NEW TECHNIQUE EFFICIENTLY TURNS ANTIBODIES IN TO HIGHLY TUNED NANO BODIES
Antibodies, in charge
of recognizing and homing in on molecular targets, are among the most useful
tools in biology and medicine. Nanobodies -- antibodies' tiny cousins -- can do
the same tasks, for example marking molecules for research or flagging diseased
cells for destruction. But, thanks to their comparative simplicity nanobodies
offer the tantalizing prospect of being much easier to produce
Antibodies are
defensive proteins deployed 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. Like 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," says study author Michael Rout, head of the
Laboratory of Cellular and Structural Biology. "We hope that our system
will make high-affinity nanobodies more available, and open up many new
possible uses for them."
In their first
studies, the team generated high-affinity antibodies, those that are capable of
most precisely binding to their targets, directed against two fluorescent
proteins that biologists often use as markers to visualize activity within
cells: GFP and mCherry. Their new system, like 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, prompting 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," says
Brian Chait, Camille and Henry Dreyfus 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, led
by graduate student Peter Fridy and postdoc Yinyin Li, started by making
antibody sequence databases from RNA isolated out of antibody-producing cells
in the llamas' bone marrow. Next, they picked out 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.
They 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 putting them to
use in experiments.
Antibodies are often
used to isolate a particular structure within a cell so scientists can remove
and examine it, and the team did just that with their new nanobodies. They
purified various cellular structures tagged with GFP or mCherry, and also
visualized these structures in situ.
All in all, their
procedure 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 conventional techniques.
This abundance opens
up new options. Scientists can select 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, exactly 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," says
Rout.
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