Future for nanobodies as alternative research tools to antibodies looks bright

Date: Nov-03-2014 In nature, antibodies are useful for locating specific molecular targets, for

instance, to help the immune system spot and attack disease microbes. These properties

also make them useful for biomedical research. Now, nanobodies - tiny cousins of

antibodies - could be even simpler to make and use, thanks to a "robust pipeline"

technique reported in a new study.

When researchers introduced nanobodies they made to cells engineered to express a tagged version of a protein in skeletal fibers known as tubulin (red), the nanobodies latched on.
Image credit: The Rockefeller University

Scientists use antibodies as basic tools in human and animal health fields like

research, diagnostics and treatment development. For instance, to understand how

normal cells work and how they differ from diseased cells, researchers might use

antibodies to target and identify specific proteins that are in the cells at

particular stages of development.

Using the results of such tests, they can then build a model of how the cell works,

and what happens when it becomes diseased. This is valuable information for developing

and testing new treatments for the disease.

Nanobodies - which were first discovered several years ago in the unique antibodies

of camels and llamas - offer an exciting alternative to antibodies as

biomedical tools because they are much smaller, and show higher affinity to their

molecular targets. Nanobodies have about one-tenth of the weight of antibodies, and

they are stable and easy to manipulate.

However, it is not easy to identify repertoires of nanobodies with sufficient high

affinity to specific targets - current methods have proved too time consuming and

difficult - and so many researchers continue to use antibodies.

Now, that might change, thanks to a new technique published in Nature

Methods and led by The Rockefeller University in New York, NY, as co-author

Michael Rout, professor and head of Rockefeller's Laboratory of Cellular and

Structural Biology, explains:

"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. We hope that our system will make high-affinity

nanobodies more available, and open up many new possible uses for them."

Team isolated nanobodies from high-affinity antibodies produced in llamas

First, the team made antibodies with high-affinity - that is highly tuned to bind

precisely to their molecular targets - and targeted them to find two fluorescent

proteins: GFP and mCherry. Biologists use these fluorescent proteins to visualize

activity inside cells.

Like conventional ways of making antibodies, their technique uses animals at first.

In this case, the team started with llamas, which are known to make antibodies that

are easily modified to make nanobodies. They immunized the llamas with the two

proteins, so their immune systems readily produced the required antibodies.

The next step was crucial in speeding up the production of nanobodies: how to

rapidly sequence the genetic code of the high-affinity antibodies - the ones that had

the greatest ability to find and bind to the proteins.

It is easy engineer bacteria to mass produce the nanobodies

Co-author Brian Chait, professor and head of Rockefeller's Laboratory of Mass

Spectrometry and Gaseous Ion Chemistry, says:

"Up until now obtaining these high-affinity sequences has been something of a holy

grail. Once those sequences are obtained, it's easy to engineer bacteria to mass

produce the antibodies."

The team started by making sequence databases from RNA they found in the antibody-making cells of the immunized llamas' bone marrow. Then, using blood samples from the

same llamas, they selected the antibodies most tightly bound to the target proteins

and chemically cut them into smaller sections. For making the nanobodies, they kept

only those sections of the antibodies that were tightly bound to the proteins.

Using mass spectrometry and a computer algorithm they called "llama magic," the

team then determined the partial amino acid sequences of the building blocks of the

nanobodies, and matched the highest affinity ones with the original RNA sequences they

found in the antibody-producing cells.

They then used the antibody cell RNA sequences that matched the sequences of the

high-affinity nanobodies to engineer bacteria to mass produce the nanobodies.

New technique generates much larger repertoire of high-affinity nanobodies

The next step was to test the new nanobodies. Scientists often use antibodies to

isolate a particular part within a cell so they can remove it and study its structure.

So this is what the team did with their new nanobodies. They purified various cell

structures tagged with GFP or mCherry, and visualized them in place.

Using their new technique, the team generated 25 types of nanobodies with high

affinity for GFP and six for mCherry. This is a much larger repertoire than ones

typically produced with conventional methods.

A large repertoire is important because it gives researchers more options: they can

choose the best nanobodies, eliminate ones that might react with other molecules as

well as the target ones, and they can also string two together and attack two places

on the same target molecule.

Prof. Rout says, "Given that we can now readily identify suites of high-affinity

nanobodies, the future for them as research tools, diagnostics and therapeutics looks

bright."

In 2012, Medical News Today learned how researchers from the Institute of

Tropical Medicine, in Antwerp, Belgium, are working on a way to use "trojan horse"

bacteria to release nanobodies to conquer sleeping

sickness.

Written by Catharine Paddock PhD

Not to be reproduced without permission.

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Courtesy: Medical News Today Note: Any medical information available in this news section is not intended as a substitute for informed medical advice and you should not take any action before consulting with a health care professional.