Review
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Ribosome display: next-generation
display technologies for
production of antibodies in vitro
Mingyue He† and Farid Khan
CONTENTS
Exploitation of antibody
diversity by
DNA recombination
Antibody selection in vitro
Antibody evolution in vitro
Cell-free ribosome display:
the next generation of
display technologies
Antibody production
Expert commentary
Five-year view
Key issues
References
Affiliations
Antibodies represent an important and growing class of biologic research reagents and
biopharmaceutical products. They can be used as therapeutics in a variety of diseases.
With the rapid expansion of proteomic studies and biomarker discovery, there is a need for
the generation of highly specific binding reagents to study the vast number of proteins
encoded by the genome. Display technologies provide powerful tools for obtaining
antibodies. Aside from the preservation of natural antibody repertoires, they are capable of
exploiting diversity by DNA recombination to create very large libraries for selection of
novel molecules. In contrast to in vivo immunization processes, display technologies allow
selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment
of antibodies with desired properties from large populations. In addition, in vitro selection
enables the isolation of antibodies against difficult antigens including self-antigens, and this
can be applied to the generation of human antibodies against human targets. Display
technologies can also be combined with DNA mutagenesis for antibody evolution in vitro.
Some methods are amenable to automation, permitting high-throughput generation of
antibodies. Ribosome display is considered as representative of the next generation of
display technologies since it overcomes the limitations of cell-based display methods by
using a cell-free system, offering advantages of screening larger libraries and continuously
expanding new diversity during selection. Production of display-derived antibodies can be
achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based
expression systems. In the near future, cell-free protein synthesis may be developed as an
alternative for large-scale generation of antibodies.
Expert Rev. Proteomics 2(3), 421–430 (2005)
†
Author for correspondence
Protein Technologies Laboratory,
Babraham Research Campus,
Cambridge CB2 4AT, UK
Tel.: +44 1223 496 306
Fax: +44 1223 496 045
[email protected]
KEYWORDS:
antibody selection and evolution,
display technologies, in vitro,
ribosome display,
single-chain antibodies
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Antibodies have widespread applications in life
science research, the biopharmaceutical industry and medical sciences. Since the first
approval of an antibody therapeutic in 1995,
they have become potential diagnostic and
therapeutic agents for the treatment of diseases
such as cancer, autoimmune disorders and
viral or bacterial infections. Currently, 20% of
pharmaceutical products are antibodies and
there are approximately 20 antibodies in
oncology trials [1]. The growth of therapeutic
antibody sales has reached 30% and this figure
may well continue to rise in the future. With
the rapid expansion of proteomic studies,
highly specific antibodies are required for
functional and expression analysis of proteins
[2,3]. Antibody microarrays, in which many
10.1586/14789450.2.3.421
thousands of specific antibodies are spotted on
a surface, have been demonstrated as a versatile
tool for profiling protein expression, monitoring signaling protein pathways [4] and probing
protein post-translational modifications [5].
Clearly, there is a need for the development
of technologies to produce monoclonal antibodies with high affinity and specificity. Traditionally, monoclonal antibodies have been produced through rodent immunization followed
by hybridoma screening [6]. However, this
in vivo method is less effective when poorly
immunogenic epitopes or self-antigens are
used. The method is rather involved and complicated for the production of human antibodies. In addition, hybridoma antibodies
have a limited affinity for their target antigens
© 2005 Future Drugs Ltd
ISSN 1478-9450
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He & Khan
due to in vivo biologic constraints that limit the maximum
binding affinity [7]. Moreover, some therapeutic applications
require antibody molecules with desirable properties such as
size, valency and stability, which are difficult to obtain by the
hybridoma method.
Display technologies provide a suitable alternative for antibody generation. These methods generate antibodies in vitro
directly from large DNA libraries without the need for in vivo
immunization. They are also powerful tools for improving
antibody affinity, specificity and stability. Since antibody selection is carried out in vitro, display methods offer the potential
to exceede the apparent in vivo affinity ceiling [7]. A number of
display methods have been developed, including cell-based systems such as phage display [8] and cell surface display [9,10] or
cell-free systems such as ribosome display [11] and mRNA display [12]. In general, all display methods use the principle of
coupling the phenotype (protein) and genotype (genetic information) for selection. For the display of antibodies, this coupling is achieved by synthesizing individual antibodies as Fab or
single-chain Fv fragments (scFv), either on the surface of an
organism (e.g., phage, bacteria or yeast) or in association (covalently or noncovalently) with their encoding mRNA or DNA
[8–12]. Thus, affinity selection of displayed antibodies allows
isolation of organisms or complexes carrying the genetic information, which can then be recovered as DNA for sequence
determination or used directly for protein production. The display process can be repeated to enrich these antibodies, which
initially present as rare species in a large library. This article
will review the generation of antibodies in vitro using display
technologies, with a focus on ribosome display.
