NATURE BIOTECHNOLOGY VOLUME 23 NUMBER 9 SEPTEMBER 2005 1105

Selecting and screening recombinant antibody 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. Automation of 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 hybridomas—a 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 libraries, 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 the basis of the antigen-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 transgenic 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 librariesThe antigen-binding site of an antibody is composed of six comple-mentarity determining regions (CDRs) or hypervariable regions—three 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 those lymphocytes that produce antigen-reactive antibodies, and triggers 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 fourth, 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. (h.hoogenboom@home.nl).

Published online 7 September 2005; doi:10.1038/nbt1126

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1106 VOLUME 23 NUMBER 9 SEPTEMBER 2005 NATURE BIOTECHNOLOGY

from the B lymphocytes of immunized animals. This collection of genes is then cloned to provide a physical link between each antibody’s 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 ofbacteriophage, 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 tissues 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 libraries11–15, includ-ing antibodies against self antigens; second, the generation of picomolar affinity antibodies by in vitro affinity maturation16–18; 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 of in vitro dis-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 antibodies24–26. 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-siae29 provides 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 peptides or enzymes, such as surface display on the bacterial cell walls of Escherichia coli, Staphylococcus aureus and Zymomonas mobilis or on spores of cer-tain Bacillus strains, has been limited by problems with the antibody’s

* *

*

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 H2Clonal 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 JH1 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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NATURE BIOTECHNOLOGY VOLUME 23 NUMBER 9 SEPTEMBER 2005 1107

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 approach bypasses most of these problems: it is based on anchoring the antibody fragment on the periplasmic face of the inner membrane of E. 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 compatible with (filamentous) phage display, combines the ease of E. 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 antibody fragments, see ref. 41.)

To establish a platform to select recombinant antibody libraries in the IgG format, the preferred format for many applications, researchers recently 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 format–based libraries may be built using vaccinia virus–based vectors42, or diversity may be introduced in vivo by using B-cell lines that hypermutate a carrier 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. 1126–1136).

Strategies to select and screen antibody librariesIndividual 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 antigen 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 display–based 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 fragment complementation

a bSteps in antibody selection Selection platformsFigure 2 Creating and selecting recombinant antibody 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 its 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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1108 VOLUME 23 NUMBER 9 SEPTEMBER 2005 NATURE BIOTECHNOLOGY

maximally sample the repertoire47. Fewer selection methods have been tested on nonphage-display systems, but both ribosome and mRNA-dis-play work particularly well with purified antigen and yeast cell–display 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 ribosome-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 antigen 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-antibody 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 of immunoliposomes 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 enzyme–based 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-lated—for 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 information 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 one of 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 multivalent display in the first step(s), for example, using helper phage variants (reviewed in ref. 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 ribosome, 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 entirely in vitro, thereby eliminating the need for cell transformation. The other advantage is that it is very amenable to mutagenesis to provide additional diversity be-tween generations (e.g., by nonproofreading polymerases), without the need to transform the cloned library into E. coli. Although there are many examples of antibody-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. cerevisiae cell surface via fusion to the α-agglutinin yeast adhesion receptor, which is located on the yeast cell wall. The display level on the cell is variable (on average about 3 × 104 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 fluorescently 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. Limiting factors such as the transformation efficiency 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 beads172–174 and then sorted by flow cytometry to yield single-nanomolar affinity 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 vivo gene 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 display–based 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. 3a–c) and all selection and amplification steps are carried out in microtiter plate for-mat56–58. 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 vitro expression67, 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 further downstream, 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 methods73–75.

Recombinant antibody library typesMAb 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 nonimmune 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, bone marrow, 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 properties21,22,77 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 VL genes of single antibody–producing cells79. Libraries 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) to reduce antigen-induced biases in the repertoire, and were the first librar-ies used to isolate anti-self antibodies—otherwise 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

NameValency ofdisplay

Typical max.library size

Selectionscope Main application Main strength Main weakness

Suitableformats

Phage MonovalentMultivalent

1010 to 1011 Versatile mAbs from natural librariesmAbs from synthetic librariesAffinity maturationStability increase

Large mAb panelsTechnically robustEasy to useAutomated

Introduction of diversity by cloning is slowLarge libraries difficult to makeNot truly monovalent

scFvFab

Fab´2dAb

Diabody

Ribosome Monovalent 1012 to 1013 Limited Affinity maturationStability increase

Intrinsic mutagenesisFastest of all systemsAmenable to automation

Small mAb panelsLimited selection scopeTechnically sensitive

scFvdAb

Yeast cell Multivalent 107 Sorting Affinity maturationExpression increase Stability increase

Fast in combination with random mutagenesisDirect screening for kinetics with cells

Sorting expertise and equipment neededTransformation efficiency

scFvFabdAb

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 in E. coli and setup for downstream engineering are used in combination with trinucleotide-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 before library 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 libraries 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 antibodies 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 antibodies—the 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 library—up to 10 nM for libraries with 107 to 108 clones, and up to 0.1 nM for the best libraries with over 1010 members. 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 vitro selection and/or optimization

Antibody/developer Target Mutagenesis StrategyIncrease in affinity/resulta Reference

Vitaxin (MEDI-522)/Medarex, Gaithersburg, MD, USA

αvβ3 Focused on CDR Screening ×80 96

Synagis (palivizumab)/MedImmune, Gaithersburg, MD, USA

Respiratorysyncytial 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 shuffling 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

TNFα 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

C11L34/University of Zurich/Switzerland GCN4 Immune library and error-prone PCR Selection on ribosomes ×65 to 40 pM 27

4M5.3/University of Illinois,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 ×285 to 30 pM/expres-sion increases

116

VL-12.3/MIT Huntington(htt) protein

Error-prone PCR/homologous recombination

Sorting using yeast ×10 177

14B7/University of Texas, Austin, Texas Anthrax toxin Error-prone PCR Sorting using bacteria ×200-fold 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 histocompatibility complex; VEGF, vascular endothelial growth factor; GCN4, general control protein 4; FITC, fluoroisothiocyanate; CEA, carcinoembryonic antigen.

