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WE LIVE in a world teeming with bacteria and viruses looking for a warm,
moist, nutrient-rich home such as the human body. Our ability to hold out
for decades against these would-be invaders is a tribute to the strength
of our immune system, of which antibodies are a key component. Antibodies
are first and foremost binding proteins, however, and they do not kill cells
themselves. Rather, by clustering on the surface of the foreign cell, antibodies
trigger complex ‘effector’ systems that do lead to the death of cells. The
molecular nature of these triggering processes is now becoming clear.

First it is worth recalling what antibodies look like and why. They
are adaptor molecules, connecting the intended victim with the potential
killer. Antibodies launch an attack by first recognising and binding to
foreign material, known as antigens. Then the antibodies bind to effector
molecules circulating in bodily fluids as well, thereby linking the foreign
material with its means of elimination. In molecular terms, the antibody
first binds to structures on the surface of the foreign material, known
as antigenic determinants. These antigenic determinants, such as proteins
on the surface of a bacterial cell, are present in many copies. The effector
molecules now recognise and bind to the array of antibodies bound to antigen
and trigger its elimination. Only clustered antibodies will trigger pathways
that lead to elimination; free antibody, which does not, is at high concentration
in blood serum.

This dual binding presents the antibody with a potential problem. Antigens
vary widely – a human being can produce antibodies against more than a million
different molecular structures – but there are very few ways of getting
rid of them. Put another way, it is not practical for nature to devise a
different molecular solution to the problem of elimination for each different
antibody molecule. The conflicting requirements for diversity and commonality
are elegantly met by the stucture of the ‘typical’ antibody
(see Figure 1).

The structure, deduced from the work that won Rodney Porter and Gerald
Edelman the Nobel Prize in 1972, consists of three units. Two of the units
are identical and are involved in binding to antigen; these units are called
the Fab, or fragment antigen binding, arms of the molecule. Regions of these
units contain sequences of amino acids that vary greatly from one antibody
to another; these variable regions give each antibody its ability to bind
uniquely to a particular antigen. The existence of two Fab arms enables
the antibody to bind antigen strongly in the normal situation where the
host meets multiple copies of antigenic determinants. The third unit – Fc,
or fragment crystalline – links the antibody to effector molecules. The
antibody molecule is made up of four chains, consisting of two identical
‘heavy’ chains spanning the Fab and Fc units and two identical ‘light’ chains
associated only with the Fab unit.

The five classes of antibodies or immunoglobulins, called immunoglobulin
G (IgG), IgM, IgA, IgD and IgE, differ in their heavy chains, labelled with
the Greek letters &ggr;, &mgr;, &agr;, &dgr;, &egr; respectively. The differences, most pronounced
in the Fc regions, enable different classes of antibodies to trigger different
effector functions once they have bound to antigen. If IgG recognises an
antigen, for example, it might lead to the activation of a cascade of protein-destroying
enzymes known as complement. On the other hand, recognition by IgE, possibly
of the same antigen, might lead to a dangerous, potentially lethal, allergic
reaction: the release of histamine from mast cells and the development of
anaphylactic shock, in which blood vessels become more permeable and smooth
muscles contract.

Structural differences in the antibodies also lead to differences in
how the antibodies of a class associate with one another. IgG and IgE usually
exist as separate molecules, or monomers, whereas IgM occurs as a pentamer.
IgA exists predominantly as a monomer in blood serum and as a dimer in secretions
in the gut, for instance.

The major antibody in serum is IgG. The Fab arms are joined to the Fc
by a flexible region known as the hinge. This allows the arms to move relative
to one another, giving the antibody a variable reach. This enables IgG to
attach to two antigens even though they are close together or far apart.
The hinge also allows Fc to move relative to the Fab arms, and this may
be important in triggering the effector molecules. This model is still only
a prediction, however. Protein crystallographers have analysed crystals
of fragments of Fc and Fab regions, and for mutant IgGs with no hinge, and
produced high-resolution images. But no one has yet worked out the complete
structure of an intact, functional IgG. So in our picture, the hinge has
been generated on a computer graphics display, using IBM WINSOM solid modelling
software, courtesy of Peter Quarendon, of IBM Winchester. This hinge links
the known structures of human Fab and Fc determined by Robert Huber, Johan
Deisenhofer and their colleagues in Munich. Like other antibodies, IgG is
organised into domains, mostly paired, based on a common pattern of folding
known as the immunoglobulin fold.

Many splendoured molecules

The antibody IgE, responsible for the symptoms of allergy as well as
its protective role, differs somewhat from IgG. The hinge is now replaced
with an extra pair of domains. This structure is more speculative than the
model of IgG, as none of its parts has been crystallised so that X-rays
could reveal how they are built. The structure is based on extrapolation
from IgG carried out by David Davies and Eduardo Padlan of the National
Institutes of Health in Bethesda, Maryland.

Armed with some understanding of the structure of antibodies, researchers
have tackled the intriguing question of how antibodies trigger effector
systems. Complement, the cascade of death-dealing enzymes, is a major system
for defence in the bloodstream. In the ‘classical’ pathway, it is activated
by antibodies clustered on a foreign particle or organism. Under certain
conditions, it can also be activated by the surface of cells devoid of antibody,
a phenomenon known as the alternative pathway. Complement consists of nine
proteins of components in the blood, defined as C1 to C9. The
early proteins in the series are linked together in a cascade process similar
to what happens when blood clots: one protein acts on the next to generate
an active protein. The active protein is usually an enzyme that clips off
a part of the next protein in the cascade to convert it from an inactive,
proenzyme, to an active enzyme. Because enzymes are catalysts that can act
on many molecules, the cascade has built-in amplification: a small number
of activated molecules can result in activation of a large number further
down the cascade. As in blood-clotting, this allows the body to respond
to trauma rapidly and effectively.

