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Selecting and screening recombinantantibody librariesHennie R Hoogenboom
During the past decade several display methods and other library screening techniques have been developed for isolating
monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely
exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are
now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with
ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automationof the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first
antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already
approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for
the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.
In humans, the immune system is capable of creating thousands of
millions of different antibodies from which suitable antigen-binding
antibodies are rapidly selected. Envious of this unsurpassed powerful
system for making binding sites, scientists have been investigating for
decades methods to recreate systems to build immunoglobulin-based
binding sites using recombinant approaches (reviewed by Winter and
Milstein1). One of the first breakthroughs came in 1989 with an inno-
vative technology that enabled the cloning of antibody genes2, thereby
bypassing hybridomasa hybrid cell produced by the fusion of an
antibody-producing lymphocyte with a tumor cell, which was the tra-
ditional means of manufacturing mAbs. In the new method, antibody
genes were cloned directly from lymphocytes of immunized animals and
expressed as a single-domain library3 of antibody heavy- or light-chain
variable regions or as a combinatorial library of antigen-binding frag-
ment (Fab) fragments in bacteria4. To screen combinatorial librar ies, a
slow and cumbersome colony-lifting and filter-based screening method
with radio-labeled antigen was then used to identify the few antigen-
reactive antibodies in libraries from millions of clones.
Within a year, a method based on the expression of functional anti-
body fragments on the surface of filamentous phage was described,
which provided a way to quickly select antibodies from libraries on thebasis of the ant igen-binding behavior of individual clones5. A few years
later this technique, called phage display, in combination with PCR-
based cloning of antibody repertoires2,4, was successfully used to isolate
murine6 and human7,8 antibodies from recombinant antibody libraries
built from natural sources, such as from animal or human B lympho-
cytes, and eventually libraries were created entirely by in vitro cloning
techniques (reviewed in ref. 9).
Fifteen years later, phage, and more recently, ribosome- and yeast-
display technologies (described below) have turned into mainstream
antibody and protein engineering platforms. Display technology has
also become one of the three major technologies for creating mAbs for
human therapy, in addition to the use of immunized t ransgenic mice
and the humanization of mAbs. This review covers the most important,
currently used selection platforms for recombinant antibody libraries,
the methods for selecting and screening different types of libraries, sev-
eral antibody affinity and stability optimization strategies and finally, the
impact of library-based approaches on antibody humanization, with a
focus on the developments (and citations) of the past few years.
Selection platforms for antibody libraries
The antigen-binding site of an antibody is composed of six comple-
mentarity determining regions (CDRs) or hypervariable regionsthree
within the light-chain variable domain (VL) and three within the heavy-
chain variable domain (VH). In the immune system, a large collection of
different antibody binding sites is created by the combinatorial assembly
of germline-encoded segments (Fig. 1). This produces a repertoire of
naive B-cell lymphocytes, each expressing a unique antibody binding site
on their surface. Exposure to antigen selects from this repertoire thoselymphocytes that produce antigen-reactive antibodies, and tr iggers the
incorporation of somatic mutations in the V genes, allowing subsequent
selection of mutations that improve the affinity of the antibody for the
antigen.
Antibodies can also be isolated from recombinant antibody libraries
in the laboratory, using one of the platforms for selection that in essence
mimics this in vivo process. Many of these selection platforms share
four key steps with the procedure for antibody generation in the in vivo
immune system: first, the generation (or cloning) of genotypic diversity;
second, the coupling of genotype to phenotype; third, the application of
selective pressure; and four th, amplification (Fig. 2a). This process first
leads to a diverse collection of recombinant antibody genes, such as those
Ablynx NV, Technologiepark 4, 9052 Ghent, Belgium. Correspondence should be
addressed to H.R.H. ([email protected]).
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from the B lymphocytes of immunized animals. This collection of genes
is then cloned to provide a physical link between each antibodys phe-
notype (antigen-binding behavior) and the encoding genotype.Rather
than screening the clones directly for antigen binding, antibody libraries
are enriched by rounds of selection with target antigens and amplifica-
tion and after a few rounds, individual clones are screened for antigen
reactivity (Fig. 2a). Box 1 and Figure 2b describe some of the latest
and most frequently used selection platforms for antibody isolation and
engineering; in Table 1 the three main platforms are compared.
Phage display. Antibody display on the surface of two types of
bacteriophage, fd and M13, is currently the most widespread method for
the display and selection of large collections of antibodies and for the
engineering of selected antibodies (reviewed in ref. 10). It owes its favor-
able status to being robust, simple to use and highly versatile; the selec-
tion process can be adapted to many specific conditions that foil most
other display platforms, including selections on whole cells and t issues
and even in animals. The most successful applications of phage antibody
display include the following: first, the de novo isolation of high-affinity
human antibodies from nonimmune and synthetic libraries1115, includ-
ing antibodies against self antigens; second, the generation of picomolar
affinity antibodies by in vitro affinity maturation1618; and third, the
discovery of antibodies with unique properties from nonimmune19,20
and immune libraries from animal or human donors21,22.
Ribosome and mRNA display. The most developed forms ofin vitrodis-
play rely on the stable formation of a complex of antibody fragment and
its encoding mRNA (for a review, see ref. 23). The mRNAs from selected
complexes are then amplified. The most successful applications of ribo-
some display are in the field of affinity maturation of antibodies2426.