Exploitation of antibody diversity by DNA recombination
Natural antibody diversity is attributed to the hypervariable
sequences or complementary-determination regions (CDR)
presented on different variable (V) regions and the combination
of heavy (H) and light (L) chains. Each V region in the H and
L chain contains three CDRs, which together form the combining site for antigen binding. Diversity is created during B-cell
development by DNA rearrangement, combinatorial assembly
of different genetic segments and somatic mutation during the
process of maturation [13]. In general, the mature B-cell population
constitutes of only approximately 107 clones [14].
However, in vitro display technologies make use of recombinant DNA techniques to exploit molecular diversity. Not only
does this preserve the original natural repertoire but it is also
capable of enlarging on the natural diversity, leading to generation of novel antibodies, which is not possible by in vivo
immune systems. Using DNA techniques such as gene fusion,
codon randomization and DNA shuffling or any combination
of these methods, very large combinatorial libraries can be constructed from various animal and human sources. For example,
randomized assembly of antibody H and L chains from different lymphoid sources (e.g., peripheral blood, bone marrow,
spleen or tonsils) has led to the generation of naive single-chain
antibody libraries [15]. The random joining of H and L chains
422
creates new diversity, but some of the pairs may not be favorably
recombined since they are not compatible, and thus do not
form folded molecules [16]. Another method of generating new
diversity is by the use of oligonucleotides to randomize antibody
CDR region(s) to create synthetic libraries [17].
Libraries containing a single framework or a consensus
sequence have also been designed in which diversity was built
up by shuffling native CDR repertoires on a single antibody
scaffold [18,19]. This approach recombines all six CDR regions,
thereby generating a large potential for novel variations. A clear
advantage of this method is that such a library is expected to
produce functional molecules since CDRs are derived from
mature B-cells, which usually express folded proteins [19]. The
choice of a single framework or consensus sequence for library
construction may also facilitate downstream antibody production (see below), thus avoiding framework sequences that have
expression problems in bacterial systems [19]. A human combinatorial antibody library (HuCAL) has been constructed by
this strategy using a number of modular consensus frameworks
to display CDR repertoires [18]. With DNA shuffling technology, it is possible to generate megalibraries through recombination of the native CDR repertoires within members of the
naturally occurring frameworks. Naive and synthetic libraries
are generally antigen independent, and thus are very useful for
unbiased selection of antibodies against any antigens [15].
Immunized antibody libraries can be generated from animals
or patients. This strategy combines the advantage of in vivo
immunization processes with in vitro DNA recombination
[20,21], creating libraries in which specific antibodies against the
antigen used for the immunization can be rapidly isolated [20].
Library diversity has a significant influence on the properties
of antibodies being selected. A larger library increases the
probability of finding antibodies with desirable characteristics.
For example, while a library size of 106–8 members produced
antibody affinities of 10–100 nM, libraries of 109–10 members
led to the isolation of antibodies with dissociation constants
(Kd) in the subnanomolar range [22].
Antibody selection in vitro
One of the advantages of display technologies is that antibodies
are selected in vitro, which allows the direct control of experimental conditions for favoring the enrichment of molecules
with desired properties. For example, by using an off-rate
selection strategy, antibodies with high affinity (Kd up to
10-12 M) have been isolated [23]. Inclusion of a reducing reagent, dithiothreitol (DTT), in the selection mixture has
generated antibodies with enhanced stability [23]. In order to
decrease antibody crossreactivity, a strategy known as epitopeblocked panning, in which undesirable epitopes were blocked
prior to selection, has led to enrichment of epitope-specific
antibodies [24]. By switching a hapten conjugated to different
protein carriers in alternative selection cycles, hapten-binding
antibodies have been produced with a reduced crossreactivity to
the carrier proteins [25]. Selection of antibodies on whole cells
or frozen tissue sections has produced antibodies capable of
Expert Rev. Proteomics 2(3), (2005)
Generation of antibodies in vitro
recognizing the protein epitope in situ [26]. Selection conditions
can also be chosen to favor the generation of stable and soluble
antibodies. For instance, selection using thermal and chemical
denaturation has generated highly stable and aggregation-resistant antibody fragments [27,28]. However, while selection in the
presence of protease(s) has yielded protease-resistant proteins,
this strategy may not be suitable for selection of antibodies due
to the proteolytic susceptibility of the peptide linker and
unstructured CDR regions [28].
