Diagnostic and Therapeutic Antibodies

452 Pages·2000·2.54 MB·English

by  Andrew J.T. George

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The Antibody Molecule 1
1
The Antibody Molecule
Andrew J. T. George
1. Introduction
The importance of antibody molecules was first recognized in the 1890s,
when it was shown that immunity to tetanus and diphtheria was caused by
antibodies against the bacterial exotoxins (1). Around the same time, it was
shown that antisera against cholera vibrios could transfer immunity to naïve
animals, and also kill the bacteria in vitro (1). However, although antitoxin
antibodies rapidly found clinical application, there was little understanding
regarding the nature of the antibody molecule. Indeed, the earliest theories
suggested that the antitoxins were derived by modification of the toxin—
intriguingly similar “antigen incorporation” theories were propounded as late
as 1930 (1).
In more recent times, thanks to the efforts of both cellular and molecular
immunologists, we have a more complete understanding of the structure,
genetics, and function of an antibody molecule. As is discussed in the rest of
this volume, this knowledge has allowed the design of improved molecules for
clinical application.
2. Structure of the Antibody Molecule
The basic structure of an antibody (immunoglobin G [IgG]) molecule is
shown in Fig. 1, and is reviewed in detail in ref.2. It consists of four chains:
two identical heavy (H) and two identical light (L) chains. The heavy
chains vary between different classes and subclasses of antibody (e.g., (cid:161)heavy
chains are found in IgE, µ in IgM, (cid:97)1 in IgG1, and so forth). These different
classes and subclasses have specialized roles in immunity. There are two types
of light chains, (cid:103) and (cid:104). These do not have different functions, but represent
alternatives that help increase the diversity of immune recognition by antibod-
From:Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies
Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
1
2 George
Fig. 1. The antibody molecule. The structure of IgG is shown, with the domains
represented by separate blocks. The hinge region contains multiple disulfide bonds;
one is shown for convenience.
ies. The four chains are held together by both noncovalent interactions and
disulfide bonds, as shown in Fig. 1.
The H and L chains are made up of a number of domains of approx 110
amino acids arranged as two layers of antiparallel (cid:96)-sheets held together by a
conserved disulfide bond. These Ig domains, which fold independently, pro-
vide a modular structure to the antibody molecule, which has been exploited in
antibody engineering studies (seeChapter 3). These domains are the archetype
of those found in members of the immunoglobulin superfamily. In addition to
the Ig domains, there is a hinge region, which has an extended structure that
provides flexibility for the molecule.
A comparison of the sequence similarity between the domains of the anti-
body molecule shows that the majority of the domains have the same sequence
between antibody molecules of the same subclass, and so are termed constant
(C) domains (C 1, C 2, and so forth on the H chain, and C on the L chain).
H H L
However, one domain in each chain has a variable sequence, and so is termed
the variable (V) domain (V and V ). Comparison of the sequence between
H L
different V regions shows that most of the variability is confined to three parts
of the molecule, termed the complementarity-determining regions (CDRs),
which come together in three-dimensional space when the molecule is folded
to form the antigen-binding site (containing six CDR regions, three from the
V domain and three from the V domain).
H L
The structure of the antibody molecule was determined, in part, by the use
of enzymes that cut the molecule into distinct fragments. Thus, papain cleaves
the molecule N terminal to the disulfide bonds in the hinge region to yield the
The Antibody Molecule 3
Fig. 2. Fragments of antibody molecule. The major fragments of an IgG molecule
are represented here, with the antigen-binding Fv (~25 kDa in size), the Fab and Fab(cid:118)
(~50 kDa), and the F(ab(cid:118)) (~100 kDa) compared to the intact IgG (~150 kDa) and the
2
Fc region.