The built-in affinity maturation feature of this display system, caused
by the error-prone process of reverse transcriptase and amplification,contributes to the efficient maturation of picomolar concentrations of
antibodies27,28.
Microbial cell display. Surface display on the yeast Saccharomyces cerevi-
siae29provides the possibility to select repertoires of cells by flow cytom-
etry. In combination with random mutagenesis methods to diversify the
VH and VL genes (see below), the yeast-display method has yielded the
highest affinity (48 fM) for any antibody30. The use of many other micro-
bial display formats that have been used successfully with pept ides or
enzymes, such as surface display on the bacterial cell walls ofEscherichia
coli, Staphylococcus aureusand Zymomonas mobilisor on spores of cer-
tainBacillusstrains, has been limited by problems with the ant ibodys
* *
*
Antibody variable regiongermline segments
DNArearrangement
Selection forantigen binding
Somatichypermutation
and affinityselection
Library of naivelymphocytes
Ag-selectedlymphocyte(s)
Maturedlymphocyte(s)
Naive/nonimmunebinding site library
Immunebinding site library
Unique Ag-bindingsite surface
Improved Ag-bindingsite surface
L2 H3
L3
H1
L1 H2
Clonal level
Library levelL2 H3
L3
H1
L1 H2
L2 H3
L3
H1
L1 H2
Low affinity
L2 H3
L3
H1
L1 H2
High affinity
+
VH
VL
D J
V J
H1 H2 H3
L1 L2 L3
VH
VL
H1 H2 H3
L1 L2 L3
VH
VL
H1 H2 H3
L1 L2 L3
+ +
V
Figure 1 Generating binding site diversity in the immune system. In the immune system, a large collection of different antibody-binding sites is created by
the combinatorial assembly of germline-encoded segments (V, D and J for the heavy-chain variable region VH, V and J for the light-chain variable region VL).
This DNA reshuffling process targets most diversity in the primary repertoire to the heart of the binding site, the two CDR3 regions of heavy and light chains,
respectively, creating a large chemical diversity that is of primary importance for the potential recognition of many different types of antigenic structures.
This is depicted in a schematic representation of a collection of antibody-binding-site surfaces (in green), for simplicity indicated with H3 and L3. The other
CDR/hypervariable regions (indicated with H1, H2, L1 and L2) are encoded in the human germ line by ~100 different functional V-gene segments; from
a structural perspective, they are located at the periphery of the binding site surface, surrounding the H3 and L3 regions (lower level of diversity is shown
in light green). Exposure to antigen (red) selects from this naive repertoire those lymphocytes that produce antigen-reactive antibodies and triggers the
incorporation of somatic mutations in the V genes (indicated by the green lines at the clonal level) and subsequent selection of mutations that improve the
affinity of the antibody for the antigen (green stars and circles at clonal and library level, respectively). Recombinant antibody libraries are made by rescue of
genes from these various V-gene pools; note that nonimmune libraries, depending on their exact construction, may contain a mix of V genes from both naive
and other B-cell sources (indicated with the dotted arrows). Ag, antigen.
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heterologous expression, secretion and fold-
ing, with proteolysis and antigen-antibody
accessibility. Therefore, many of these display
and screening systems, although elegant in
nature31,32, are not widely used today for anti-
bodies. However, a recently described approachbypasses most of these problems: it is based
on anchoring the antibody fragment on the
periplasmic face of the inner membrane ofE.
coli followed by disruption of the outer mem-
brane, incubation with fluorescently labeled
antigen and sorting of the protoplasts. This
very promising and versatile display method
is directly compat ible with (filamentous) phage display, combines the
ease ofE. coli-based library constructions with the power of cell sorting,
and therefore, is likely to become widely used.
Other selection platforms. Directed evolution platforms recently devel-
oped for antibody fragments include retroviral display34, display based
on protein-DNA linkage35,36, microbead display by in vitro compart-
mentalization37, in vivo-based growth selection based on the protein
fragment complementation assay (PCA)38 or other systems39 and even
single-molecule sorting40. Although each of these methods will have
specific theoretical advantages, to date, their validation with antibody
fragment libraries has been limited, and their advantages over more
established systems (e.g. regarding the truly monovalent nature of the
method, eukaryotic expression advantages, increase in library size or
selection efficiency) remain to be demonstrated. (For a more in-depth
discussion of library-display technologies, including PCA and two-
hybrid systems, that are available but have not yet been used in combi-
nation with ant ibody fragments, see ref. 41.)
To establish a platform to select recombinant antibody libraries in
the IgG format, the preferred format for many applications, researchersrecently displayed small libraries of IgGs on the surface of mammalian
cells. After homologous integration of a single-gene copy in each cell,
the population was sorted by flow cytometry to obtain a clone with
sevenfold affinity improvement (W.D. Shen, Amgen, personal commu-
nication). In the future, bigger combinatorial IgG formatbased libraries
may be built using vaccinia virusbased vectors42, or diversity may be
introduced in vivo by using B-cell lines that hypermutate a carr ier anti-
body gene constitutively43 or upon induction44 or that harbor induc-
ible hypermutable enzymes involved in this process in nature45. Some
of these newer selection and diversification methods may open novel
applications for the directed evolution of antibodies and other proteins
(see also accompanying review on p. 11261136).
