Inhaled Nanobodies: A Potential Treatment for COVID-19

Nanobodies are small, highly specific fragments of antibodies produced by llamas and other camelids. Dr. Yi Shi, assistant professor in the department of cell biology at the University of Pittsburgh and colleagues are exploring the potential of nanobodies as a treatment for COVID-19. The team recently published findings from preclinical studies on the use of a nanobody called Pittsburgh inhalable Nanobody-21 (PiN-21) that targets the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein.

Technology Networks had the pleasure of speaking with Dr. Yi Shi, to learn more about the use of nanobodies to treat COVID-19. Shi discusses the key differences between antibodies and nanobodies, highlights some of the advantageous properties of nanobodies and the benefits related to aerosolized delivery.

Laura Lansdowne (LL): What is the difference between antibodies and nanobodies?

Yi Shi (YS): There are several differences between antibodies and nanobodies. So, antibodies are a specific component of the immune system, basically for all mammals. Nanobodies are unique in that they are only produced by members of the Camelidae family, which includes llamas, alpacas and camels. Nanobodies are much smaller than conventional antibodies – approximately 10% of the size. and they have outstanding properties in terms of stability and solubility compared to antibodies which can be exploited for drug development.

In terms of production, human antibodies usually have to be produced from human cells, which can be an expensive process, but for nanobodies, because of their small size, they can be produced using microbes such as E. coli or by using yeast cells, decreasing the production cost. It is also easier to scale up the production of nanobodies, which means they're more compatible in instances where you require them quickly and in vast quantities, such as a pandemic when millions of doses are potentially needed.

LL: In terms of dosage, is there a difference in the amount you would need to administer compared to antibodies?

YS: Well, that depends on the potency, the in vivo preclinical results and specific antibodies used. Antibodies with different potencies would likely require different doses. But one of the unique properties of nanobodies, as I mentioned, is stability. They are extremely stable, which means that when treating infections, instead of using intravenous delivery methods, nanobodies could be directly inhaled by aerosolization.

LL: So, they are quite amenable to that type of administration?

YS: Yes, exactly, this delivery approach is more direct compared to intravenous injection. For intravenous injection, a very small proportion (generally believed to be less than 0.2%) of the antibodies reach the intended target – in this case, the pulmonary infection sites deep in the lung. This is due to the need to overcome numerous hurdles, including the plasma and pulmonary barriers. If you could generate aerosolization the administration, efficiency is potentially much better. Another benefit is that this type of administration can also be conducted in both inpatient and outpatient settings.

Aerosolization is possible as I mentioned previously, because the nanobodies are so stable. It's really important that the biomolecules are stable because otherwise, the surface tension could easily aggregate the protein, negatively impacting bioactivity.

LL: How does the process for discovering these nanobodies work?

YS: We rely on animal immunization. In the case of SARS-CoV-2, we immunize a Camelidae with receptor binding domain (RBD) derived from the virus’ spike protein. We then use the immunized animal and allow for in vivo nanobody maturation. After about 50 to 60 days after immunization, we “boost”, and the animal produces a high-affinity antibody that binds to the immunized antigen, in this case, the RBD derived from the spike protein. We then use mass spectrometry (MS)-based proteomic technology to isolate the nanobodies that bind to the RBD from the immunized llama serum. We can then determine the amino acid composition of the nanobody protein and can reverse translate it into DNA, allowing us to produce the protein using E. coli.

LL: Are there any key challenges in terms of producing the nanobodies? For example, the chance of false positives?

YS: The false positive rate of a “true binder” is actually very small. And that's how we managed to identify some of the most potent neutralizing nanobodies that arise in < ng/mL concentrations. While this concentration is low, once they are identified, the production of the nanobodies is straightforward to carry out in a laboratory setting. As I said previously, once we know the amino acid sequence, we can reverse translate it and produce very small DNA pieces, which are introduced into the E. coli cells. Overnight E. coli will start to produce nanobodies in bulk quantities – it’s fast and it's very simple.

LL: In terms of storage, can they be stored at room temperature?

YS: Compared to monoclonal antibodies, these nanobodies are thermostable. We have conducted some experiments on the “best” nanobody we discovered and found that it can be stored at room temperature for at least six weeks, they are pretty stable.

LL: Are there any advantages to using nanobodies to treat COVID-19, compared to antibodies in terms of emerging SARS-CoV-2 variants and tackling those?

YS: With aerosolization, the nanobodies are distributed throughout both the upper and the lower respiratory tract, which is very important.

In the case of monoclonal antibodies and perhaps vaccines too, it's difficult to protect the upper respiratory tract, which means that a portion of vaccinated patients, even though they are vaccinated, could still be able to transmit the virus, so I think that's a very encouraging use. In terms of variants, we have generated a large repertoire of nanobodies, binding different regions on the virus’ spike protein, and then through structural studies looking at atomic-resolution details of these nanobodies to observe the impact of the major variants of concern on the neutralizing antibodies, and the result was very interesting. The take-home message is that they seem to be different compared to monoclonal antibodies, in that most of these neutralizing nanobodies are highly resistant to the circulating variants of concern.

LL: Could this process be used to produce antibodies against other viruses?

YS: This technology really showcases what potent neutralizing nanobodies could do. So, I would imagine that there are many other exciting applications by which nanobodies could be used, for the treatment of infectious diseases and in many other pathologies.

LL: In terms of your work developing nanobodies to treat COVID-19, what are the next steps?

YS: We recently evaluated the preclinical efficacy of a nanobody using a Syrian hamster model of moderate to severe COVID-19. We chose this model because they are highly sensitive; once the hamster is inoculated with the virus, they quickly develop the COVID-19 phenotype in terms of lung infection and a reduction in weight. We tested our lead nanobody candidate, Pittsburgh inhalable Nanobody 21 or “PiN-21”. After the successful delivery of the PiN-21 aerosols, the animals' weight loss was quickly reversed. And on top of that, there was (a six-order magnitude) reduction of the virus in the lungs and viral pneumonia was prevented.

Now we have completed at least one study using the sensitive COVID-19 Syrian hamster model, we are going to quickly move to non-human primate studies, which are much more expensive, but necessary to support the transition to clinical trials.

Dr. Yi Shi was speaking to Laura Lansdowne, Managing Editor for Technology Networks.