Antibodies are essential research tools, but producing them is often a lengthy and expensive process. Nanobodies, which are a type of antibody produced by Camelids (an animal family that includes camels, llamas, and alpacas), offer many advantages over traditional antibodies.
They are smaller, are stable at higher temperatures, and can be expressed in bacteria, yeast, plants, and mammals. Not only does this make their production easier and cheaper, but it means they can be utilised in many research contexts that normal antibodies can’t.
Svend Kjaer, deputy head of the Crick Structural Biology Science Technology Platform (STP), has had a long-standing interest in utilising nanobodies in research. “We had been looking at various possibilities for starting nanobody production. With help from head of translational science Alison Maloney, we identified a farm at the University of Reading that had a herd of llamas. We have now leased two of their llamas, Arla and Curtis, to produce our own nanobodies in collaboration with different research groups at the Crick”.
The ‘body shop’
In order to generate nanobodies that will bind to a particular target, or antigen, the llamas are injected three times with the antigen over a few weeks, which causes their immune system to start producing the specific nanobodies. Svend and his team isolate immune cells from a small blood sample and then purify the genetic material that encodes the millions of different nanobodies produced by the llamas
They next use a screening technique called ‘phage-display’ to identify a set of nanobodies that bind to the target antigen with high specificity.
This production pipeline can be adapted to any relevant project: “One of the first projects we got involved with was generating nanobodies against the COVID-19 spike protein” explains Svend. “We generated a genetic library that contained 75 million different clones and from this isolated over 100 unique nanobodies, which interact with different parts of the spike protein”.
“In collaboration with Mary Wu, who is part of the High Throughput Screening STP at the Crick, we realised some of these nanobodies had neutralising activities, meaning they blocked viral reproduction. As you can produce them very easily, we have been supplying these nanobodies to be used in neutralising antibody tests, as part of the Crick Legacy Study of COVID-19 immune protection”.
Having an in-house production pipeline also meant Svend and his team could adapt to emerging COVID-19 variants: “it’s given us a lot of flexibility, as once you have this library of nanobody sequences you can go back and select ones that bind to different parts of the target protein. For example, we went back into our existing battery of nanobodies and found some that also targeted the Omicron variant.”
“It's another component in your toolbox”
The Structural Biology STP has also been collaborating with several other groups at the Crick, in order to unlock the potential of nanobodies in scientific research. One of these has been with senior group leader Jean-Paul Vincent and his lab, who study how cells organise themselves to form tissues.
They were interested in generating an antibody to detect Notum - a protein that is highly expressed in colorectal cancer – that could be used in diagnostic screens for cancer, yet had been unsuccessful using traditional methods. “We had been trying for a long time to make an antibody against Notum” explains Jean-Paul. “Svend had this opportunity to use the llamas, and it turns out the llamas were very good at making nanobodies against this protein”.
As in the COVID-19 project, Jean-Paul and his group found these nanobodies have impressive qualities. “Nanobodies can potentially be used as a treatment, by specifically inhibiting a disease-causing molecule. The advantage of nanobodies is that they’re small and they’re much more easily produced than antibodies on a large scale. Using a nanobody will allow us to design something that is much more easily manufactured”.
For Jean-Paul, a huge advantage in developing these nanobodies is also their ability to also be used for investigative research. “As a tool for research, a key benefit of nanobodies is that once you have the genetic code it can be inserted into another organism or cell line, and the nanobody will be produced there.
This allows you to manipulate the biological environment in many different ways, such as by specifically trapping or modifying other proteins. You can also add a dye to nanobodies and use them for imaging. Their small size allows you to get much closer to the protein you want to visualise, which is useful for newer super-resolution imaging techniques”.
As Jean-Paul emphasises, nanobodies are going to be a useful research tool long-term. “In the end, everything in biology is about proteins interacting with one another, and with nanobodies you can engineer an interaction network. These types of applications are going to be expanding and wouldn’t be possible with normal antibodies”.
“We can learn together”
Thanks to the production pipeline created by Svend and his team, it is now much more feasible for researchers at the Crick to utilise nanobodies in their work. “We’ve established a workflow here so that now from blood to nanobodies is a relatively smooth process. Once a nanobody collection has been created by the structural biology team, it’s a process of working closely with the researchers, in order to identify the ideal nanobody for that application. We keep the loop short so we can learn together”.