells.

Although the COVID-19 pandemic is unprecedented in scope, researchers have met this challenge by developing a suite of preventive and therapeutic strategies to combat the virus. Due to the urgent nature of the pandemic, investigational preventive and therapeutic agents are being administered to individuals in a “real world” setting, while also being tested simultaneously in clinical trials.

Among preventive approaches,
COVID-19 vaccines were developed at a record pace, using new mRNA technology, which amplifies the host’s immune response to the SARS-CoV-2 spike protein to boost host protection. Additional vaccines using more conventional technologies are also being deployed. Of the therapeutic approaches, neutralizing antibodies present in convalescence plasma are being investigated, as are pharmacological agents, such as the anti-inflammatory glucocorticoid dexamethasone.

In order to control the pandemic, preventive vaccines have been a major focus, and over
4.6 billion doses have been administered worldwide. However, despite immense progress, concerns remain about potential “breakthrough” infections, which are infections that occur in vaccinated individuals. Breakthrough infections happen as a result of virus “variants”, which harbor mutations. Mutations are especially critical when they occur in the viral spike protein, the region used by the vaccine to mount the host’s immune response. Moreover, the vaccine may be less effective in vulnerable populations including frail geriatric adults. As a result, therapeutic strategies continue to be explored. This article will review two such therapeutic avenues, broadly neutralizing antibodies (NAbs) and nanobodies.

Broadly neutralizing antibodies against SARS-CoV-2


One therapeutic approach for combating SARS-CoV-2 infection is to administer antibodies to COVID-19 patients, to “neutralize” or disable the circulating virus. This involves high-affinity binding of the NAb to the virus. This process  generally occurs via the
receptor-binding domain (RBD) on the virus’s spike protein, prohibiting the virus from entering the host cell. NAbs can be isolated from convalescent plasma, i.e., plasma collected from patients that have recovered from infection, and manufactured as a biopharmaceutical. Isolating antibodies from plasma is limited by availability of plasma containing the antibodies. Thus, NAbs are present only transiently in convalescent plasma, and individuals vary in the type and efficacy of the antibodies they produce. An alternative is to clone NAbs from the memory B cells, which are long-lived, obtained from the peripheral blood of convalescent patients.

Before the pandemic began, Professor
Cascalho at the Department of Surgery and Department of Microbiology and Immunology at the University of Michigan and colleagues were working on B cell responses in connection with multiple disease states, including infection, cancer and transplant medicine. “We felt we were uniquely placed to contribute to our understanding of the immune response to SARS-CoV-2,” Cascalho recalled of her motivation to get involved in the early days of the pandemic.

Since then, she and her team have worked to isolate, clone and
characterize NAbs from convalescent blood B cells to study the landscape of antibody evolution. They succeeded and isolated monoclonal antibodies, which neutralize SARS-CoV-2 with high potency. However, the potency of these NAbs may be lost if viral mutations occur at the antibody–virus interface, as this lowers antibody affinity – a feasible scenario in increasing number of emerging viral variants.

“One way around this issue is to develop broadly NAbs, which retain their potency against a range of viral variants,” explained Professor
Cascalho. “Antibodies are the host’s first line of defense against an infectious agent, such as SARS-CoV-2. They are therefore an essential component of the immune response a host mounts against infection. We found that most COVID-19 patients produced antibodies that neutralized the virus. Some antibodies were exceptionally good and were among the most potent reported at the time, with half-maximal inhibitory concentrations in the femto molar range,” Cascalho added.

B cells evolve over the course of infection in a dynamic manner, a process called
somatic hypermutation. This process occurs by mutating the sequence of the variable domain (IgV) of antibodies produced by B cells. As the IgV domain mutates, it diversifies the generated antibodies, varying their affinity against the infectious agent. “In COVID-19 patients, we found antibodies gained affinity during infection through somatic hypermutation and thus became more effective against SARS-CoV-2,” explained Cascalho.

However, Cascalho explained that they had observed a subset of antibodies that had lower affinity for the virus, but were effective against a broader range of variants, including the
Wuhan Hu-1 founder strain and various spike protein mutants, such as D614G, which contributes to the higher infectivity of the delta variant. “This subset of broadly NAbs was highly mutated, and, interestingly, developed within the timeframe of acute SARS-CoV-2 infection, a period of only a few weeks. In contrast, during chronic HIV infection, it takes three years to develop broadly NAbs. We’re not sure yet why broadly NAbs develop rapidly during SARS-CoV-2 infection. Additionally, we know T cells are also involved in the immune response to clear virus-infected cells, but are still elucidating the details.”