The later proteins of the cascade are laid down together in a giant
complex in the membrane of a foreign cell. There, they form pores or holes
that prove fatal to the foreign cell. Intermediate proteins can also contribute
to the death: the clipped-off fragments can increase the flow of blood near
the site of infection, bringing more antibody and complement. They can also
cause white blood cells to leave blood vessels and move into tissues to
reach the site of invasion. One activated protein can bind directly to the
membrane of a foreign cell, where it becomes a recognition signal for host
white cells which then destroy the foreign cell. Clearly, the body must
confine the effects of such a powerful system to a local site and prevent
it from spreading. The body achieves this by packing the blood with high
concentrations of molecules that inhibit various stages of the pathway.

So the complement cascade is a beautifully intricate and delicately
poised system for destroying cells. How is it triggered? The critical interaction
is that of complement C1 with clustered antibody. C1
is a complex of three proteins, referred to as C1qC1r₂C1s₂.
C1q binds to antibody, which activates C1r. Activated
C1r in turn activates C1s and triggers the cascade.
So the first event is the binding of C1q to antibody, an event
portrayed in Figure 2.

C1q is a most unusual molecule; research by Ken Reid and
Rodney Porter of the University of Oxford suggests that it is made up of
six units and looks rather like a bunch of tulips. The extended stalks are
made up of a triple helix of collagen molecules, as found in skin, bone
and cartilage, for example. The heads form compact, folded, globular units.
These globular units bind to a simple motif on the Fc part of the antibody
IgG.

The hexameric nature of C1q is in a sense the molecular key
that explains why antibodies will trigger complement when they are clustered
on the surface of a cell, but not when they are free in the serum. A protein
inhibitor bound to C1 normally prevents its activation, and although
C1 will bind single antibody molecules in the serum, the interaction
is too weak to displace the C1 inhibitor. When antibodies are
clustered, however, several ‘heads’ of the C1 molecule can bind,
making the link much stronger. Now, the inhibitor is displaced and C1
activated. The binding of C1q to clustered antibodies also seems
to produce a signal that enhances the activation of C1, but we
do not yet understand how.

We have a rough idea of how C1q bound to an array of antibodies
might look from studies by Egidijus Uzgiris and Roger Kornberg and their
colleagues. They looked at antibodies bound in two dimensions to the grid
of an electron microscope. No one knows how close this artificial situation
is to that found at the surface of a cell. One interesting feature of the
model, however, is that the antibody molecules are dislocated; that is,
the Fab arms are bent out of the plane of the Fc so that the plane formed
by the arms is roughly at right angles to that of Fc. This emphasises the
flexibility of antibodies and the importance of thinking of them as three-dimensional
objects and not flat Y or T shapes on paper.

Cell eats cell

The body has devised another way of killing. White blood cells such
as monocytes, lymphocytes and neutrophils employ two main mechanisms to
kill target cells coated with antibodies. One is phagocytosis, where the
white cell engulfs or swallows the foreign cell and then destroys it internally
using an array of toxic agents. The second approach is called cellular cytotoxicity:
the white cell appears to inject toxic agents into the foreign cell. Both
mechanisms are triggered by the Fc parts of the bound antibody molecules
interacting with proteins on the surface of the white cell known as Fc receptors.
These Fc receptors also have other roles. They are found on specialised
cells called trophoblasts, where they transport antibody from mother to
fetus, and on mast cells, where the binding of clustered IgE can cause the
cells to release histamine. The resulting flood of histamine can help the
body to reject parasites, but it is more often associated with the unwelcome
symptoms of allergy.

Researchers are beginning to understand how an antibody molecule may
link a white blood cell such as a monocyte to a cell infected, for example,
by influenza virus, and so lead to the infected cell’s death (see Figure
3). Again, the antibody is dislocated, with the Fab arms bent out of the
plane of the Fc. Dislocation is almost inevitable when two cells seek to
interact with the Y-shaped antibody: one cell binds at the extremities of
the arms and the other at the midpoint (corresponding to the bottom of the
hinge in the IgG molecule).

Why do we believe that the Fc receptor binds to this site on the antibody
IgG? The best piece of evidence comes from the new technique of protein
engineering, in which researchers make precise changes in the gene coding
for a given protein. This process, known as site-directed mutagenesis, results
in precise changes in the amino acid sequence of the protein made from the
gene. Earlier studies by Dennis Burton, Jenny Woof and their colleagues
at Sheffield had implicated a specific sequence of amino acids in the hinge
(leucine. leucine. glycine. glycine) in the binding of the receptor. An
antibody with an altered sequence (leucine. aspartate. glycine. glycine)
could not bind to the receptor. Greg Winter and Alex Duncan at the Laboratory
of Molecular Biology at Cambridge engineered the aspartate in this antibody
to a leucine. The new antibody, tested in Burton’s laboratory, could bind
normally to the receptor.

So protein engineering is a powerful tool allowing us to understand
how antibodies work at the molecular level. It is also powerful in that
we can now contemplate the ‘design’ of antibodies to treat various human
ills. We can now engineer in or out the sites responsible for the triggering
the effector systems: death by antibody by design.

Dr Dennis Burton is a Jenner Fellow of the Lister Institute of Preventive
Medicine, Dr Peter Artymiuk is a Royal Society University Research Fellow,
and Dr Geoffrey Ford is a Wellcome Trust Senior Lecturer. They all work
at the Krebs Institute for Biomolecular Research at the University of Sheffield.