De novo design of antigens that are exposed to a display
library can also guide selection of antibody-combining sites.
For example, catalytic antibodies carrying a specific enzymatic
activity have been selected using designed antigen analogs [29].
Display technologies have also been used successfully to produce antibodies against toxic and unstable proteins and even
self-antigens [30].
Antibody evolution in vitro
In combination with DNA mutagenesis, display technologies
offer a powerful means of protein evolution in vitro. This
approach has been utilized for antibody maturation to improve
binding characteristics. For example, successive rounds of mutation followed by selection under appropriate conditions have
led to identification of antibody variants with enhanced affinity,
specificity or stability [31]. Antibody in vitro maturation has an
advantage over in vivo somatic maturation in that it overcomes
constrains of the natural limit, generating antibodies with
affinities beyond that set by the in vivo biologic process. For
instance, whereas the in vivo somatic maturation process generally produces antibodies with a limited affinity of 10-9–10-10 M
for their target antigens, an in vitro affinity maturation strategy
has isolated antibodies with a Kd of 5 × 10-14 M [32], which is at
least a 10,000-fold improvement. This significant increase in
affinity suggests that antibody in vitro maturation is a promising
approach for the generation of therapeutic antibodies since it
has been demonstrated that the affinity of an antibody has a
major effect on the efficiency of in vivo targeting [33].
In vitro evolution can be applied to improve the thermal
stability of antibodies, which is an important therapeutic and
diagnostic parameter, since these antibodies need to retain full
activity at 37oC in patient serum for hours or days without precipitation or degradation. Elevated temperatures have also been
used in selection conditions in which antibodies have been
evolved with increased thermal stability [28].
The in vitro evolution strategy can also be used in humanization of rodent antibodies. Antibody humanization has been
developed to reduce immunogenicity of rodent antibodies [34],
which provides a useful route to explore existing rodent antibodies as diagnostic and therapeutic agents. One approach is by
antibody reshaping, which involves changing of the solventexposed residues in a murine framework to their human
homologs, followed by display and selection for those maintaining the original antibody function. This approach has been
demonstrated as a rapid and effective method as it allows simultaneous assessment of all mutated molecules that best preserve
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the original antibody properties from a designed combinatorial
library, overcoming the major drawback of other humanization
approaches, which require separate construction and analysis of
individual antibody variants [35,36].
Cell-free ribosome display: the next generation of
display technologies
Cell-based display methods such as phage display and cell surface display have been widely used for selection and evolution
of antibodies in vitro. However, they suffer from a relatively
limited library size due to the restriction of cell transformation
efficiency (generally 107–10 members). Ribosome display has
been developed to avoid the transformation step by generating
antibody–ribosome–mRNA (ARM) complexes for selection in
a cell-free system (FIGURE 1). The formation of ARM complexes
is achieved through the stalling of a ribosome at the end of
translation, which links individual antibody fragments to their
corresponding mRNA and allows simultaneous isolation of the
functional antibody and its encoding mRNA through affinity
capture with a ligand. The ribosome-bound mRNA is then
recovered as DNA by reverse transcription (RT)-PCR
(FIGURE 2). Similar to other display methods, this process can be
reiterated to enrich high-affinity antibodies. Both prokaryotic
and eukaryotic ribosome display systems have been established
and successfully utilized for in vitro selection and evolution of
proteins including antibodies [11].
