Recombinant Antibodies for Cancer Therapy. Methods and Protocols

456 Pages·2002·3.96 MB·English

by  Martin Welschof

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Generation of Antibody Molecules 3
1
Generation of Antibody Molecules
Through Antibody Engineering
Sergey M. Kipriyanov
1. Introduction
Twenty-five years ago, Georges Köhler and César Milstein invented a means of
cloning individual antibodies, thus opening up the way for tremendous advances in the
fields of cell biology and clinical diagnostics (1). However, in spite of their early
promise, monoclonal antibodies (MAbs) were largely unsuccessful as therapeutic
reagents resulting from insufficient activation of human effector functions and
immune reactions against proteins of murine origin. These problems have recently
been overcome to a large extent using genetic-engineering techniques to produce chi-
meric mouse/human and completely human antibodies. Such an approach is particu-
larly suitable because of the domain structure of the antibody molecule (2), where
functional domains carrying antigen-binding activities (Fabs or Fvs) or effector func-
tions (Fc) can be exchanged between antibodies (seeFig. 1).
On the basis of sequence variation, the residues in the variable domains (V-region)
are assigned either to the hypervariable complementarity-determining regions (CDR)
or to framework regions (FR). It is possible to replace much of the rodent-derived
sequence of an antibody with sequences derived from human immunoglobulins with-
out loss of function. This new generation of “chimeric” and “humanized” antibodies
represents an alternative to human hybridoma-derived antibodies and should be less
immunogenic than their rodent counterparts. Furthermore, genetically truncated ver-
sions of the antibody may be produced ranging in size from the smallest antigen-
binding unit or Fv through Fab' to F(ab') fragments. More recently it has become
2
possible to produce totally human recombinant antibodies derived either from anti-
body libraries (3) or single immune B cells (4), or from transgenic mice bearing
human immunoglobulin loci (5,6).
From:Methods in Molecular Biology, vol. 207:Recombinant Antibodies for Cancer Therapy: Methods and Protocols
Edited by: M. Welschof and J. Krauss © Humana Press Inc., Totowa, NJ
3
4 Kipriyanov
Fig. 1. Domain organization of an IgG molecule. Antigen-binding surface is formed by
variable domains of the heavy (V ) and light (V ) chains. Effector functions are determined by
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constant C 2 and C 3 domains. The picture is based on the crystal structure of an intact IgG2
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anti-canine lymphoma MAb231 (2) (pdb entry 1IGT). The drawing was generated using a
molecular visualization program RasMac Molecular Graphics, version 2.7.1 (R. Sayle,
Biomolecular Structure, Glaxo Research and Development, Greenford, Middlesex, UK).
2. Cloning the Antibody Variable Regions
Significant progress has been made in the in vitro immunization of human B cells
(7) and in the development of transgenic mice containing human immunoglobulin loci
(for review, see refs. 5,8). Recombinant DNA technology can also be employed for
generating human MAbs from human lymphocyte mRNA. The genetic information
for antibody variable regions is generally retrieved from total cDNA preparations
using the polymerase chain reaction (PCR) with antibody-specific primers (9,10). As
a source of immunoglobulin-specific mRNA, one can use hybridoma cells (11),
human peripheral blood lymphocytes (PBL) (3), and even a single human B cell (4,12).
Using the latter approach, it is possible to avoid the cumbersome hybridoma technol-
ogy and obtain human antibody fragments with the original V /V pairing. Single
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bacterial colonies expressing antigen-specific antibody fragments can be identified by
colony screening using antigen-coated membranes (13). Novel high-throughput selec-
tion technologies allow screening thousands of different antibody clones at a time
(14). The appropriate V /V combination may also be selectively enriched from a
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phage-displayed antibody library through a series of immunoaffinity steps referred to
as“library panning”(15,16).
