Nanobodies are small, extremely particular fragments of antibodies produced by llamas and different camelids. Dr. Yi Shi, assistant professor within the division of cell biology on the College of Pittsburgh and colleagues are exploring the potential of nanobodies as a remedy for COVID-19. The staff not too long ago printed findings from preclinical research on using a nanobody known as Pittsburgh inhalable Nanobody-21 (PiN-21) that targets the receptor-binding area (RBD) of the SARS-CoV-2 spike (S) protein.
Expertise Networks had the pleasure of talking with Dr. Yi Shi, to study extra about using nanobodies to deal with COVID-19. Shi discusses the important thing variations between antibodies and nanobodies, highlights a number of the advantageous properties of nanobodies and the advantages associated to aerosolized supply.
Laura Lansdowne (LL): What’s the distinction between antibodies and nanobodies?
Yi Shi (YS): There are a number of variations between antibodies and nanobodies. So, antibodies are a selected part of the immune system, mainly for all mammals. Nanobodies are distinctive in that they’re solely produced by members of the Camelidae household, which incorporates llamas, alpacas and camels. Nanobodies are a lot smaller than typical antibodies – roughly 10% of the scale. they usually have excellent properties by way of stability and solubility in comparison with antibodies which could be exploited for drug growth.
When it comes to manufacturing, human antibodies normally must be produced from human cells, which could be an costly course of, however for nanobodies, due to their small measurement, they are often produced utilizing microbes comparable to E. coli or by utilizing yeast cells, lowering the manufacturing price. It’s also simpler to scale up the manufacturing of nanobodies, which implies they’re extra suitable in cases the place you require them rapidly and in huge portions, comparable to a pandemic when thousands and thousands of doses are probably wanted.
LL: When it comes to dosage, is there a distinction within the quantity you would wish to manage in comparison with antibodies?
YS: Properly, that is determined by the efficiency, the in vivo preclinical outcomes and particular antibodies used. Antibodies with totally different potencies would possible require totally different doses. However one of many distinctive properties of nanobodies, as I discussed, is stability. They’re extraordinarily steady, which signifies that when treating infections, as an alternative of utilizing intravenous supply strategies, nanobodies might be instantly inhaled by aerosolization.
LL: So, they’re fairly amenable to that sort of administration?
YS: Sure, precisely, this supply method is extra direct in comparison with intravenous injection. For intravenous injection, a really small proportion (typically believed to be lower than 0.2%) of the antibodies attain the supposed goal – on this case, the pulmonary an infection websites deep within the lung. That is because of the want to beat quite a few hurdles, together with the plasma and pulmonary obstacles. In the event you may generate aerosolization the administration, effectivity is probably significantly better. One other profit is that this sort of administration will also be performed in each inpatient and outpatient settings.
Aerosolization is feasible as I discussed beforehand, as a result of the nanobodies are so steady. It is actually vital that the biomolecules are steady as a result of in any other case, the floor stress may simply combination the protein, negatively impacting bioactivity.
LL: How does the method for locating these nanobodies work?
YS: We depend on animal immunization. Within the case of SARS-CoV-2, we immunize a Camelidae with receptor binding area (RBD) derived from the virus’ spike protein. We then use the immunized animal and permit for in vivo nanobody maturation. After about 50 to 60 days after immunization, we “enhance”, and the animal produces a high-affinity antibody that binds to the immunized antigen, on this case, the RBD derived from the spike protein. We then use mass spectrometry (MS)-based proteomic expertise to isolate the nanobodies that bind to the RBD from the immunized llama serum. We are able to then decide the amino acid composition of the nanobody protein and may reverse translate it into DNA, permitting us to supply the protein utilizing E. coli.
LL: Are there any key challenges by way of producing the nanobodies? For instance, the possibility of false positives?
YS: The false optimistic charge of a “true binder” is definitely very small. And that is how we managed to establish a number of the most potent neutralizing nanobodies that come up in < ng/mL concentrations. Whereas this focus is low, as soon as they’re recognized, the manufacturing of the nanobodies is easy to hold out in a laboratory setting. As I mentioned beforehand, as soon as we all know the amino acid sequence, we will reverse translate it and produce very small DNA items, that are launched into the E. coli cells. In a single day E. coli will begin to produce nanobodies in bulk portions – it’s quick and it is quite simple.
LL: When it comes to storage, can they be saved at room temperature?
YS: In comparison with monoclonal antibodies, these nanobodies are thermostable. Now we have performed some experiments on the “finest” nanobody we found and located that it may be saved at room temperature for at the least six weeks, they’re fairly steady.
LL: Are there any benefits to utilizing nanobodies to deal with COVID-19, in comparison with antibodies by way of rising SARS-CoV-2 variants and tackling these?
YS: With aerosolization, the nanobodies are distributed all through each the higher and the decrease respiratory tract, which is essential.
Within the case of monoclonal antibodies and maybe vaccines too, it is tough to guard the higher respiratory tract, which signifies that a portion of vaccinated sufferers, despite the fact that they’re vaccinated, may nonetheless be capable to transmit the virus, so I believe that is a really encouraging use. When it comes to variants, we have now generated a big repertoire of nanobodies, binding totally different areas on the virus’ spike protein, after which via structural research taking a look at atomic-resolution particulars of those nanobodies to watch the impression of the most important variants of concern on the neutralizing antibodies, and the consequence was very fascinating. The take-home message is that they appear to be totally different in comparison with monoclonal antibodies, in that the majority of those neutralizing nanobodies are extremely proof against the circulating variants of concern.
LL: Might this course of be used to supply antibodies towards different viruses?
YS: This expertise actually showcases what potent neutralizing nanobodies may do. So, I might think about that there are lots of different thrilling functions by which nanobodies might be used, for the remedy of infectious illnesses and in lots of different pathologies.
LL: When it comes to your work creating nanobodies to deal with COVID-19, what are the following steps?
YS: We not too long ago evaluated the preclinical efficacy of a nanobody utilizing a Syrian hamster mannequin of reasonable to extreme COVID-19. We selected this mannequin as a result of they’re extremely delicate; as soon as the hamster is inoculated with the virus, they rapidly develop the COVID-19 phenotype by way of lung an infection and a discount in weight. We examined our lead nanobody candidate, Pittsburgh inhalable Nanobody 21 or “PiN-21”. After the profitable supply of the PiN-21 aerosols, the animals’ weight reduction was rapidly reversed. And on prime of that, there was (a six-order magnitude) discount of the virus within the lungs and viral pneumonia was prevented.
Now we have now accomplished at the least one research utilizing the delicate COVID-19 Syrian hamster mannequin, we’re going to rapidly transfer to non-human primate research, that are far more costly, however essential to assist the transition to medical trials.
Dr. Yi Shi was talking to Laura Lansdowne, Managing Editor for Expertise Networks.