Strategies to select and screen antibody libraries
Individual clones of a recombinant single-chain Fv (scFv) or Fab library
theoretically can be directly screened for antigen binding, for example,
using binding assays based on ELISA or filter-based screening. Screening
is limited by the number of clones that can be examined, hence in many
applications the frequency of antigen-reactive clones is too low, and
the libraries too large (with tens of millions to billions of clones) to
do this efficiently. The connection between genotype and phenotype
in phage- or ribosome-display libraries provides a means to select for
clones binding to a desirable antigen, thereby increasing the frequency of
antigen-reactive clones, enriching the clones with best binding affinity,
or the clones with certain predefined binding characteristics. Typically
many more clones can therefore be sampled compared with screen-
ing procedures. Many different selection methods and experimental
approaches have been developed that separate clones that bind from
those that do not (Fig. 3).
Selection procedures. For phage-display libraries, selection involves
exposure to ant igen to allow antigen-specific phage antibodies to bind
their targets during biopanning. This is followed by recovery of antigen-bound phage and subsequent infection in bacteria. Although ideally,
only one round of selection would be required, nonspecific binding
limits the enrichment that can be achieved per selection round and
therefore, in most cases, recursive rounds of selection and amplification
are needed to select the best binders from the library (Fig. 2a).
Phage displaybased selections are now a relatively standard procedure
in many molecular biology laboratories (a more detailed description of
these procedures is provided elsewhere10,46 and references therein). For
more complex selections such as those using cells or tissues, it can be
instructive to use enrichment studies with control phage antibodies to
optimize the efficiency of the selection method and to compare different
selection approaches, and then tune the selection strategy accordingly to
Phage display
Protein-mRNAlink via:
Protein-DNAdisplay
Growthselection via:
Display on:
Microbeadvia in vitro
compartmentalization
Coupling of genoto phenotype
Selective pressureon phenotype
Screening Amplification
+
Antibody gene pool
Displayed library
Selected antibody lead
Synthetic DNA
Cloning ofgenetic diversity
B-cells
Selectioncycle
Mutagenesisand
selectioncycle
-ribosome display
-mRNA display
-Yeast-Bacteria-Mammalian cells-Retroviruses-.....
-Yeast 2-hybrid-Protein fragmentcomplementation
a bSteps in antibody selection Selection platformsFigure 2 Creating and selecting recombinantantibody libraries. (a) First, antibody diversity
is generated from synthetic V genes or cloned
from B cells. Next, antibody phenotype (boxes
in green, blue and orange) is coupled to its
genotype (wavy line) via a phenotype-genotype
link (green) packaged in a host (purple) (shown
here schematically for phage display). As a
result, each host particle expresses (or displays)
a unique antibody on i ts surface. The repertoire
of antibodies displayed on these host particles
is subjected to The process is repeated and
eventually antibodies binding to antigen are
confirmed by screening. (b) Different selection
platforms for conventional antibodies. Color code
as for a (see text for details and citations).
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maximally sample the repertoire47. Fewer selection methods have been
tested on nonphage-display systems, but both ribosome and mRNA-dis-
play work part icularly well with purified antigen and yeast celldisplay
libraries are preferentially selected by cell sorting.
Selection antibody libraries for target binding. A variety of protocols
have been described for selecting antibodies from phage- and r ibosome-
display libraries with improved binding affinity or kinetics toward the
target antigen. For example, by using limited and decreasing amounts of
antigen, the selection favors clones with lower Kd; by using long washing
steps after the incubation of target antigen, library clones with improved
off-rate are selected; and by using very short incubation times prefer-
entially, clones with improved on-rate are selected. In yeast display, the
optimization of ant igen concentration and time for dissociation of anti-
body-antigen complexes enables cell sorting with flow cytometry on the
basis of small differences in the kinetic parameters of antigen-an tibody
interactions, at least for monomeric antigens48. Antibodies may also be
selected with or for a particular functional activity, such as for recep-
tor cross-linking49, signaling or gene transfer (reviewed in refs. 10 and
50). Special selection procedures have been developed to identify phage
antibodies that catalyze certain chemical reactions51 or that bind to and
are internalized by cells52; the latter property is useful for targeting ofimmunoliposomes and other payloads.
Screening antibody libraries for target binding. The outcome of any selec-
tion procedure is a mixture of antibody clones with different target-binding
properties that then need to be individually screened. Antigen-binding
of poly- or monoclonal phage antibodies is tested using typical anti-
body-binding assays, ranging from ELISAs to immunoprecipitation.
The best screening assays are fast, robust, amenable to automation (e.g.,
in 96- or 384-well format) and use the display host (phage or yeast cells)
or the soluble antibody fragment equipped with tags for detection and
purification. The diversity of the clones present in the selected antibody
library (which is tracked by restriction enzymebased fingerprinting or
by high-throughput DNA sequencing of selected clones) can be used as
a guideline to define at what stage to screen the library. When finding
drug candidates among the selected antibody leads, it may be advanta-
geous to screen for biological function using biochemical or cell-based
bioassays because binding affinity and potency are not always corre-
latedfor example, this applies when identifying antibodies that neu-
tralize an interaction, agonize or antagonize receptor binding. Screening
(or selection) procedures can also be tailored to identify antibody leads
that cross-react with the murine antigen or bind to different isoforms
of the antigen. In ribosome/mRNA display, selected populations are
first cloned and individual antibodies are expressed either by the host
cell in vivo or by translation in vitro. The preferred screening method in
yeast display is flow cytometry, which under the right conditions yields
informat ion on affinity, expression level and epitope binding53.