Unique advantages of ribosome display
Without the need for cell transformation, ribosome display can
therefore screen a much larger library. This is because uncloned
PCR fragments are used as the library in which a very large size
can be easily generated. For example, a ribosome display library
of 1012–14 members can be rapidly produced by a few PCR
reactions, whereas cell-based methods require at least 10,000
transformations to achieve a similar sized library [37]. For a
ribosome display library, the restriction is perhaps dependent
on the number of functional ribosomes in the reaction, which
can be up to 1014/ml in rabbit reticulocyte lysate [38]. The use
of PCR fragments in ribosome display also provides a simple
tool for efficient introduction of new diversity into the selected
DNA pool during subsequent cycles [23,31]. Inclusion of
mRNA
Translation of mRNA
lacking stop codon
Ribosome
Spacer
VH V Antibody
L fragment
ARM complex
Figure 1. Formation of the ribosome complex.
ARM: Antibody–ribosome–mRNA complex; VH/VL: Variable region of
antibody heavy (H) and light (L) chain, respectively.
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He & Khan
Qβ RNA-dependent RNA polymerases in the cell-free system
can also introduce random mutations into mRNA templates
[39]. This unique capability of consecutively diversifying DNA
followed by a functional selection offers an efficient means for
searching sequence space, providing an ideal system for in vitro
antibody maturation.
Recently, ribosome display has been adapted to display
polypeptides composed of un-natural or chemically modified
amino acids (pure translation display), which further expands
the diversity with novel chemical properties into displayed
proteins [40]. This also suggests that ribosome display can be a
powerful tool to bridge the gap between chemistry and biology. Since cell-free systems tolerate toxic, proteolytically sensitive or unstable proteins, ribosome display is ideally suited to
display such proteins that are otherwise difficult to express by
cell-based systems.
method generated ribosome complexes through deletion of the
3´ terminal stop codon from DNA. It also included a number of
additional components, such as protein disulfide isomerase,
vanadyl ribonucleoside complexes and anti-ssrA antisense oligonucleotide in the translation mixture in order to promote protein
folding, stabilize mRNA and inhibit the action of ssrA RNA,
respectively [42]. To avoid the possible disruptive effect of DTT
on folding of antibody domains through reduction of disulfide
bridges, transcription and translation were performed separately
in an uncoupled E. coli S30 translation system without DTT,
which was used successfully to display mRNA libraries generated
by in vitro transcription [42]. Recent modification of E. coli S30
ribosome display has produced even more stable ribosome complexes by introducing a protein–mRNA interaction, leading to
an improvement in the selection efficiency [43].
Eukaryotic systems
Ribosome display systems
At the same time, a eukaryotic ribosome display system was
also developed for selection of active single-chain antibody fragThe first published description of ribosome display was termed ments in a coupled rabbit reticulocyte lysate system that was
‘polysome display’, which used a coupled Escherichia coli S30 initially terrmed ‘ARM display’ (FIGURE 2B) [38]. Deletion of the
system for peptide selection [41]. In this method, a synthetic stop codon from the PCR fragment was again used to generate
DNA library encoding random ploypeptides was used to gener- eukaryotic ribosome complexes. A distinctive feature of ARM
ate polysome complexes by employing chloramphenicol to stall display is the use of a novel recovery procedure in which
the translating ribosomes. Polysome complexes displaying RT-PCR was performed directly on the ribosome complexes
interacting peptide epitopes were then captured with an immo- without the need to dissociate them first (see details below).
bilized antibody by panning on microtiter wells. The recovered The success of the in situ RT-PCR was achieved through the
complexes were dissociated with EDTA to release the bound design of primers hybridizing slightly upstream of the 3´ end,
mRNA for DNA recovery by RT-PCR [41]. This procedure was avoiding the ribosome-covered region [38]. This novel procelater modified to display folded single-chain antibody frag- dure simplifies the recovery process and also avoids material
ments [42]. FIGURE 2A outlines the display cycle. The modified losses incurred by disrupting complexes for mRNA isolation.
ARM display has been demonstrated by
rapidly enriching specific ARM comDNA cloning
plexes from a mutant library [38] and a sucT7
PCR
and expression
cessful selection of human antiprogeserone
antibodies with high affinities from a
transgenic mouse library [20]. A modified
RT-PCR
Cell-free
version has later been described in which
transcription
3´
oxidized/reduced
glutathione
and
5´
& translation
Qβ RNA-dependent RNA polymerase
mRNA
were included in the translation mixture to
isolation
improve protein folding and introduce
3´
mutations [40]. Recently, a wheat germ sys5´
tem has also been developed for ribosome
display of folded proteins [44].