Generation of Antibody Molecules 5
3. Genetically Engineered Monoclonal Antibodies
3.1. Chimeric Antibodies with Human Constant Regions
The first generation of recombinant monoclonal antibodies consisted of the rodent-
derived V-regions fused to human constant regions (Fig. 2). It is thought that the most
immunogenic regions of antibodies are the conserved constant domains (17). Because
the antigen-binding site of the antibody is localized within the variable regions, the
chimeric molecules retain their binding affinity for the antigen and acquire the func-
tion of the substituted constant regions. The human constant regions allow more
efficient interaction with human complement-dependent cytotoxicity (CDC) and anti-
body-dependent cell-mediated cytotoxicity (ADCC) effector mechanisms. Rituximab
(Rituxan; IDEC Pharmaceuticals, San Diego, and Genentech, Inc., San Francisco, CA)
is a chimeric anti-CD20 MAb containing the variable regions of the CD20-binding
murine IgG1 MAb, IDEC-2B8, as well as human IgG1 and kappa constant regions
(18,19). Rituximab was the first monoclonal antibody to be approved for therapeutic
use for any malignancy. Its approval was based on a single-agent pivotal trial in
patients with indolent B-cell lymphoma, in which 166 patients were enrolled from 31
centers in the United States and Canada. Administration of this antibody induced
remissions in 60% of patients with relapsed follicular lymphomas, including 5%–10%
complete remissions (20).
As a further step to reduce the murine content in an antibody, procedures have been
developed for humanizing the Fv regions.
3.2. Antibody Humanization (Reshaping)
3.2.1. Humanization by CDR Grafting
CDRs build loops close to the antibody’s N-terminus, where they form a continu-
ous surface mounted on a rigid scaffold provided by the framework regions. Crystallo-
graphic analyses of several antibody/antigen complexes have demonstrated that
antigen-binding mainly involves this surface (although some framework residues have
also been found to take part in the interaction with antigen). Thus, the antigen-binding
specificity of an antibody is mainly defined by the topography and by the chemical
characteristics of its CDR surface. These features in turn are determined by the con-
formation of the individual CDRs, by the relative disposition of the CDRs, and by the
nature and disposition of the side chains of the amino acids comprising the CDRs (21).
A large decrease in the immunogenicity of an antibody can be achieved by grafting
only the CDRs of xenogenic antibodies onto human framework and constant regions
(22,23) (Fig. 2). However, CDR grafting per se may not result in the complete reten-
tion of antigen-binding properties. Indeed, it is frequently found that some framework
residues from the original antibody need to be preserved in the humanized molecule if
significant antigen-binding affinity is to be recovered (24,25). In this case, human V
regions showing the greatest sequence homology to murine V regions are chosen from
a database in order to provide the human framework. The selection of human FRs can
be made either from human consensus sequences or from individual human antibodies.
In some rare examples, simply transferring CDRs onto the most identical human
6 Kipriyanov
Fig. 2. Humanization of an IgG molecule. The mouse sequences are shown in white and the
human sequences are shown in gray. In a chimeric antibody, the mouse heavy- and light-chain
variable region sequences are joined onto human heavy-chain and light-chain constant regions.
In a humanized antibody, the mouse CDRs are grafted onto human V-region FRs and expressed
with human C-regions.
V-region frameworks is sufficient for retaining the binding affinity of the original
murine MAb (26). However, in most cases, the successful design of high-affinity CDR-
grafted antibodies requires that key murine residues be substituted into the human
acceptor framework to preserve the CDR conformations. Computer modeling of the
antibody is used to identify such structurally important residues that are then included
in order to achieve a higher binding affinity. The process of identifying the rodent
framework residues to be retained is generally unique for each reshaped antibody and
can therefore be difficult to foresee.
Such approach was successfully used for humanizing a MAb 4D5 against the prod-
uct of protooncogene HER2 (27). HER2 is a ligand-less member of the human epider-
mal growth factor receptor (EGFR) or ErbB family of tyrosine kinases. HER2
overexpression is observed in a number of human adenocarcinomas and results in
constitutive HER2 activation. Specific targeting of these tumors can be accomplished
with antibodies directed against the extracellular domain of the HER2 protein. The
MAb 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech,
San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with
trastuzumab results in a number of phenotypic changes, such as downmodulation of
the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance,
restored E-cadherin expression levels, and reduced vascular endothelial growth factor
production. Interaction of trastuzumab with the human immune system via its human
IgG1 Fc domain may potentiate its anti-tumor activities. In vitro studies demonstrate
that trastuzumab is very effective in mediating antibody-dependent cell-mediated
cytotoxicity against HER2-overexpressing tumor targets (28). Trastuzumab treatment
of mouse xenograft models results in marked suppression of tumor growth. When
given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab
treatment generally results in statistically superior anti-tumor efficacy compared with
either agent given alone (28).