Fab (fraction antigen binding) and Fc part of the molecule. Pepsin cuts the C
terminal of the cysteines to produce a F(ab(cid:118)) fragment. This can be mildly
2
reduced to produce the Fab(cid:118)fragment (seeFig. 2). Other fragments that can be
produced by proteolytic cleavage include the Fv (V and V domains). The
H L
production of these fragments was instrumental in our understanding of the
structure–function relationship of the immunoglobulin molecule: thus the anti-
gen-binding property of the molecule was shown to reside in the Fab fragment,
4 George
with the Fab and Fab(cid:118) being monovalent and the F(ab(cid:118)) being bivalent. How-
2
ever, none of these parts of the molecule are capable of recruiting effector
function. That property resides in the Fc portion.
3. Functions of the Antibody Molecule
The antibody molecule has two major functions. The first is to act as the
antigen receptor for B cells. Thus, binding of antigen by surface immunoglo-
bulin on a B cell is a vital step in the triggering of the cell for activation (and in
delivering antigen to the MHC class II processing pathway). The second func-
tion is to act as the antigen-specific soluble effector molecule in the humoral
arm of the immune response. It is this function that is the topic of this volume.
Antibodies can exert their effector functions in one of three ways. The first is
to simply bind to their target antigen and neutralize it. Thus, antiviral antibod-
ies can bind to molecules on the virus surface that are essential for binding to
and infection of target cells; this can result in steric blocking of these mol-
ecules and so prevent infection. Similarly, antitoxin antibodies can act in a
similar manner. The other two ways in which antibodies can work rely on the
molecule-recruiting effector functions, either as complement or cells bearing
receptors for the Fc part of the molecule.
3.1. Complement
The complement system consists of a series of proteins arranged in a series
of pathways. In terms of antibodies there are three pathways of importance: the
classical pathway, the alternative pathway, and the lytic pathway. The classical
pathway is initiated by the binding of the first component of the pathway, C1,
to the Fc of antibody molecules that have bound their antigen. This causes
activation of C1, which can then activate the next component of the pathway,
C4, by chopping it up into the fragments C4a and C4b. C4b can associate with
C2, which is then cleaved by C1 into C2a and C2b. The complex formed of
C4bC2b is then capable of proteolytic cleavage of C3 into C3a and C3b.
The alternative pathway similarly serves to cleave C3 into C3a and C3b.
This pathway can be activated in several ways. However, in the context of
antibody-mediated activation, it serves as an amplification pathway. This is
because the pathway is initiated by C3b. Thus, once C3b is generated by the
classical pathway, it activates the alternative pathway, which then cleaves C3
to produce more C3b, thus providing a positive feed-forward pathway.
The lytic pathway is also initiated by C3b. This activates C5 by cleavage
into C5a and C5b. The membrane attack complex (C5b678(C9)n) is then
assembled. This forms a pore in membranes, and can lead to death of targeted
cells by osmotic lysis.
The Antibody Molecule 5
Table 1
Fc(cid:97)(cid:97)(cid:97)(cid:97)(cid:97)R
Name CD Distribution Affinity Specificity
Fc(cid:97)R1 CD64 Monocytes High (10–8M) IgG1 = IgG3 > IgG4
Fc(cid:97)RII CD32 Monocytes, Low (~10–6M) IgG1 = IgG3 > IgG2,IgG4
neutrophils,
eosinophils,
platelets,
B cells
Fc(cid:97)RIII CD16 Neutrophils, Low (~10–6M) IgG1 = IgG3
eosinophils,
macrophages,
NK cells
The effector functions of the complement system include lysis via the mem-
brane attack complex, opsonization (through receptors for C3b and C4b on
leukocytes) and the proinflammatory effects of anaphylotoxins (C5a, C3a,
C4a), which are chemotactic and also cause the release of vasoactive molecules
by mast cells and basophils.
3.2. Binding to FcR+ Cells
Many cell types express receptors for different classes of immunoglobulin.
These recognize determinants on the Fc part of the molecule. In the case of IgG
there are three major types of FcR on human leukocytes, as shown in Table 1.
The function of these molecules depends on the cell type expressing them, and
other features of the interaction, such as the affinity of the interaction. Thus,
CD16 (Fc(cid:97)RIII), when expressed on natural killer (NK) cells, directs antibody-
dependent cellular cytotoxicity (ADCC) against antibody-coated target cells.