Cascalho is pursuing additional research avenues in COVID-19 research: “We just published a
paper that leveraged directed evolution to develop neutralizing nanobodies and have a non-peer reviewed preprint examining adjuvants for a recombinant SARS-CoV-2 protein vaccine.” Cascalho is satisfied that she is contributing to a deepening of our understanding of the immune response to SARS-CoV-2 and developing potentially therapeutic antibodies. However, she notes larger collaborative studies and more institutional support are needed: “We have the know-how to advance science, respond fast to challenges, of which SARS-CoV-2 is a great example, and to create great therapeutics. In fact, we had our antibody sequences within four months of starting the research, in the early Summer of 2020. But because we lack industry ties those discoveries remained in limbo and did not fulfill their potential. We hope that institutions learn how to better support their scientists, helping to move discoveries to a clinical setting.”

Where Do We Go From Here: A Reflection of COVID-19

In less than two years, much has been discovered about SARS-CoV-2 and its variants, and to date several vaccines have been created. Capitalizing on several decades of progress on new vaccine technologies, viral immunology, structural biology and protein engineering research, along with clinical trial operations, seven vaccines have undergone the process of rapid development, evaluation, manufacturing and deployment. Download this app note to explore various solutions designed to improve COVID-19 detection and the development of vaccines and therapeutics.

Download App Note

Nanobodies: Miniaturized but versatile SARS-CoV-2 neutralizing agents


Although serviceable, antibody-based therapeutics, such as NAbs, face
production challenges and are temperature sensitive. Nanobodies, a smaller variant of full-sized antibodies, have emerged as an alternative and are being pursued as a potential COVID-19 treatment. “Nanobodies are functional antigen-recognizing molecules, which were first isolated in camelids, the family of mammals that includes camels, alpacas and llamas,” explained Associate Professor Wai-Hong Tham, joint head of the Infectious Diseases and Immune Defence Division at WEHI. “Nanobodies are so-called due to their smaller size, since they lack the light chains present in full-sized antibodies. The smaller size imparts many advantages to nanobodies, which renders them attractive as therapeutics. This includes very strong affinity to target antigens and high stability to temperature and pH. Production is cost-effective and high-yielding in bacterial, yeast and mammalian expression systems compared to conventional antibodies. Perhaps most importantly for respiratory diseases, their high stability opens up the possibility of developing them as inhaled biotherapeutics,” Tham elaborated.

Early in the pandemic, nanobodies emerged as potent
neutralizing agents against SARS-CoV-2 by interfering with RBD binding to ACE2, including the development of multivalent candidates. However, these studies only demonstrated efficacy and strong binding of the nanobody to the SARS-CoV-2 RBD in vitro. A significant hurdle to in vivo applications of nanobodies is their small size, which increases renal elimination and shortens half-life. Additionally, the virus can escape neutralization by nanobodies due to variant evolution. To address these challenges, Tham and her team worked to develop a nanobody cocktail technology in vivo by testing efficacy in mice. “We know SARS-CoV-2 mutates, including at the RBD, which can lead to antigen escape. The chance of escape increases with an individual antibody or nanobody,” explained Tham. “In contrast, a neutralizing cocktail made up of multiple viral RBD-targeting nanobodies decreases the rate of viral escape. If one nanobody fails to bind to the mutated RBD, another nanobody from the cocktail may retain high binding affinity.”

Tham leveraged
phage display, a high-throughput screening method, to identify nanobodies from alpacas immunized with recombinant (non-infectious) SARS-CoV-2 spike and RBD. “We isolated 50 nanobody candidates with binding affinities ranging from about 0.1 to 19 nM and viral neutralization activities ranging from 3 nM to 36 µM,” reported Tham. Next, the four most promising nanobodies were fused to the human IgG1 Fc domain to increase the molecular weight and improve pharmacokinetics. “Fusion to the Fc domain did not impair binding affinity of the nanobodies to SARS-CoV-2 RBD. In fact, we found that chimeras retained the ability to bind to wild-type RBD as well as a wide variety of RBD variants, spanning 22 mutations at 16 residues,” Tham summarized. Lastly, testing in vivo was conducted in wild-type mice infected with SARS-CoV-2 by inhalation. “We found that prophylaxis with our nanobody cocktail significantly lowered the burden of SARS-CoV-2 in the lung of mice with SARS-CoV-2 infection.”

Tham’s approach has several advantages. The technology can be adapted rapidly to target a mutating SARS-CoV-2 virus. “We have characterized a library of over 50 nanobodies, so we have the ability to decipher which library candidates will remain effective against any emerging global variant of concern. Furthermore, phage display is a high-throughput technology. Thus, if none of the nanobodies from our current library are effective against an emerging variant, we can screen through millions of other nanobodies, to identity candidates that would recognize the novel mutant epitope.”

Additionally, the strategy could be adapted to develop broadly neutralizing nanobodies. “There are
conserved epitopes between the SARS-CoV-1 and SARS-CoV-2, which is already targeted by one of her leading neutralizing nanobodies. This is most definitely an important future approach,” Tham concluded.

Meet The Author
Masha Savelieff, PhD
Masha Savelieff, PhD
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