Turning Antibodies into Precisely Tuned Nanobodies

Nanobodies can do the same functions, for instance, tagging molecules for research or flagging diseased cells for destruction. However, because of their comparative simplicity, nanobodies offer the enticing possibility of being much easier to make.

Regrettably, their potential has not been fully realized, because scientists have lacked an effective way of identifying the nanobodies most closely tuned to their targets. However, a new system, developed by researchers at Rockefeller University and their collaborators, and described November 2, 2014, in the journal Nature Methods, has the potential to make nanobodies dramatically more accessible for all kinds of research.

Antibodies are defensive proteins implemented by the immune system to identify and neutralize invaders. But their power can be harnessed in other ways as well, and they are used in biology and medicine for visualizing cellular processes, attacking diseased cells and delivering specific molecules to specific places. Similar to their larger cousins, nanobodies can also be used for these tasks, but their small size makes nanobodies much easier to grow in bacterial factories. They can also access hard to reach places that may be off limits to larger molecules.

“Nanobodies have tremendous potential as versatile and accessible alternatives to conventional antibodies, but unfortunately current techniques present a bottleneck to meeting the demand for them,” said study author Dr. Michael Rout, head of the laboratory of cellular and structural biology at Rockefeller University (New York, NY, USA). “We hope that our system will make high-affinity nanobodies more available, and open up many new possible uses for them.”

In their first research project, the scientists generated high-affinity antibodies, those that are capable of most precisely attaching to their targets, directed against two fluorescent proteins that biologists often use as markers to visualize activity within cells: green fluorescent protein (GFP) and mCherry. Their new system, similar to existing ones for generating antibodies, begins with an animal, in this case llamas housed in a facility in Massachusetts.

Llamas were chosen because the antibody variants they produce are easily modified to generate nanobodies, which are only one-tenth the weight of a regular antibody. The llamas were immunized with GFP and mCherry, triggering their immune systems to generate antibodies against these foreign proteins, known as antigens.

“The key was to figure out a relatively fast way of determining the genetic sequences of the antibodies that bind to the targets with the greatest affinity. Up until now obtaining these high-affinity sequences has been something of a holy grail,” said Brian Chait, professor and head of the laboratory of mass spectrometry and gaseous ion chemistry. “Once those sequences are obtained, it’s easy to engineer bacteria to mass produce the antibodies.”

The researchers began by generating antibody sequence databases from RNA isolated out of antibody-producing cells in the llamas’ bone marrow. Next, they captured the tightest binding GFP and mCherry antibodies from blood samples from the same llamas, and chemically cut these into smaller pieces, keeping only the antigen-binding section to create nanobodies.

The scientists then determined partial sequences of the amino acids that made up the protein of the nanobodies with a technique known as mass spectrometry. Using a computer algorithm called “llama magic,” developed by David Fenyö and Sarah Keegan of New York University School of Medicine, they matched up the composition of the highest affinity nanobodies with the original RNA sequences. With the sequences, they could engineer bacteria to mass produce the nanobodies before using them in their research.

Antibodies are frequently employed to isolate a specific structure within a cell so that scientists can remove and study it, and the investigators did just that with their new nanobodies. They purified various cellular structures tagged with GFP or mCherry, and also visualized these structures in situ.

In total, the technology generated 25 types of nanobodies capable of precisely targeting GFP and six for mCherry, a far more diverse set of high affinity nanobodies than is typically possible with traditional techniques. This profusion opens up new avenues for treatments. Scientists can choose only the best ones, eliminating nanobodies that by chance cross-react with other molecules, or string together two nanobodies that attach to different spots on the same target molecule to generate a super-high-affinity dimer, precisely as the researchers demonstrated for the GFP nanobodies. This super-high-affinity could be a powerful feature when delivering therapeutic or diagnostic molecules because it would lower the required dosage, and so reduce unwanted side effects. “Given that we can now readily identify suites of high affinity nanobodies, the future for them as research tools, diagnostics and therapeutics looks bright,” stated Dr. Rout.

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