The same molecule on monocytes promotes phagocytosis. In addition to pro-
moting phagocytosis, FcRs are involved in clearance of immune complexes
and antibody-coated debris by the reticuloendothelial system, and in the
release of mediators by basophils and mast cells (which bind IgE by high-
affinity Fc(cid:161)R). They can also have a role in antigen presentation and activation
of B cells.
3.3. Classes and Subclasses of Antibodies
As we discussed, antibodies can have different Fc regions depending on
their class or subclass. Different classes and subclasses have different func-
tions in immunity, as they show different abilities to recruit effector mecha-
nisms. In addition, some antibody classes have specialized functions, e.g., IgA
6 George
Fig. 3. Human (cid:103) gene locus. The (cid:103) gene locus consists of 40 V gene segments,
(although there is some variation in the population regarding the exact number), 4 J
segments, and a constant segment. During rearrangement one of the V segments is
recombined with one of the J segments at random. The figure is diagrammatic. In
reality the gene segments are more widely separated by introns. For an accurate map
seeref.3.
molecules can be dimerized to form (IgA) , which are secreted onto mucosal
2
surfaces and are an important component of host defenses at these sites. IgM
molecules are produced early in the immune response, before affinity matura-
tion (see Subheading 4.) has occurred. In order to compensate for the low
affinity of primary antibodies, IgM is found as a pentamer, with five compo-
nents similar to the archetypal Y-shaped IgG joined by disulfide bonds and an
additional J chain. This structure increases the valency of the molecule (from 2
to 10 antigen-binding sites) and so increases the avidity of its interaction with
antigen.
4. Genetics of Antibodies
In order to produce an antibody molecule with its variable domains and con-
stant domains, the B cell has to undergo complex DNA rearrangements. These
allow the vast diversity of antibody specificities to be produced, while retain-
ing constant regions capable of recruiting effectors. Mice and humans have
three immunoglobulin loci; the heavy chain, (cid:103) light-chain, and (cid:104) light-chain
loci. Each locus has a number of different gene segments. We first consider the
(cid:103) locus. This consists of a number of V-region gene segments (in the human
40), J gene segments (5 in the human), and a single constant-gene segment (see
Fig. 3)(3). During B-cell development, the V and J gene segments recombine
at random, such that one V segment is juxtaposed to one J segment, with the
intervening DNA being lost. This recombinatorial diversity means that 200
(40 × 5) different combinations of V and J segments can be obtained in the
human(cid:103) chain.
A similar arrangement is seen in the human (cid:104)locus with 30 V and 4 J seg-
ments (although the mouse (cid:104) locus has very little diversity, with just 2 V
T
h
e

A
n
t
ib
o
d
y

M
o
le
c
u
le
Fig. 4. Heavy-chain locus. The heavy-chain locus differs from the (cid:103)locus by having additional D segments that are rearranged
with the V and J segments. It also has multiple genes encoding different constant regions, corresponding to the different classes
and subclasses of immunoglobulin. These are rearranged during the process of class switching. Each of the genes for the constant
region contains multiple exons, as illustrated for µin the expanded section at the bottom of the figure, rather than the one shown.
For more detailed maps seeref.3.
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8 George
segments). The heavy-chain locus has an additional source of diversity in the
D segments (27 in humans), which are between the V (51 in humans) and J (6
in humans) (Fig. 4)(3–5). The heavy chain needs to undergo V-D-J recombi-
nation. The potential number of V(D)J recombinations in the human is there-
fore 8262 for the H chain locus, 200 for (cid:103), and 120 for (cid:104). This then allows for
combinatorial diversity, because any H chain can be paired with any light chain,
giving 1,652,400 possible different H-(cid:103) and 991,440 H-(cid:104) pairings.
Additional diversity can still be obtained by the imprecise nature of the join-
ing process between the gene segments, resulting from both untemplated nucle-
otide addition, which adds random coding sequences at the junction of the
segments, and by variations in the exact site of splicing of the DNA.