Ribosome
Escherichia coli S30 system
Ribosome
disruption
complexes
Selection
5´
3´
Antigen
Figure 2A. Escherichia coli ribosome display cycle.
RT: Reverse transcription; T7: T7 promoter.
424
Key factors affecting ribosome display
Constructs for ribosome display
In general, DNA constructs for ribosome
display require a promoter such as T7 and
a translation initiation sequence such as
Shine–Dalgarno for E. coli S30 extract or
Kozak sequence for eukaryotic systems
(FIGURE 3). A consensus sequence for protein
initiation in both E. coli and eukaryotic
Expert Rev. Proteomics 2(3), (2005)
Generation of antibodies in vitro
cell-free systems has been described [45],
and it is thus possible to design a single
DNA cloning
T7
and expression
PCR
consensus sequence for ribosome display
in both prokaryotic and eukaryotic systems. To generate a connection to the
ribosome and enable the complete exit of
In situ
Cell-free
RT-PCR
transcription
the displayed protein from the ribosomal
& translation
tunnel, a spacer domain of at least 23–30
5´
3´
amino acids in length is required at the
C-terminus [11]. The nucleotide sequence
encoding the spacer domain also provides
a sequence for the design of primers in the
Antigen
RT-PCR recovery stage. A variety of spacSelection
Ribosome
ers have been used successfully, including
complexes
the constant region of immunoglobulin
(Ig) κ chain, gene III of filamentous Figure 2B. Eukaryotic antibody–ribosome–mRNA display cycle.
phage M13, and the CH3 domain of RT: Reverse transcription; T7: T7 promoter.
human IgM [11], streptavidin and glutathione-S-transferase [46]. The length of a spacer has been uncoupled systems allow the optimization of the transcription
shown to affect display efficiency. For instance, a 116-amino and translation step separately. It also controls the amount of
acid spacer from gene III was more efficient than its shorter mRNA added in the reaction mixture.
partners [47]. Constructs designed for E. coli S30 display also
require incorporation of sequences containing stem–loop Folding of ribosome-bound antibody
structures at both 5´ and 3´ ends of the DNA to stabilize The folding of a newly synthesized antibody fragment into a
mRNA against degradation by high RNase activities in the well-defined structural conformation on the ribosome is critical
E. coli cell-free system (FIGURE 3) [41].
for the selection of functional antibodies. It has been demonstrated that nascent polypeptides fold cotranslationally on riboMonoribosome or polysome display
somes within a living cell or in a cell-free system and that moleThe choice of a cell-free system is generally dependent on the cular chaperones are involved in the folding process [49]. The
origin and properties of the proteins to be displayed and its ribosome itself or ribosomal RNA from both prokaryotic and
subsequent application. Some proteins may demonstrate eukaryotic sources can act as chaperones to promote protein
improved expression in a particular cell-free system than others. folding [50]. Active ribosome-bound enzymes have been demonThe use of a cell-free system may also affect the formation of strated in a cell-free system after the C-terminus was extended,
ribosome complexes either as monoribosome or polysome com- suggesting that the protein is capable of folding into its funcplexes. Polysomes carry more than one ribosome translating a tional 3D conformation while attached to the ribosome. The
single mRNA molecule, whereas monoribosomes have only one successful selection of functional ribosome-displayed antibody
ribosome on each mRNA. The formation of polysomes raises fragments also confirms the correct folding of nascent antithe possibility of polyvalent attachment of incomplete nascent bodies [11,47]. In fact, the use of a cell-free system provides a flexproteins or peptides and selection of low-affinity combining ible and versatile environment for adding molecular chaperones
sites after multipoint attachment (avidity effects). While it has and selection reagents to produce correctly folded proteins [42].
been shown that rabbit reticulocyte lysate can produce mainly
monoribosome complexes [101], E. coli S30 usually generates DNA recovery after selection
polysomes [41]. However, by controlling the ratio between input After selection, ribosome complexes are subjected to RT-PCR
mRNA templates and ribosome numbers, it is possible to use to recover the attached mRNA (FIGURE 2). A highly efficient prothe E. coli system to generate monoribosome complexes [48].
