Designing Antibodies

210 Pages·1993·5.5 MB·English

by  Ruth Mayforth (Auth.)

Designing Antibodies
Ruth D. Mayforth
Chicago, Illinois
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Library of Congress Cataloging-in-Publication Data
Mayforth, Ruth D.
Designing Antibodies / Ruth D. Mayforth.
p. cm.
Includes index.
ISBN 0-12-481025-X (pbk.)
1. Monoclonal antibodies. 2. Anti-idiotypic antibodies.
3. Immunotherapy. I. Title.
[DNLM: 1. Antibodies—genetics. 2. Antibodies—therapeutic use.
3. Drug design 4. Genetic Engineering. QW 575 M468d 1993]
QR186.85.M38 1993
616.07'9-dc20
DNLM/DLC
for Library of Congress 92-48391
CIP
PRINTED IN THE UNITED STATES OF AMERICA
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Preface
Progress in designing antibodies has been both fast and spectacular.
One challenging aspect of designing antibodies is that advancements
rely on the expertise of investigators from a variety of different fields,
including molecular biology, immunology, biochemistry, chemistry,
pharmacology, and medicine. An expansive volume of literature on
designing antibodies has been accumulating in these fields over the
past few years. This book compiles and integrates this literature to pro-
vide useful, readily accessible information. This is not intended to be a
step-by-step laboratory manual. Rather, its aim is to describe the tech-
niques used in designing antibodies, the kinds of antibodies that have
been generated through modern techniques, and their applications in
medicine and science. It is hoped that, in addition to the excitement of
watching research in this area unfold, many of the creative and innova-
tive approaches reviewed in this book will be modified or will stimulate
new ideas that will further the research and application of designer
antibodies.
Antibodies themselves are not a recent discovery. Antibodies were
first defined functionally in the 1890s as a serum substance capable of
conferring passive immunity to other animals. Forty years later, it was
discovered that the γ globulin fraction of serum proteins contained
antibody reactivity. It was evident that antibodies were important in
defending an animal against foreign pathogens, yet scientists were hin-
dered from capitalizing on the properties of antibodies until two impor-
tant immunological breakthroughs were made. One was the develop-
ment of hybridoma technology in 1975, through which hybridomas
(immortalized antibody-secreting cells) can be generated against any
antigen (ligand) and can secrete virtually limitless quantities of antigen-
reactive monoclonal antibodies. These homogeneous antibody prepara-
tions (compared to the heterogeneous, polyconal mixtures obtained
from the serum of immunized animals) have enabled scientists to study
and characterize the structure and function of antibodies in great
depth. Another significant milestone was the elucidation of the mecha-
nism of antibody gene rearrangement. These immunological discover-
vii
viii Preface
ies, together with recent advances in genetic engineering and biological
chemistry, have empowered scientists to exploit many of the advan-
tageous properties of antibodies, such as an antibody's high degree of
selectivity and affinity for its ligand and the potentially vast number of
different antibodies (more than 1010-1011). Already, a number of anti-
bodies have been designed as biomolecular tools in research, pro-
phylaxis, diagnosis, and therapy.
Antibodies can be designed by manipulating either the antibody
protein or its genes, or by constructing an antigen that should induce
the production of antibodies of the desired specificity. Not only can
desirable features be incorporated into an antibody, but undesirable
properties can be eliminated through these techniques as well. For ex-
ample, in designing antibodies for human therapy, specific changes in
an antibody's genes can be incorporated to minimize the antibody's
harmful or undesirable side effects.
The first two chapters review antibody structure, function, bio-
synthesis, and technology, setting the framework for the remainder of
the book, which has been developed around the strategies employed to
design antibodies with certain properties. In Chapter 3, antibody genes
are manipulated to generate antibodies with a desired characteristic,
such as rodent/human chimeric and humanized antibodies. Antibodies
can be conjugated to other effector molecules and specifically target
certain cells (such as cancer cells) for destruction; these antibody-
effector molecule conjugates (e.g., immunotoxins) are discussed in
Chapter 4. Chapter 5 reviews antibodies (called anti-idiotypic antibod-
ies) that have been designed to mimic antigens, a feature that is partic-
ularly suited to vaccine development and hormone receptor mimicry.
Finally, the calculated design of an antigen can induce the generation of
antibodies with enzymatic properties; these catalytic antibodies are re-
viewed in Chapter 6. It has been exciting to watch advancements in
antibody design unfold, as their present and forseeable impact in sci-
ence and medicine has been phenomenal.