Generation of Antibody Molecules 7
3.2.2. Humanization by Resurfacing (Veneering)
A statistical analysis of unique human and murine immunoglobulin heavy- and
light-chain variable regions revealed that the precise patterns of exposed residues are
different in human and murine antibodies, and most individual surface positions have
a strong preference for a small number of different residues (29,30). Therefore, it may
be possible to reduce the immunogenicity of a nonhuman Fv, while preserving its
antigen-binding properties, by simply replacing exposed residues in its framework
regions that differ from those usually found in human antibodies. This would human-
ize the surface of the xenogenic antibody while retaining the interior and contacting
residues that influence its antigen-binding characteristics and interdomain contacts.
Because protein antigenicity can be correlated with surface accessibility, replacement
of the surface residues may be sufficient to render the mouse variable region “invis-
ible” to the human immune system. This procedure of humanization is referred to as
“veneering” because only the outer surface of the antibody is altered, the supporting
residues remain undisturbed (31).
Variable domain resurfacing maintains the core murine residues of the Fv sequences
and probably minimizes CDR-framework incompatibilities. This procedure was suc-
cessfully used for the humanization of murine MAb N901 against the CD56 surface
molecule of natural killer (NK) cells and MAb anti-B4 against CD19 (26,32). A direct
comparison of engineered versions of N901 humanized either by CDR grafting or by
resurfacing showed no difference in binding affinity for the native antigen (26,30).
For the anti-B4 antibody, the best CDR-grafted version required three murine residues
at surface positions to maintain binding, while the best resurfaced version needed only
one surface murine residue (26). Thus, even though the resurfaced version of anti-B4
has 36 murine residues in the Fv core, it may be less immunogenic than the CDR-
grafted version with nine murine residues in the Fv core because it has a pattern of
surface residues that is more identical to a human surface pattern.
3.3. Choice of Constant Region
The construction of chimeric and humanized antibodies offers the opportunity of
tailoring the constant region to the requirements of the antibody. IgG is preferred class
for therapeutic antibodies for several practical reasons. IgG antibodies are very stable,
and easily purified and stored. In vivo they have a long biological half-life that is not
just a function of their size but is also a result of their interaction with the so-called
Brambell receptor (or FcRn) (33). This receptor seems to protect IgG from catabolism
within cells and recycles it back to the blood plasma. In addition, IgG has subclasses
that are able to interact with and trigger a whole range of humoral and cellular effector
mechanisms. Each immunoglobulin subclass differs in its ability to interact with Fc
receptors and complement and thus to trigger cytolysis and other immune reactions.
Human IgG1, for example, would be the constant region of choice for mediating
ADCC and probably also CDC (34,35). On the other hand, if the antibody were
required simply to activate or block a receptor then human IgG2 or IgG4 would prob-
ably be more appropriate. For example, the humanized versions of the immunosup-
pressive anti-human CD3 MAb OKT3 were prepared as IgG4 antibodies (36,37).
8 Kipriyanov
However, all four human IgG subclasses mediate at least some biological func-
tions. To avoid the unwanted side effects of a particular isotype, it is possible to
remove or modify effector functions by genetic engineering. For example, amino acid
substitutions in the C 2 portion of an anti-CD3 antibody led to the retention of its
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immunosuppressive properties, but markedly reduced the unwanted biological side
effects associated with Fc receptor binding (38–40). An alternative strategy has recently
been described whereby potent blocking antibodies could be generated by assembly
the C 2 domain from sequences derived from IgG1, IgG2, and IgG4 subclasses (41).
H
3.4. Alternative Strategies for Producing “Human” Antibodies
Other strategies for the production of “fully human” antibodies include phage
libraries(42,43) or transgenic mice (5,8), both utilizing human V-region repertoires.
3.4.1. Mice Making “Human” Antibodies
Several strains of mice are now available that have had their mouse immunoglobulin
loci replaced with human immunoglobulin gene segments (6,44,45). Transgenic mice
are able to produce functionally important human-like antibodies with very high
affinities after immunization. Cloning and production can be carried out employing
the usual hybridoma technology. For example, high-affinity human MAbs obtained
against the T-cell marker CD4 are potential therapeutic agents for suppressing adverse
immune activity (44). Another human MAbs with an affinity of 5 × 10–11 M for
human EGFR was able to prevent formation and eradicate human epidermoid carci-
noma xenografts in athymic mice (46). However, during affinity maturation, the anti-
bodies from transgenic mice accumulate somatic mutations both in FRs and CDRs
(45). It means that they are no longer 100% identical to inherited human germline
genes and can, therefore, be potentially immunogenic in humans (47). Besides, “human
antibodies” from mice can be distinguished from human antibodies produced in human
cells by their state of glycosylation, particularly with respect to their Gal 1–3Gal resi-
(cid:1)
due, against which human serum contains IgG antibody titers of up to 100 (cid:2)g/ml. It
has been argued that an antibody containing such residues would not survive very long
in the human circulation (48).