cedure would allow rare species to be recovered, increasing the
Both coupled and uncoupled cell-free systems (rabbit sensitivity and enrichment. Currently, two recovery methods
reticulocyte lysate, wheat germ and E. coli S30 extract) have are commonly employed: prokaryotic ribosome disruption [42]
been successfully developed for ribosome display of antibodies and eukaryotic in situ RT-PCR [38]. Prokaryotic ribosome disand other proteins [11]. A coupled system uses DNA as the ruption dissociates ribosome complexes by EDTA treatment
template on which transcription is immediately followed by followed by isolation of the released mRNA and RT-PCR
translation, and is thus simpler and more efficient. It also amplification [42], while eukaryotic in situ RT-PCR is peravoids problems of mRNA degradation and secondary struc- formed directly on the ribosome complexes without using disture effects, which prevents protein synthesis. An uncoupled ruption and purification steps (FIGURE 4) [38]. As in situ RT-PCR
cell-free system translates mRNA, which is produced either by recovers DNA directly from ribosome complexes, it is possible
in vitro transcription or isolated from native sources. The to characterize the binding of ribosome-displayed antibodies
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425
He & Khan
DNA library of 2 × 1013 molecules, a peptide was selected that bound specifically to
A. Construct for E. coli ribosome display
streptavidin with a Kd of 4 nM, an affinity
that is 1000-fold greater than peptides
T7
SD Gene to be displayed
Spacer
X (no stop codon) selected by cell-based display methods [37].
Active enzymes have also been displayed
3´ stem loop
5´ stem loop
and enriched by using either substrate
analogs or inhibitors [54]. Recently, riboB. Construct for eukaryotic ARM display
some display has been successfully used to
T7 Kozak
Gene to be displayed
Spacer
display full-length membrane proteins for
X (no stop codon)
identification of protein–protein interaction partners, suggesting that membrane
Figure 3. DNA constructs for ribosome display. (A) Construct for Escherichia coli ribosome display.
SD is the binding site for E. coli ribosome. (B) Construct for eukaryotic ARM display. T7 and Kozak
proteins are soluble when they are attached
sequences are indicated.
to the entire ribosome complexes [43].
ARM: Antibody–ribosome–mRNA; SD: Shine–Dalgarno sequence; T7: T7 promoter; X: Stop codon has
In proteomic applications, ribosome disbeen removed.
play has been adapted to display a genomic
cDNA library from Staphylococcus aureus,
quantitatively through detection of the attached mRNA by the leading to a comprehensive identification and recovery of antiTaqMan® method. The use of in situ RT-PCR also facilitates genic proteins that could be potential vaccine candidates. This
demonstrates, for the first time, that ribosome display can be
automation of the entire ribosome display process.
Comparison of E. coli S30 and rabbit reticulocyte lysate sys- used on a whole genome. This study has found that a large fractems has showed that the prokaryotic ribosome disruption pro- tion of the identified peptides could not be displayed by the
tocol led to a poor recovery and enrichment from rabbit cell-based E. coli display system [55]. Ribosome display can also
reticulocyte lysate ribosome complexes [51]. Similarly, a compari- be combined with protein microarrays to allow library-versusson of the two recovery methods on rabbit reticulocyte lysate library screening for high-throughput generation of antibodies
ribosome complexes revealed that prokaryotic ribosome disrup- and genomic discovery of protein–protein interactions.
To explore protein domains as alternative scaffolds or antition was fivefold less efficient than eukaryotic in situ RT-PCR
body mimics, ribosome display has been used in combination
[64]. This suggests that the prokaryotic ribosome disruption
method is ineffective on eukaryotic ribosome complexes. A new with folding-based selection strategies. This has led to the genmethod has been described to disrupt rabbit reticulocyte lysate eration of novel protein scaffolds with improved solubility and
protease resistance [56]. Recently, ankyrin repeat proteins have
ribosome complexes by heating at 70oC [52].
Recently, a highly sensitive RT-PCR procedure has been been designed as scaffolds for ribosome display of diversified
developed that is capable of recovering single complementary libraries, from which highly specific binding molecules, with
DNA (cDNA) molecules from ribosome complexes [UNPUB- affinities in the low nanomolar range against several proteins,
have been selected [57].
LISHED OBSERVATIONS]. This development not only improves the
recovery efficiency but also makes it possible to clone cDNA
in vitro from ribosome complexes to produce antibodies in a Antibody production
fully cell-free process [UNPUBLISHED OBSERVATIONS].
Antibody fragments selected by display technologies are usually
produced in heterologous hosts in relatively large quantities.