I am very grateful to the following people for critically reviewing
portions of the text and for their comments and suggestions: Jeffrey
Bluestone, Mark Duban, Loren Joseph, Jose Quintans, Andrea Sant,
Hans Schreiber, Mark Scott, Steven Seung, Ursula Storb, and Howard
Tager. I am especially indebted to Sheri Chamberlain, Cindy Go, and
Mark S. Scott for their help in producing some of the figures and to
Jerry Santos, Pamela Blunt, and my father, Richard Mayforth, for help
with typing portions of the manuscript.
Ruth D. Mayforth
I DI
Antibody Overview
Introduction
In this chapter, an overview of the humoral immune system and of
antibody structure, function, and biosynthesis is presented. Its aim is
to set the stage for a discussion of recent developments in antibody
technology, which is the focus of the remainder of this book.
The Humoral Immune System
A number of features of antibodies are particularly remarkable, making
them amenable to a number of scientific and medical applications. Anti-
bodies bind antigens (their ligands, which generally can be thought of
as foreign macromolecules) with a high degree of specificity and can
discriminate between two very closely related antigens. Another striking
characteristic of antibodies is their diversity. Human beings can produce
at least 107 (and potentially even more than 1011) antibodies with different
specificities. (The genetic mechanisms responsible for generating this
vast repertoire are quite extraordinary and are described later in this
chapter.) Antibody diversity is so great that virtually any foreign macro-
molecule can be recognized. The diversity of antibodies, combined with
their specificity, makes them ideal biomolecular tools for scientific, diag-
nostic, and therapeutic purposes.
The human immune system can be divided into two major compo-
nents: the humoral immune system and the cell-mediated immune sys-
tem. Each human has about 2 x 1012 lymphocytes (types of white blood
cells). There are two kinds of lymphocytes, T cells and B cells, which
are represented in approximately equal numbers. Both B cells and T
cells express antigen-specific receptors on their cell surface, called the
immunoglobulin receptor (IgR) and the T-cell receptor (TCR), respec-
1
2 1 Antibody Overview
tively. These receptors are clonally distributed—that is, all of the immuno-
globulins that a given B-cell clone expresses are identical and have exactly
the same specificity for antigen. When stimulated, B cells can also secrete
their immunoglobulins. Immunoglobulins (or antibodies) are an im-
portant component of the humoral immune system. T cells form part of
the cell-mediated immune system. T cells can be divided into two groups:
cytotoxic (CD8+) T cells mediate cytotoxicity and helper (CD4+) T cells
"help" generate an antibody response to T-cell-dependent antigens
and provide the B cells with necessary lymphokines (biologically active
polypeptides secreted by lymphocytes). In general, proteins are T-cell-
dependent antigens while polysaccharides are T-cell-independent antigens.
Immunoglobulins are synthesized by B lymphocytes and can be either
membrane-bound or secreted. Membrane-bound immunoglobulins form
part of the IgR on B cells. When this IgR recognizes and binds its antigen,
the B cell is stimulated to proliferate (divide and expand) and differentiate
into antibody-secreting cells and memory B cells. T helper cells aid in the
proliferation and differentiation of antibody-secreting cells (plasma cells)
by supplying necessary lymphokines. A B-cell clone and its daughter
cells undergo repeated cell divisions and greatly expand in number.
Each of them synthesizes antibodies with exactly the same antigenic
specificity (although some of the daughter cells may mutate and express
slight variants of the antibody, as discussed later). This is referred to
as clonal expansion. Fundamental to this process is the "selection" of a
preexisting antigen-specific B-cell clone by the foreign antigen. Once
selected, the B cell clonally expands, and it and its progeny secrete their
antigen-specific antibodies. This is the basic tenet of the clonal selection
theory proposed by Macfarlane Burnet in the late 1950s (1956, 1959,
1962). The important point to stress is that each B-cell clone develops with
no a priori knowledge of the antigen and expands after it has encount-
ered antigen.
The diversity of the antibody repertoire ensures that virtually any
foreign macromolecule that is encountered will be recognized by at least
one (and usually more than one) B-cell clone. Each B-cell clone secretes
antibodies of exactly the same antigenic specificity, or monoclonal antibod-
ies. In a typical immune response, antigens are recognized in slightly
different ways by the antibodies of a number of different B-cell clones.
For example, as many as 5,000-10,000 B-cell clones with unique specifici-
ties can recognize the antigen dinitrophenol. This is called a polyclonal
response.