3.4.2. Human Antibodies from Phage Libraries
A rapid growth in the field of antibody engineering occurred after it was shown that
functional antibody fragments could be secreted into the periplasmic space and even
into the medium of Escherichia coli by fusing a bacterial signal peptide to the
antibody’s N-terminus (49,50). These findings opened the way for transferring the
principles of the immune system for producing specific antibodies to a given antigen
into a bacterial system. It was now possible to establish antibody libraries in E. coli
that could be directly screened for binding to antigen.
In order to screen large antibody libraries containing at least 108 individual mem-
bers, it was necessary to develop a selection system as efficient as that of the immune
system, in which the antibody receptor is bound to the surface of a B lymphocyte.
Generation of Antibody Molecules 9
After binding its antigen, the B lymphocyte is stimulated to proliferate and mature into
an IgG-producing plasma cell. A similar selection system could be imitated in micro-
organisms by expressing antibodies on their surface. Millions of microorganisms could
then be simultaneously screened for binding to an immobilized antigen followed by
the propagation and amplification of the selected microorganism. Although protein
display methods have been developed for eukaryotic systems, e.g., retroviral (51),
baculoviral(52), yeast (53,54) and even cell-free ribosome display (55,56), the most
successful surface expression system has been created using filamentous bacterioph-
ages of the M13 family (57). The phage display was originally reported for scFv frag-
ments (15), and later for Fab fragments (58) and other antibody derivatives such as
diabodies(59). Now it became possible to generate antibody libraries by PCR cloning
the large collections of variable-region genes, expressing each of the binding sites on
the surface of a different phage particle and selecting the antigen-specific binding sites
by in vitro screening the phage mixture on a chosen antigen. The phage display tech-
nology could be used to select antigen-specific antibodies from libraries made from
human B cells taken from individuals either immunized with antigen (60), or exposed
to infectious agents (61), or with autoimmune diseases (3), or with cancer (62). More-
over, it was demonstrated that antibodies against many different antigens could be
selected from “naive” binding-site library, prepared from the V and V IgM-V-gene
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pools of B cells of a non-immunized healthy individuals (16,63). It was also shown
that libraries of synthetic antibody genes based on human germline segments with
randomized CDRs behave in a similar way to “naive” antibody libraries (64,65). It
became, therefore, possible to use primary (“naive” or “synthetic”) antibody libraries
with huge collections of binding sites of different specificity for in vitro selection of
“human” antibody fragments against most antigens, including nonimmunogenic mol-
ecules, toxic substances and targets conserved between species (for review, seerefs.
42,66).
However, for some therapeutic applications whole IgGs are the preferred format as
a result of their extended serum half-life and ability to trigger the humoral and cellular
effector mechanisms. This necessitates recloning of the phage-display derived scFvs
or Fabs into mammalian expression vectors containing the appropriate constant domains
and establishing stable expressing cell lines. The specificity and affinity of the anti-
body fragments are generally well retained by the whole IgG, and, in some cases, the
affinity may significantly improve due to the bivalent nature of the IgG (67,68). In the
past few years, four phage-derived antibodies have begun clinical trials (69).
4. Recombinant Antibody Fragments
The Fv fragment consisting only of the V and V domains is the smallest immuno-
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globulin fragment available that carries the whole antigen-binding site (Fig. 1). How-
ever, Fvs appear to have lower interaction energy of their two chains than Fab
fragments that are also held together by the constant domains C 1 and C (70). To
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stabilize the association of the V and V domains, they have been linked with pep-
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tides(71,72), disulfide bridges (70) and “knob-into-hole” mutations (73) (Fig. 3).
10 Kipriyanov
Fig. 3. Monovalent immunoglobulin fragments. Fab, Fv, disulfide-stabilized Fv (dsFv), and
Fv fragments with remodeled V /V interface (“knob-into-hole” Fv) consist of two separate
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chains, while the single V domain and single chain Fv (scFv) fragments are made from a
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single gene.