Applications of ribosome display
Full-length antibodies can be generated by fusion of antibody
Ribosome display has been successfully applied for in vitro anti- constant regions to display-derived fragments carrying the
body selection, evolution and humanization [11,31,47,53]. Differ- V regions. A variety of expression systems have been estabent libraries have been used to select antibodies including lished, including bacterial and mammalian cell culture and
human antibodies with high affinity and specificity [11,31,47,53]. transgenic animals and plants [58]. Each system has advantages
Through repeated rounds of mutation and in vitro selection, and limitations (see below). The choice of an expression system
ribosome display has effectively isolated antibody variants with is dependent on the intended application and, indeed, the ecoimproved affinity (Kd as low as 10-12 M), specificity and stabil- nomics of production. Recent progress in cell-free protein synity [11,53]. It has also isolated novel antibodies that can recognize thesis has made this method a promising tool for large-scale
conformational epitopes or have catalytic activities [54]. production of antibodies [54].
Recently, ribosome display has been utilized for antibody
Bacterial expression offers a cheap and rapid means to proreshaping, which rapidly identifies humanized antibodies from duce therapeutic proteins [59]. Expression levels up to 2 g/l of
a shuffled DNA library [36].
some antibody fragments have been achieved [60]. Antibody
In addition to antibodies, ribosome display has been used to fragments produced in E. coli have been used for in vivo tumor
select for peptides, ligand-binding molecules, receptors and targeting and imaging [58]. Although full-length antibodies
enzymes [11,53,54]. For example, using a very large synthetic have been successfuly expressed in E. coli [61], large-scale
426
Expert Rev. Proteomics 2(3), (2005)
Generation of antibodies in vitro
production remains to be developed. E. coli-synthesized proteins are known to have little or no post-translational modifications; however, recent improvements in bacterial strain engineering have made it possible to achieve post-translational
modifications in E. coli [59,62].
Mammalian cell lines are suitable for generating full-length
antibodies with effector functions and serum stability similar to
those of naturally occurring antibodies. Despite the detection
of different glycosylation patterns, there are no clinical data
suggesting that these differences have any adverse effect on antibody activity in vivo or induce antibody responses [58]. A major
disadvantage of using mammalian cell culture is the high cost
of the manufacturing process.
Transgenic animals and plants are alternative systems for
large-scale production of full-length therapeutic antibodies and
fragments [58]. Transgenic chickens and calves have shown
promise. However, the success rates of obtaining live births are
low and the development of transgenic systems is still in its
infancy. The use of transgenic plants has the added advantages
of being inexpensive and free of human pathogens, but the
current limitation is the long initial lead-time for production.
Also, the suitability of plant-derived antibodies for therapeutic
application remains to be tested [58].
Cell-free protein synthesis is a promising system for antibody
production. It exploits the cellular protein synthesis machinery
to direct protein synthesis outside intact cells using exogenous
mRNA or DNA as a template. Thus, it is simple and rapid. A
cell-free system also provides an open and flexible environment
that can be controlled and adjusted for producing correctly
folded proteins. With the improvement in expression levels,
which is now comparable to or better than cell-based expression systems, cell-free protein synthesis is becoming an alternative tool for large-scale production of proteins [54]. Eukaryotic
cell-free systems are also capable of performing a variety of
post-translational modifications that are important for protein
function and therapeutics. Antibody fragments with native
activity have been produced in cell-free systems [54]. However,
it still remains to be seen whether full-length antibodies can be
produced in a cell-free system.
display not only screens much larger populations but also
allows continuous expanding of new diversity during selection,
providing an ideal system for rapid selection and evolution of
antibodies in vitro. Ribosome display is particularly useful
when selection of antibodies requires construction of new or
large libraries. Thus far, only scFv fragments have been demonstrated for selection, although it remains to be seen whether Fab
fragments can be displayed on the ribosome.
Production of the selected antibodies is an important downstream process. Although a variety of expression systems are
available, the choice requires careful consideration, taking
account of expression level, antibody structure, ease and cost of
purification and production. Antibodies can be produced either
as full-length molecules or fragments (Fab or scFv) or fusions
with other proteins. Full-length antibodies have effector functions and increased half-life, while Fab and scFv have advantages
of good penetration into tumours and rapid clearance from the
blood. Mammalian cell cultures or transgenic animals should be
considered for generation of full-length antibodies, whereas bacterial expression systems are preferred for production of Fab or
scFv fragments. Alternatively, cell-free protein synthesis may be
chosen for producing antibody fragments.