One interesting (and still not well understood) feature of the immune
system is that it has memory. The second time that a given antigen is
encountered, the response is significantly faster and greater in magni-
The Humoral Immune System 3
tude than in the primary response. The following factors contribute to
heightened secondary immune responses. Some of the cells that were
recruited in the primary response are thought to become long-lived
memory B cells that can quickly be recruited the next time antigen is
encountered. Also, some of the daughter clones can make small point
mutations in their antibody genes, which may result in antibodies
with even higher affinities than the parent antibody. (This process is
called somatic hypermutation or affinity maturation and is discussed
further in the section on antibody biosynthesis.) These factors make a
secondary immune response stronger and more rapid, and provide the
theoretical basis for vaccinating individuals against highly infectious
diseases.
Antibodies help defend the body from foreign invaders in a variety
of ways. First, antibodies can directly neutralize the antigen by forming
antigen-antibody complexes that are cleared from the circulation. In
addition, antibodies can bind the antigen on pathogens such as bacteria,
coating the bacteria with antibody. These "opsonized" bacteria are more
efficiently phagocytosed by macrophages, since the macrophages have
Fc receptors that bind the Fc ("fragment crystalline"; see later discussion)
portions of the coating antibodies. Furthermore, antibodies that have
bound antigen on the surface of a cell can activate the complement
system, resulting in the lysis of the cell. The complement system is
composed of a series of plasma proteins that, when activated, initiates
a sequential cascade of events. The final step in this cascade is the
formation of protein pore complexes in the membrane of the target
cell that lyses it. Thus, neutralization, opsonization, and complement
activation are three defensive strategies used by antibodies to protect
their host against foreign invaders.
The defensive arsenal of the humoral immune system is rather impres-
sive. As previously mentioned, there are as many as 1011 unique antibod-
ies in a human being. The concentration of antibodies in human serum
is 15 mg/ml, which calculates to be 3 x 1020 secreted immunoglobulin
molecules per person! Furthermore, each B cell expresses approximately
105 immunoglobulin molecules of identical specificity on its surface,
which means that about 1017 membrane-bound IgRs also scan the body
for antigen. To maximize the chances of encountering antigen, lympho-
cytes go on a number of "reconnaissance missions," recirculating from
lymphoid tissues (such as lymph nodes and spleen) through the blood
and back again to the lymphoid tissues. At any given time, there are
approximately 1010 lymphocytes in human blood with a mean transit
time of approximately 30 min (Pabst, 1988), translating to an exchange
rate of almost 50 times per day.
4 1 Antibody Overview
Antibody Structure
Introduction
Immunoglobulins are multifunctional glycoproteins found only in verte-
brates. These molecules bind antigen through the variable domains at
their amino-terminal (NH ) end and initiate a variety of effector functions
2
(such as complement activation, Fc receptor binding, and placental trans-
fer) through the constant region domains at their carboxy-terminal
(COOH) tails. Immunoglobulins are composed of four polypeptide
chains. Two of the chains are identical heavy chains and two are identical
light chains. The light chain consists of about 220 amino acids and has
a molecular weight of about 25 kD (kilodaltons). The heavy chain is
made up of approximately 450-575 amino acids (depending on the class
of the heavy chain) with a molecular weight of about 51-72 kD. As shown
in Figure 1.1, a schematic representation of the monomeric antibody
molecule resembles a Y or a T. Each arm of the Y or T contains one com-
plete light chain and the amino-terminal end of the heavy chain, while
the base is comprised of the carboxy-terminal end of the heavy chain.
The heavy and light chains are composed of a series of building blocks
of globular domains that are each about 110 amino acids long. These
domains, called immunoglobulin domains, have a characteristic tertiary
structure of two roughly parallel jS-pleated sheets that are joined by a
disulfide (S-S) bond. Other proteins also share these immunoglobulin
domains, which are discussed later in more detail. Each light chain has
two domains, while each heavy chain has either four or five domains.
The first 110 amino acids of the amino-terminal portions of both the
heavy and light chains exhibit relatively high amino acid sequence vari-
ability and are hence designated the variable domains (V and V , re-
H L
spectively). They contain the antigen-binding sites or hypervariable
regions that are complementary to, and thus bind, the antigenic deter-
minants. The remainders of both chains are more conserved in amino
acid sequence and are designated the constant regions (C and C ).