4.1. Monovalent Antibody Fragments
4.1.1. Single Chain Fv Fragments (scFv)
Peptide linkers of about 3.5 nm are required to span the distance between the
carboxy terminus of one domain and the amino terminus of the other (72). Both orien-
tations, V -linker-V or V -linker-V , can be used. The small scFvs are particularly
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interesting for clinical applications (for review, see ref. 74). They are only half the
size of Fabs and thus have lower retention times in nontarget tissues, more rapid blood
clearance, and better tumor penetration. They are also potentially less immunogenic
and are amenable to fusions with proteins and peptides.
Unlike glycosylated whole antibodies, scFv can be easily produced in bacterial cells
as functional antigen-binding molecules. There are two basic strategies to obtain
recombinant antibody fragments from E. coli. The first is to produce antibody proteins
as cytoplasmic inclusion bodies followed by refolding in vitro. In this case the protein
is expressed without a signal sequence under a strong promoter. The inclusion bodies
contain the recombinant protein in a non-native and non-active conformation. To obtain
functional antibody, the recombinant polypeptide chains have to be dissolved and
folded into the right shape by using a laborious and time-consuming refolding proce-
dure (for review, seeref.43). The second approach for obtaining functional antibody
fragments is to imitate the situation in the eukaryotic cell for secreting a correctly
folded antibody. In E. coli, the secretion machinery directs proteins carrying a specific
signal sequence to the periplasm (75). The scFv fragments are usually correctly pro-
Generation of Antibody Molecules 11
cessed in the periplasm, contain intramolecular disulfide bonds, and are soluble. How-
ever, the high-level expression of a recombinant protein with a bacterial signal peptide
inE. coli often results in the accumulation of insoluble antibody fragments after trans-
port to the periplasm (76,77).
It is now recognized that aggregation in vivo is not a function of the solubility and
stability of the native state of the protein, but of those of its folding intermediates in
their particular environment (78,79). The degree of successful folding of antibody
fragments in the bacterial periplasm appears to depend to a large extent on the primary
sequence of the variable domains (80,81). The overexpression of some enzymes of the
E. coli folding machinery such as cytoplasmic chaperonins GroES/L, periplasmic disulfide-
isomerase DSbA as well as periplasmic peptidylprolyl cis,trans-isomerases (PPIase)
PpiA and SurA did not increase the yield of soluble antibody fragments (82–84). In
contrast, the coexpression of either bacterial periplasmic protein Skp/OmpH or PPIase
FkpA increased the functional yield of both phage-displayed and secreted scFv frag-
ments (84,85). Modifications in bacterial growth and induction conditions can also
increase the proportion of correctly folded soluble scFv. For example, lowering the
bacterial growth temperature has been shown to decrease periplasmic aggregation and
increase the yield of soluble antibody protein (78,86). Additionally, the aggregation of
recombinant antibody fragments in the E. coli periplasm can be reduced by growing
the induced cells under osmotic stress in the presence of certain nonmetabolized
additives such as sucrose (87,88) or sorbitol and glycine betaine (89). Moreover,
inducing the synthesis of recombinant antibody fragments in bacteria under osmotic
stress promotes the formation of domain-swapped scFv dimers (89).
Single-chain Fv antibody fragments produced in bacteria provide new possibilities
for protein purification by immunoaffinity chromatography. Their advantages include
lower production costs, higher capacity for antigen on a weight basis, and better pen-
etration in a small-pore separation matrix. Such recombinant immunosorbent proved
to be useful for the one-step purification of a desired antigen from complex protein
mixtures (90). Another interesting possible application is the purification or separa-
tion of toxic compounds, which cannot be used for immunization of animals, using
antibodies selected from phage-displayed antibody libraries.
4.1.2. Disulfide-Stabilized Fv Fragments (dsFv)
Another strategy for linking V and V domains has been to design an intermolecular
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disulfide bond (Fig. 3). The disulfide-stabilized (ds) Fv fragment appeared to be much
more resistant to irreversible denaturation caused by storage at 37°C than the unlinked
Fv. It was more stable than the scFv fragment and a chemically crosslinked Fv (70).
The two most promising sites for introducing disulfide bridges appeared to be V 44-V 100
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connecting FR2 of the heavy chain with FR4 of the light chain and V 105-V 43 that
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links FR4 of the heavy chain with FR2 of the light chain (91).
4.1.3. Single Antibody-Like Domains
To obtain even smaller antibody fragments than those described earlier, antigen-
binding V domains were isolated from the lymphocytes of immunized mice (92).
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However, one problem of the V domains is their “sticky patch” for interactions with
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