Automating the process. Although automation seems particularly
straightforward for in vitro display-based approaches (e.g., ribosome
display and microbead display by in vitro compartmentalization37,
Several different molecular selection strategies for isolating and
engineering human antibodies are currently in use. Described here
are the three best established platforms.
Phage display. To express an antibody fragment on the surface
of the phage particle, its encoding gene is fused in-frame to oneof the phage coat proteins and cloned in a vector that can be
packaged as a phage particle. Different display systems can lead to
monovalent (single copy) or to multivalent (multiple copy) display
of an antibody, depending on the type of anchor protein and display
vector used. The most popular system is to use monovalent display,
which is convenient for selecting antibodies of higher affinity,
achieved by using a direct fusion or a disulfide-bridged link to
a minor coat protein, pIII, and by using phagemids into which
antibody libraries are easier to clone than phage vectors. Libraries
with over 1010 clones can be made using recombination-based
protocols11,159, but more frequently are made by conventional
transformations with genetically stable phagemid vectors. Selection
efficiency is improved by using mult ivalent di splay in t he first
step(s), for example, using helper phage variants (reviewed inref. 46), inducible promoters160,161 or bivalent display162,163.
Multivalent display is also used for selecting antibodies that
mediate receptor-mediated endocytosis164 , panels of antibodies for
target discovery165 and to rapidly select antibody-antigen pairs on
the tip of the phage166 .
Ribosome and mRNA display. In ribosome display167 , the link
between antibody and encoding mRNA is made by the ri bosome,
which at the end of translating this mRNA is made to stop
without releasing the polypeptide168,169. The ternary complex
as a whole is used for the selection. In mRNA display, there is
a covalent bond between antibody and mRNA established via
puromycin as an adaptor molecule170 . These display methods
are carried out enti rely in vitro, t hereby eliminating the need
for cell transformation. The other advantage is that it is very
amenable to mutagenesis to provide additional diversity be-
tween generati ons (e.g., by nonproofreading polymerases),
without t he need to t ransform the cloned li brary into E. coli.
Although there are many examples of anti body-ribosome display,
the mRNA-display format has been used more exclusively for
single-domain protein171 and has only recently been used for
conventional antibodies23 .
Yeast cell display. Antibodies are displayed on the yeast S.
cerevisiaecell surface via fusion to the -agglutinin yeast
adhesion receptor, which is located on the yeast cell wall. The
display level on the cell is variable (on average about 3 10 4
fusions per cell for a scFv), but the intrinsic avidity of this display
system is counteracted by the power of cell sorting. By staining
the cells with both f luorescently labeled antigen and anti-epitope
tag reagent, the yeast cells can be sorted according to the level
of antigen binding and antibody expression on the cell surface.Limi ting factors such as the transformation eff iciency of yeast
and the cell sorting speed are currently being tackled. This
approach has been used to build a nonimmune human scFv
library172 with over 109 clones, which is efficiently selected
with magnetic beads172174 and then sorted by flow cytometry
to yield single-nanomolar affini ty antibodies172 . Combinations
of phage with yeast-display platforms have been described174 ,
and recent approaches include the use of the yeast cells mating
system to create combinatorial diversity estimated to be 1014 in
size175,176 and in another study the use of the yeast homologous
recombination system for in vivogene diversification177 .
Box 1 Selection platforms
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which in theory avoids complicated host cell and phage manipulations,
in practice these methods rely on PCR which is highly sensitive to con-
tamination and nonspecific amplification. Therefore to date most prog-ress has been made by adapting the more robust phage displaybased
selection and screens to robotics54.
Automation of the selection process is required when handling many
antigens in parallel, for example, when generating thousands of anti-
bodies for use in proteomic screens or for antibody arrays55. In these
approaches, antigens are immobilized to a surface (Fig. 3ac) and all
selection and amplification steps are carried ou t in microtiter plate for-
mat5658. The materials used for such selections are most often produced
by antigen-derived, surface-exposed linear peptides59,60 or recombinant
approaches54,55,61. Devices have also been built to carry out semi-auto-
mated cell selections using capillary flow chambers62. Automation of
the screening process has been used to test thousands of different anti-
body leads15,63.These high-throughput screening platforms consist of
a combination of robotic colony-pickers and workstations, incubators
for high-throughput expression, fluid-handling robots for perform-
ing ELISAs, high-throughput cloning and high-throughput purifica-
tion, detection devices, PCR-machines, sequencing apparatus and data
handling systems and software to integrate the data from all steps64.
Further efforts to streamline the screening procedures65,66, including
miniaturization, in vitroexpression67, multiplexing and signal detection
and data processing, will increase the throughput of these screening
systems. Finally, high-density gridding of bacteria followed by protein
array screening68 and filter-based colony screening69,70 have been used
to bypass the selection step and directly screen antibody libraries.
In the future, protein microarrays may also become particularly use-
ful for high-throughput analysis of antibody specificity71 and affinity72.
Arguably, the greatest bottleneck in screening today is analysis furtherdownstream, including kinetic analysis and in vitro/in vivo functional
and bioactivity analysis. Functional and potency assays often require the
recloning of antibody genes for expression in the IgG format, which is
also amenable to high-throughput methods7375.
Recombinant antibody library types
MAb libraries can be based on immune fragments (that is, biased
towards certain specificities present in immunized animals6 or natu-
rally immunized, or infected, humans8) or naive fragments (not biased
toward specificities found in the immune system). The latter type of
fragment can be derived from n onimmune natural or semi-synthetic
sources.