H L
Each light chain has one variable domain and one constant domain
(V + C ), while each heavy chain has one variable domain and three
L L
or four constant domains, depending on its class [V + C 1 + C 2 +
H H H
C 3(+ CH4)] (see Fig. 1.1).
H
Both covalent and noncovalent interactions hold the four immunoglob-
ulin chains together. Covalent disulfide bonds between the carboxy-
terminus of the light chain and the carboxy-terminal portion of either
the V or C 1 domains link these chains together. (In some IgAs, a
H H
disulfide bond joins the two light chains together instead.) The V do-
H
main has a hydrophobic face that interacts with the V domain. The C 1
L H
5
Antibody Structure
antigen binding sites
Figure 1.1 Immunoglobulin structure. Antibodies are glycoproteins composed of two
identical disulfide-linked (S-S) heavy chains and two identical light chains. In most classes
of antibodies, as shown here, each light chain is linked to a heavy chain through a
disulfide bond. Antibodies are made up of a series of modular structural motifs called
immunoglobulin domains, which are made up of a stretch of approximately 110 amino
acids (represented in the figure as oval-shaped for the C domains or partially oval shaped
for the V domains). Depending on the class of the antibody, there are four or five
immunoglobulin domains in the heavy chain. IgG, Ig A, and IgD have V , C 1, C 2,
H H H
and C 3 domains and have a hinge region between C 1 and C 2. IgM and IgE have an
H H H
additional domain, C 4, but lack a hinge region. There are two immunoglobulin domains
H
in each light chain: V and C . Each antibody has two identical antigen-binding sites,
L L
one on each arm. There are three hypervariable regions (also called complementarity
determining regions) in each variable domain, represented in the figure as zig-zag lines
on the ends of the V domains. The six hypervariable regions (three from one V and
H
three from one V domain) form a pocket that makes up the antibody's antigen-binding
L
site. The effector functions of an antibody molecule (e.g., complement-mediated lysis,
antibody-dependent cell-mediated cytotoxicity, and placental transfer) are mediated
through the constant-region domains at the carboxy-terminal end of the antibody mole-
cule. The glycosylation patterns vary depending on the isotype. All IgG subclasses have
one N-linked oligosaccharide on their C 2 domain. The other immunoglobulin isotypes
H
have two to six N-linked oligosaccharides, and IgAl and IgD have O-linked oligosaccha-
rides as well.
and C domains also associate through hydrophobic interactions. The
L
two heavy chains are similarly held together through disulfide bonds and
hydrophobic interactions. The hydrophobic interactions are especially
important in keeping the C 3 domains juxtaposed. The position of the
H
6
1 Antibody Overview
Cys residues that form the disulfide bonds depends on the isotype. For
IgG, the heavy chains are connected through disulfide bonds near the
carboxy-terminus of the hinge region.
Closer inspection of the amino acid sequences of the variable regions
reveals that sequence variability is not scattered randomly throughout
the V domains. Rather, the variability in V domains is localized to three
discrete hypervariable regions that are separated by relatively constant
"framework" regions (Wu and Kabat, 1970). The hypervariable regions
are also called complementarity-determining regions (CDRs) because they
create the pockets or grooves that are complementary to and bind the
antigenic determinants. (One should keep in mind that the hypervariable
regions are defined on the basis of amino acid variability rather than
antigen-binding function. Although the actual antigen-binding site is
comprised largely of these hypervariable amino acids, funcitonal studies
have demonstrated that, depending on the antibody, a few framework
residues can also be involved in the binding.) Structurally, the hypervari-
able sequences of both the heavy and light chains are clustered near the
amino-terminal end of the molecule and project outward from the ß-
pleated sheets that make up the V domains. All antibody molecules
contain two antigen-binding sites, each consisting of three light-chain
CDRs and three heavy-chain CDRs. The antigen-binding site on the
antibody is called the paratope, and the complementary region on the
antigen that is bound by the antibody is called the epitope or antigenic
determinant (see Table 1.1 and Fig. 1.2). The unique stereochemical
conformation created by the combination of hypervariable sequences of
a given pair of light and heavy chains determines the specificity of the
antibody.
Table 1.1
Epitopes, Paratopes, and Idiotopes
Term Location Ligand Synonyms
Epitope Determinant on Paratope Antigenic determinant
antigen Antibody binding site
Paratope Antibody Epitope Hypervariable regions
hypervariable Complementarity-determining
regions regions (CDRs)
Antigen binding site
Antibody combining site
Idiotope Antibody V regions Paratope of an Possibly either epitope or paratope
anti-idiotypic
antibody