The diversity seen in the naïve B-cell repertoire is, therefore, largely the
result of recombinatorial diversity (using different V[D]J segments), combina-
torial diversity (different H and L chains), and junctional diversity. This diver-
sity is sufficient to allow the selection of the low-affinity antibodies during the
primary immune response. However, to obtain high-affinity antibodies seen
during the secondary immune response a further process occurs, that of
somatic mutation. This is seen in the germinal centers of lymph nodes and
involves essentially random mutation of the V(D)J gene segments that encode
the variable domains of the antibody. Some of these mutations lead to antibod-
ies that have a higher affinity for the antigen than the parental molecules; these
are selected. As a result of this process of random mutation, followed by selec-
tion, affinity maturation of the antibody response occurs.
The final process that we need to consider is class switching; the process by
which an antibody of one class changes to a different class or subclass. This
event involves the heavy-chain locus. Downstream of the V, D, and J gene
segments are a number of genes encoding for the constant regions of the differ-
ent antibody classes and subclasses (Fig. 4). Thus in mouse, the heavy-chain
genes are in the order µ-(cid:98)-(cid:97) -(cid:97) -(cid:97) -(cid:97) -(cid:161)-(cid:95). Naïve B cells express IgM and
3 1 2b 2a
IgD, using a process of differential splicing of the primary RNA transcript so
that in the mRNA the recombined V(D)J sequence is spliced to either the µor
(cid:98) genes. When class switching occurs there is a recombination event whereby
the gene encoding for the new antibody isotype is spliced into the position
previously occupied by the µ gene, losing all the intermediate DNA.
5. Antibody–Antigen Interactions
5.1. Affinity
The interaction between an antibody and antigen is formed by noncovalent
interactions; ionic bonds, hydrogen bonds, van der Waal’s forces, and hydro-
phobic interactions (for reviews seerefs.6–8). One important consequence of
The Antibody Molecule 9
this is that the interaction is reversible and can be represented by the equilib-
rium interaction:
A + B(cid:65)C (1)
(cid:64)
where A is the antigen, B is the antibody, and C the complex of antibody and
antigen (for simplicity we will assume a monovalent interaction, in which one
molecule of A interacts with one molecule of B, as would be seen if B were a
Fab fragment).
If, therefore, one mixes antigen and antibody together, the reaction will ini-
tially go with a relatively fast rate from left to right. As the reactants (A and B)
are consumed the reaction will slow down. At the same time, as the concentra-
tion of the produce (C) builds up the reaction from right to left increases. Even-
tually equilibrium is reached, i.e., the reaction from left to right is proceeding
at the same rate as the reaction from right to left. At this time the concentra-
tions of A, B, and C remain constant. However, it is important to realize that
the association of A and B to form C is continuing, as is the dissociation of C to
form A and B. While the reaction is in equilibrium, any one molecule of anti-
body (or antigen) may find itself changing from the free state to being bound in
the complex and back again.
The affinity of an antibody for its antigen is a measure in which the equilib-
rium of the reaction shown in Eq. 1 lies. For a high-affinity interaction the
equilibrium is further over to the right than a low-affinity interaction. This can
be expressed by the concentration of A, B, and C at equilibrium:
[C]
——– = K (2)
a
[A][B]
Note that [A] and [B] are the concentrations of free antigen and antibody at
equilibrium, not the starting concentrations. The term K is the association equi-
a
librium constant, and has terms M–1. The higher K , the higher the concentra-
a
tion of C at equilibrium and the higher the affinity of the interaction.
Immunologists often prefer to think of affinity in terms of the dissociation
equilibrium constant (K ). This is the reciprocal of K :
d a
1 [A][B]
K = — = ——— (3)
d
K [C]
a
The K has units M; the higher the affinity the lower K . The K is useful
d d d
because it gives the concentration of free antibody at which half the antigen is
bound in a complex (in other words when [A] = [C]; substituting one for the
other in Eq. 3 gives K = [B]). As in many cases, we use vast excess of anti-
d