Antibodies from immune antibody libraries. These libraries are con-
structed with VH and VL gene pools that are cloned from source B-
cells (from diverse lymphoid sources including peripheral blood, bonemarrow, spleen or tonsils) by PCR-based2,4 or similar76 cloning tech-
nologies, cloned into an appropriate vector for expression as a random
combinatorial antibody library, and subsequently selected for and/or
screened. Compared with the yield using hybridoma technology, many
more antibodies can be derived from a recombinant immune library
made with the material of a single immunized donor, and in vitro selec-
tion can enrich for rare antibody specificities. Further, human immune
or disease-associated antibody libraries have identified antibodies with
very interesting proper ties21,22,77 unlikely to be present in nonimmune
or synthetic libraries. These libraries also facilitate the investigation of
the humoral immune system at a molecular level78. If required, natural
pairings of heavy and light chain can be maintained by in-cell PCR-link-
ing of the V genes, or by parallel amplifying in high-throughput the VH
and VLgenes of single antibodyproducing cells79. Librar ies made with
this procedure may form a better reflection of the composition of the
natural immune response compared with random combinatorial librar-
ies with artificially paired chains in immune or nonimmune libraries,
but it remains to be seen whether this translates into antibodies with
higher affinity and more potency or with a lower immunogenicity when
used in humans.
Antibodies from nonimmune and semisynthetic libraries. This type
of library is comprised of antibody fragments from a source of genes
that is not explicitly biased to contain clones binding to antigen; as such
they are useful for selecting antibodies against a wide variety of antigens
(see below). Nonimmune (or naive) libraries are derived from natural,
unimmunized, rearranged V genes (e.g., from the IgM B-cell pool) toreduce antigen-induced biases in the repertoire, and were the first librar-
ies used to isolate anti-self antibodiesotherwise difficult to obtain by
immunization.
Synthetic antibody libraries are constructed entirely in vitro using
oligonucleotides that introduce areas of complete or tailored degeneracy
into the CDRs of one or more V genes. Synthetic diversity bypasses
the natural biases and redundancies1 of antibody repertoires created
in vivo and allows control over the genetic makeup of V genes and the
introduction of diversity.The first reports of synthetic antibodies in
1992 (refs. 80,81) were followed by many different design strategies,
including mimicking the natural pattern of diversity in the immune
system by randomizing CDR positions at the center of the binding site
Table 1 Comparing the main selection platforms for antibodies
Name
Valency of
display
Typical max.
library size
Selection
scope Main application Main strength Main weakness
Suitable
formats
Phage Monovalent
Multivalent
10 10 to 1011 Versatile mAbs from natural
libraries
mAbs from synthetic
libraries
Affinit y maturation
Stability increase
Large mAb panels
Technically robust
Easy to use
Automated
Introduction of diversity
by cloning is slow
Large li braries diff icult
to make
Not t ruly monovalent
scFv
Fab
Fab 2dAb
Diabody
Ribosome Monovalent 1012 to 1013 Limited Affinity maturation
Stability increase
Intrinsic mutagenesis
Fastest of all systems
Amenable to
automation
Small mAb panels
Limited selection scope
Technicall y sensiti ve
scFv
dAb
Yeast cell Multivalent 107 Sorting Affinity maturation
Expression increase
Stability increase
Fast in combination
with random
mutagenesis
Direct screening for
kinetics with cells
Sorting expertise and
equipment needed
Transformation
efficiency
scFv
Fab
dAb
dAb, domain antibody
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and using a set of V-gene germline segments to provide a low level of
diversity in peripheral regions11. Alternatively, CDR positions known to
be involved in antigen binding can be identified on structural grounds
and randomized in the context of an antibody with known structure82,
or CDR positions chosen for randomization based on the relatively high
mutation frequency of these positions in natural antibody repertoires,
and then randomized in a set of frequently used V germline segments83.
In another design consensus, V-gene segments optimized for expression
inE. coli and setup for downstream engineering are used in combination
with tr inucleotide-mediated diversity in the CDR3 regions14. Libraries
with improved quality and/or downstream characteristics of antibodies
may also be built by selecting expressed V genes for functionality beforelibrary construction84,85.
Semi-synthetic libraries have combinations of natural and synthetic
diversity; they are often created to increase natural diversity while main-
taining a certain level of functional diversity. For example, such libraries
have been created by shuffling natural CDR regions86 or by introduc-
ing naturally rearranged and highly functional CDR3 sequences from
human B-cells with synthetic CDR1 and CDR2 diversity15. Examples of
library designs are schematically depicted in Figure 4.
If sufficiently large and diverse, all these types of librar ies are a source
of antibodies against a large number of different antigens, including self,
nonimmunogenic and toxic antigens, and for this reason these libraries
are now extensively used in the industry and in academia (reviewed in
refs. 87,88). Medium-affinity an tibodies are readily isolated from rela-
tively small nonimmune libraries or from synthetic antibody libraries
diversified in just one or two CDRs or in just one of the two chains,
which reflects the situation in transgenic mice with restricted antibody
repertoires89,90. The most current and successful antibody libraries,
however, display (natural or synthetic) diversity in multiple CDRs and
routinely yield single-digit nanomolar and sometimes subnanomolar
affinity ant ibodiesthe latter having affinities equal to the affinities of
antibodies regularly isolated from immunized mice or from recombi-
nant immune libraries. In general, antibody affinities from these libraries
are proportional to the size of the libraryup to 10 nM for libraries with
107 to 108 clones, and up to 0.1 nM for the best libraries with over 1010members. More importantly, these libraries, in association with high-
throughput screening, deliver panels with thousands of antibodies that
bind distinct epitopes on the same target antigen15,63. Antibody leads
with the highest potencies can then be identified. A subnanomolar affin-
ity is also readily obtainable when selecting even relatively small librar-
ies with ribosome display, in which the V genes are mutated between
selections, although the complexity of selected antibody panels appears
limited24,25,91,92.