Five-year view
Display technologies allow selection of proteins in vitro, avoiding problems of the in vivo biologic processes, and thus provide
a suitable approach for producing antibodies for medical applications as well as proteomics studies. In the next 5 years, novel
antibody libraries will be designed for further exploitation of
natural and synthetic antibody sequence space, generating very
large, diversified, combining sites for selection. Antibody crystal structures may also be used as a guide to assist design of
effective libraries. Novel protein domains will be explored as
alternative scaffolds to build up diversity for selection of highly
A. E. coli ribosome display
5´
3´
B. Eukaryotic ARM display
5´
3´
Expert commentary
Display technologies are powerful tools for the generation of
antibodies with desired affinity and specificity. Each method
has its own advantages and disadvantages. Phage display is the
most commonly used method that allows display of antibodies
in both Fab and scFv format. In addition, phage libraries can be
selected through in vivo targeting on molecules at their original
location and conformation [63]. Cell surface display technologies provide the possibility of using cell sorting for efficient
screening of antibody fragments with improved properties [32].
However, these cell-based display technologies require cloning
of DNA, which limits the library size to be displayed. Ribosome display overcomes this limitation by using a cell-free system to express uncloned PCR fragments. Due to the ease with
which a very large library can be rapidly generated, ribosome
www.future-drugs.com
Ribosome
disruption
RT-PCR on
ARM complex
3´
5´
mRNA
isolation
5´
3´
RT-PCR using
purified mRNA
Figure 4. DNA recovery procedures. (A) Escherichia coli ribosome
display. (B) Eukaryotic ARM display.
ARM: Antibody–ribosome–mRNA complex; RT: Reverse transcription.
427
He & Khan
specific binding molecules (antibody mimics). It is expected
that approaches for high-throughput generation of antibodies
will continue to develop. These include automation of the display procedures and combination of a display method with
protein microarrays for large-scale screening of antibodies
against genomic or disease targets. While phage display and
cell-surface display methods will continue to be developed for
the generation of antibodies, ribosome display will be regarded
as the next generations of display technologies as it overcomes
the limitation of cell-based methods, allowing screening of
greater library diversity in vitro.
Therapeutic antibodies or antibody mimics will be selected
directly from novel libraries. Further improvement of the
selected antibodies will be through in vitro antibody maturation. Antibodies that are functional in an array format will
be chosen to create antibody microarrays for diagnostic and
proteomic applications.
Acknowledgement
Research at the Babraham Institute (Cambridge, UK) is
supported by the Biotechnology and Biological Sciences
Research Council (BBSRC).
Key issues
• Display technologies provide powerful tools for rapid generation of highly specific antibodies for diagnostic, therapeutic and
proteomic applications.
• Using recombinant DNA techniques, display technologies exploit natural antibody diversity, creating novel molecules for selection.
• Display technologies select antibodies under in vitro-defined selection condition(s), favoring enrichment of antibody molecules with
novel properties such as increased affinities beyond the natural affinity limitation.
• Display technologies can be combined with DNA mutagenesis for antibody evolution in vitro, producing variants with improved
affinity, specificity and stability.
• Cell-free ribosome display overcomes the limitation of cell-based display methods, providing an efficient tool for selection and
evolution of antibodies in vitro. Ribosome display technology has been successfully used to select antibodies with high affinity,
specificity and stability. Automation of the display process for high-throughput production is possible.
• A variety of expression systems are available for large-scale generation of display-selected antibodies. Each system has its own
advantages and limitations. Cell-free protein synthesis may become an alternative system for production of antibodies.
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Affiliations
•
Mingyue He, PhD
Senior Scientist, Protein Technologies Laboratory,
Babraham Research Campus,
Cambridge, CB2 4AT, UK
Tel.: +44 1223 496 306
Fax: +44 1223 496 045
[email protected]
•
Farid Khan, PhD
Research Scientist, Protein Technologies
Laboratory, Babraham Research Campus,
Cambridge, CB2 4AT, UK
Tel.: +44 1223 496 253
Fax: +44 1223 496 045
[email protected]
Expert Rev. Proteomics 2(3), (2005)