There seems to be no major differences in performance when compar-
ing the best nonimmune and synthetic antibody phage-display libraries
in use today, with regard to the frequency of binders and top affinity of
selected clones. However, the success of the drug discovery process is
Table 2 Examples of antibodies subjected to, or obtained by,in vitroselection and/or optimization
Antibody/developer Target Mutagenesis Strategy
Increase in affinity/
resulta Reference
Vitaxin (MEDI-522 )/Medarex,
Gaithersburg, MD, USA
v3 Focused on CDR Screening 80 96
Synagis (pali vizumab)/MedImmune,
Gaithersburg, MD, USA
Respiratory
syncytial virus
Focused on CDR Screening 100 off-rate;
5 on-rate
94
RFB4/National Cancer Institute,
Bethesda, MD, USA
CD22 Hot-spot mutagenesis Screening 15 175
b4/12/Scripps Research Institute/
La Jolla, CA, USA
gp120 CDR walking Selection on phage 420 to 15 pM 16
C6.5/University of California,
San Francisco
c-erbB2 CDR3 mutagenesis Selection on phage 1,230 to 13 pM 17
L19/University of Siena, Italy Fibronectin CDR3 mutagenesis Selection on phage 1,317 to 54 pM 99
G8/University of Maastricht,
The Netherlands
MHC peptide Chain shuff ling and CDR3
mutagenesis
Selection on phage 18 to 14 nM 174
Fab-12/Genentech,
S. San Francisco, CA, USA
VEGF CDR3 mutagenesis Selection on phage 100 potency 176
1121/ImClone System/New York VEGF receptor Chain shuffling Selection on phage >30 to 0.1 nM 18
Humira (adalimumab)/
Abbott Laboratories,
Deerfield, IL, USA
TNF Guided selection Selection on phage 0.3 nM 106
A4.6.1b/Genentech/S. San Francisco, CA, USA
VEGF Framework-region library of mAb Selection on phage 125 102
H6/University of Zurich/Switzerland GCN4 Error-prone PCR and DNA shuffling Selection on ribosomes 500 to 5 pM 28
C1 1L3 4/Universi ty of Zuri ch/Swi tzerland GCN4 I mmune l ibrary and error-prone PCR Sel ecti on on ri bosomes 6 5 t o 4 0 pM 2 7
4M5.3 /University of Il linois,
Urbana, IL, USA
FITC Error-prone PCR and DNA shuffling Sorting using yeast 1,800 to 48 fM 30
smE3/Massachusetts Institute of
Technology (MIT), Cambridge
CEA Error-prone PCR and DNA shuffling Sorting using yeast 2 85 to 3 0 pM/expres-
sion increases
116
VL-12.3/MIT Huntington
(htt) protein
Error-prone PCR/homologous
recombination
Sorting using yeast 10 177
1 4B7 /Uni versi ty of Texas, Aust in, Texas Ant hrax t oxi n Error-prone PCR Sort ing usi ng bact eri a 2 00 -f ol d improvement
to 21 pM
33
a indicates increase in affinity unless otherwise specified. bAvastin (bevacizumab) is a humanized version of this murine mAb. MHC, major hi stocompatibi lit y complex; VEGF, vascularendothelial growth f actor; GCN4, general control protein 4 ; FITC, fluoroisothiocyanate; CEA, carcinoembryonic antigen.
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influenced by several impor tant differences among libraries, including
antibody format and display levels, sequence diversity in selected anti-
body populations, average expression levels, tendency to multimerize,
compatibility with expression screening (e.g., automation) and with
affinity maturation and finally the ease of conversion to other antibody
formats, display or selection systems.
Affinity maturation of antibodies.Although initial antibody leads from
display libraries or from hybridomas often have a number of desirable
characteristics, their potency is sometimes insufficient for therapeutic
applications or for use in sensitive diagnoses. Frequently, an antibodys
potency is governed by its affinity for antigen, and an affinity increase
may help to increase pharmacokinetic and safety profiles and reduce
dosing, toxicity and cost of therapy. Indeed, for many well-studied
cases, increased affinities have translated into improved biological effi-
cacy (Table 2 and citations therein). For some applications, there is nostrict correlation between affinity and efficacy above a certain affinity
threshold, such as in some virus neutralizations93,94 and in tumor tar-
geting, where engineered high-affinity antibodies have been shown to
not necessarily have superior tumor targeting efficacy compared with
low-affinity variants, but on the contrary, may display diminished pen-
etration into solid tumors95
In the immune system, antibodies are affinity matured in a stepwise
fashion by incorporating mutations and selecting variants under increas-
ing selective pressures. In a first and simplest form ofin vitro affinity
maturation, small libraries with focused diversity at a small number of
residues that are most likely to interact with ant igen, the CDRs, are built
using oligonucleotides and PCR. These libraries are then screened to
identify variants with improved affinity, and mutations conferring the
highest affinities are combined in a single clone96. Randomization may
also be introduced at positions frequently mutated in vivo, which are
most likely to generate improved affinity (hot-spot mutagenesis97) or
influence affinity based on structural analysis. Focused mutagenesis has
the advantage that only very small libraries need to be screened initially,
but the disadvantage is that extensive screening of variant combinations
is required to find clones with the desired affinity or kinetic charac-
teristics. Further, the screening assay is the bot tleneck: its throughput
will determine how many different residues can be efficiently sampled
in each library, and the whole approach is crucially dependent on its
capability to discriminate relatively small differences in affinity between
clones.
The second approach involves the display of millions of antibody
variants and selection under conditions that favor clones with improved
affinity or binding kinetics, in a procedure that mimics the in vivo matu-ration process. This procedure is much faster and allows sampling of a
much larger sequence space with up to billions of variants, limited only
by transformation efficiency or scale of the production of the in vitro
(ribosome) display library. This approach has yielded impressive affin-
ity gains for selected an tibody fragments: 1,000-fold improvements in
potency are not uncommon and affinities as low as 48 fM have been cited
(Table 2), indicating that this in vitroprocedure does not suffer from the
kinetic and affinity limits inherent in the immune system98.
Many mutagenesis and selection strategies have been used to provide
subnanomolar affinities (Fig. 4). Broadly applicable mutagenesis strat-
egies target the CDRs or the whole V gene with high or low levels of
randomization, respectively. When clones from (semi)synthetic libraries
a Immobilized Ag
b Biotinylated Ag
c Recombinant Ag
d Bacteria displaying Ag
e Subcellular fractions
f Cells displayingantigen/internalizing Ag
g Alternating selectionson Ag+/ cells
h Subtractive cell sorting
i Tissues with Ag
j Proximity to other Ag
k In vivo selection
l Trypsin digestion
m Mild reduction(release phage only)
n Mild reduction(release phage +Ag)
o Competitive elution
SS
SS
Figure 3 Methods for in vitroselection for
binding. Selections from display libraries have
been carried out using several methods (or
any combination of them). (a) Antigen (Ag)
immobili zed onto solid supports, columns, pi ns
or cellulose/poly(vinylidene fluoride) membranes/
other filters, deposited on BIAcore sensorchips or
immobili zed indi rectly via capture;
(b) bioti nylated antigen (bioti n (red) is captured
via streptavidin-coated beads (gray)); (c) diverserecombinant antigens, includi ng antigens
incorporated into paramagnetic liposomes
(left ) and immunoadhesins (right); (d) fixed
prokaryotic cells displaying the (recombinant)
antigen; (e) enriched subcellular f ractions or
membrane fractions; (f) transfected or tumor
cells to select f or binding or internalization;
(g) alternating antigen-displaying cells and
depleting antigen-negative cells; (h) subtractive
selection (e.g., using sorting procedures
shown by a flow cytometric analysis of a cell
population with sorting window (in red)); (i)
enrichment on tissues (e.g., on appropriately
prepared tissue slides); (j) proximity to another
bound ligand (green); and finally, (k) injection
into living animals and recovery of the relevantcells/tissues. Elution conditions can also be
used to drive the selection towards the desired
population, for example, (l) via trypsin-digestion
of a proteolytically sensitive phage, via mild
disulfi de-bridge reduction to release (m) phage
(CysDisplay) or (n) antigen and phage, or (o)
via competitive elution with a ligand binding to
the antigen and displacing the relevant phage
antibody. (For citations, see text and refs.
10,46.) KatieRis
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tissue-specific antigens123. Combining this approach with expression
cloning, immunoprecipitation and mass spectrometry has already led to
the identification of novel target molecules on malignant cells124126.
Intracellular selection. Intracellular antibodies (or intrabodies), when
folded properly, are valuable tools for studying biological processes and
for blocking proteins inside cells127. Individual scFv antibodies can be
evolved directly for stable cytoplasmic expression by growth selection
in bacteria128, although it may be faster to functionally identify pools
of phage-selected antibodies that have been recloned and expressed
intracellularly in mammalian cells129,130. A modified yeast two-hybrid
selection strategy was previously described that can directly select and
isolate several functional antigen-binding intrabodies127,131,. More
recently, libraries have been engineered to contain a high percentage
of functional intrabodies using scFv frameworks132 or antibody heavy-
chain variable domains133selected directly in the intracellular environ-
ment , which were then employed to build single-framework intrabody
libraries. Although not yet applied to the screening of large libraries oraffinity optimization, combinations with fully automated two-hybrid
systems may eventually yield a platform suitable for generat ing antibod-
ies of medium affinity to panels of antigens for large-scale functional
proteomics projects, as recently suggested by work done with other bind-
ing proteins134.
Future developments
Library technology has led to one human antibody so far approved
for therapy and many more antibodies in clinical and preclinical trials
(Table 3). Although a full discussion on the immunogenicity of these
and other engineered antibodies135 is beyond the scope of this review, it
is accepted in the field that the risk of immunogenicity may be reduced
by using antibodies that are as human as possible. With t ime, library
designs and affinity-maturation strategies may be even more tuned
towards the ideal antibody composition: an as-close-to-human germ-
line sequence with optimal affinity yet with a minimal number of T-cell
epitopes and a human-like heavy chain CDR3 (ref. 136). Library designs
may go even further by reducing the difficulties in downstream develop-
ment by avoiding potentially problematic amino acids137 in the variable
regions (e.g., methionine oxidation, asparagine deamidation or aspartate
isomerization). Fortunately, as has been demonstrated recently138,139, a
reduction in diversity to just four or even two well-chosen amino acids,
does not necessarily limit library performance. Also, future selection
procedures against instability and aggregation behavior may help to
reduce potential immunogenicity and increase solubility.
There are now many different molecular selection strategies for iso-
lating and engineering human antibodies. The three main selection
platforms (phage, ribosome/mRNA and microbial cell display) are
somewhat complementary in their use, but they all fall short of the
ideal: a selection system that provides within a few days a large panelof antibodies to a large number of epitopes on the target antigen of
choice, a range of selected affinities, a certain level of stability and
expression and a precisely targeted sequence diversity. To build a better
antibody molecular evolution machine, we need to make further refine-
ments in several areas: first, improve methods and predictive designs
for introducing diversification into antibody genes to build libraries
with a higher quality in functionality and biophysical properties; sec-
ond, build normalized selection and amplification strategies to reduce
biases towards nondesirable variables (e.g., reduce advantages due to
PCR, infection, growth, multimerization); third, combine affinity and
expression maturation for populations rather than individual clones to
increase throughput and simultaneously maintain or improve multiple
Table 3 Examples of therapeutic antibodies derived from recombinant antibody libraries
Name Target Indication Company Clinical phase
Humira
(adalimumab)
TNF Autoimmune diseases Abbott/CambridgeAntibody Technology (CaT)
Approved for arthritis
(in phase 2/3 for others)
Numax
(MEDI-524)
Respiratory syncitial virus RSV prophylaxis MedImmune Phase 3
ABT-874 Interleukin 12 Multiple sclerosis Abbott/CaT Phase 2
CAT-192B
(belimumab)
Transforming growth factor 1 Systemic sclerosis Genzyme/CaT Phase 2
LymphoStat-B B-cell activating factor Lupus/rheumatoid arthritis Human Genome Sciences/CaT Phase 2
MT201 Epithelial cell adhesion molecule Breast and prostate cancer Micromet Phase 2
HGS-ETR1 TRAIL-R1 Non-Hodgkin lymphoma Human Genome Sciences/CaT Phase 2
CAT-213 Eotaxin1 Allergic rhinitis CaT Phase 2
MYO-029 Growth differentiation factor-8 Muscular dystrophy Wyeth/CaT Phase 1
ABthrax Protective antigen Anthrax Human Genome Sciences/CaT Phase 1 finished
HGS-ETR2 TRAIL-R2 Solid tumors Human Genome Sciences/CaT Phase 1
CAT-354 Interleukin 13 Asthma CaT Phase 1
1D09C3 MHC class II Non-Hodgkin lymphoma GPC Biotech/MorphoSys Phase 1
IMC-11F8 EGFR Solid tumors ImClone Phase 1
IMC-1121b VEGFR-2 Solid tumors ImClone Phase 1
GC-1008 Transforming growth factor Idiopathic pulmonary fibrosis Genzyme/CaT Preclinical
IMC-A12 Insulin-like growth factor receptor Solid tumors ImClone Preclinical
MOR102 Intracellular adhesion molecule 1 Autoimmune diseases MorphoSys Preclinical
DX-2240 Tie-1 Cancer Dyax Preclinical
AZD3102 Undisclosed Alzheimer disease AstraZeneca/Dyax Preclinical
GMCSFR Granulocyte-macrophagecolony-stimulating factor
receptor
Autoimmune diseases CaT/AMRAD Preclinical
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1114 VOLUME 23 NUMBER 9 SEPTEMBER 2005 NATURE BIOTECHNO LO GY
parameters; and four th, integrate all these in a high-throughput screen
that allows multiple biophysical parameters to be tracked for thousands
of antibody leads in parallel. Computational kinetic models would
help to set quantitative biophysical goals140 and protein modeling and
design141 would help build improved libraries.
Although synthetic diversity yields a structural diversity significantly
greater than that observed in nature142, this approach has not yet been
exploited to target epitopes not readily recognized by naturally derived
antibodies, such as narrow cavities and carbohydrate ant igens. Although
such features were already successfully achieved using engineered pro-
teins based on single-binding protein domains (see review by p. 1126
1136; ref. 41), future modeling-based and experimentally assisted de
novobinding-site design may also lead to libraries of conventional mAbs
with a propensity either to bind such types of epitopes143or to bind to a
chosen antigen144 with an extraordinary configuration145, structure146
or as has been recently shown, a biosensor incorporated into the bind-
ing site147.
By not only learning from nature but also liberating antibodies from
the many restrictions imposed by nature, we have amassed a large col-
lection of selection platforms that now make it possible to engineer
antibodies with exquisite and unusual binding affinities, binding kinetics
and sequence/biophysical characteristics. These platforms have maturedto the point where we can glimpse the promised land that Paul Ehrlich
wrote about over 100 years ago148: the land which [] will yield rich
treasures for biology and therapeutics.
ACKNOWLEDGMENTS
I thank many colleagues including Jane Osbourn and Lutz Jermutus, Clive Wood,
Zhenping Zhu, Patr ick Bauerle, Herren Wu, David Chen and Lex Bakker for sharing
unpublished results and am grateful to Mark Alfenito for reviewing the manuscript.
COMPETING INTERESTS STATEMENT
The author declares that he has no competing financial interests.
Published online at http://www.nature.com/naturebiotechnology/
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