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influenced by several important 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 antibody’s 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 no strict 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 of in vitro affinity maturation, small libraries with focused diversity at a small number of residues that are most likely to interact with antigen, 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 bottleneck: 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 antibody 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 vitro procedure 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 vitro selection for binding. Selections from display libraries have been carried out using several methods (or any combination of them). (a) Antigen (Ag) immobilized onto solid supports, columns, pins or cellulose/poly(vinylidene fluoride) membranes/other filters, deposited on BIAcore sensorchips or immobilized indirectly via capture;(b) biotinylated antigen (biotin (red) is captured via streptavidin-coated beads (gray)); (c) diverse recombinant antigens, including antigens incorporated into paramagnetic liposomes (left) and immunoadhesins (right); (d) fixed prokaryotic cells displaying the (recombinant) antigen; (e) enriched subcellular fractions or membrane fractions; (f) transfected or tumor cells to select for 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 relevant cells/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 disulfide-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.) K

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are used, CDRs not diversified in the primary library are targeted99,100. Residues that affect affinity may also be determined experimentally (e.g., by alanine, shotgun or homology scanning) or identified on the basis of structural knowledge and then randomized.

The most successful strategies target the CDR3 regions in the center of the binding site (see also ref. 88 for citations), in combination with resi-dues at the periphery identified by a process of error-prone mutagenesis, ribosome display–based selection and high-throughput sequencing (L. Jermutus, Cambridge Antibody Technology, UK, personal communica-tion). Usually, the affinity gain is due to mutations in the CDR loops that provide new contacts, influence the positioning of side chains contacting the antigen or replace low-affinity contact residues with those with more favorable binding kinetics . Once the affinity is below ~10–20 pM, it can be further increased through subtle structural changes in the binding site (e.g., through mutations in the framework region—parts of the variable regions that are not hypervariable—that modulate CDR loop flexibility or affect the orientation of the VH and VL domains28,30,101).

Antibody libraries for humanization. The vast majority of hybridoma-derived antibodies used for research are murine, despite advances made in display and transgenic mice technologies that allow production of human antibodies. Although this is gradually changing, humanization of murine antibodies remains a widely exploited technology to obtain antibodies suitable for human therapy. Humanization can be accom-plished readily by combining rational design and molecular evolution to yield antibodies with occasionally improved affinities.

In antibody humanization by CDR-grafting, besides replacement of the six murine CDRs with human CDRs, framework residues are also often grafted to maintain affinity. Framework residues that are likely to influence binding of the humanized antibody can be subjected to random mutagenesis and then the best variants selected102. Similarly, focused libraries in which selected framework or CDR residues are mutated can be screened and the best clones combined to yield variants with higher affinities than the original murine antibodies103.

An alternative technique, not based on any rational design or struc-tural information but instead based on the shuffling of the six mono-clonal antibody CDRs with pools of corresponding individual human frameworks, can also be used to select and screen antigen-binding vari-ants104. An antibody with similar binding or biological effects to a murine molecule, yet containing a higher human sequence content, can also be selected by sequential replacement of the chains of a murine antibody

with human chains105–107. Maintaining the CDR3 regions at the heart of the binding site in this ‘guided selection’108 often retains most of the antigen binding105,109. Alternatively, the portions of the CDRs that are likely to play a dominant role in the antibody-antigen interaction can be identified experimentally and tested in the context of a human antibody library with high germline content (M. Alfenito, KaloBios, Palo Alto,CA, USA, personal communication).

Applications to mAb therapeutics and researchApart from enabling the discovery of mAbs that bind specific targets with high affinity, selection strategies can also be applied to improve other properties, such as enhanced stability, resistance to proteases, aggregation behavior and expression level in heterologous systems, and even antibody-mediated catalysis. In addition, antibody libraries are proving to be useful research tools, enabling the discovery of novel target antigens associated with the exterior and interior of certain cells and tissues. tissues. A third application reviewed here is the use of selec-tion methods to express antibodies intracellularly and current progress in selecting antibody libraries directly in this format.

Selecting for biophysical properties. Selection for higher thermody-namic stability of a displayed protein has been achieved with phage display using a variety of selection procedures, including temperature stress110, proteolytic digestion111, cycles of denaturation under reduc-ing conditions112 and heat denaturation and cooling113. Single-chain Fvs with improved stability have also been evolved by removing the intradomain disulphide bond and applying a reducing redox potential during ribosome display26. Finally, yeast cell sorting has been used to select antibodies with increased expression level and stability114–116. The results show that there are many, often V gene–specific, molecu-lar solutions to improve stability or expression. These approaches provide a means to select well-behaved V genes for libraries, and to optimize the characteristics of selected antibody fragments for het-erologous expression117.

Selecting for target discovery. The availability of very large repertoires of different binding sites in combination with in vitro selection proce-dures for antibodies has opened up applications in the field of target dis-covery. Recently, direct cell panning or subtractive selection techniques were used to build panels of antibodies selective for cancer or endothelial cell–surface antigens118–121, internalizing cell-surface antigens122 and

L2 H3

L3

H1L1 H2

Immunelibraries

Nonimmunelibraries

a c

b

d e f gSynthetic and semi-synthetic libraries

h i j k lAffinity maturation libraries

Figure 4 Binding-site diversity in recombinant antibody libraries. In the left panel, (a) recombinant immune22 and (b) nonimmune libraries12,13,85 are cloned from naturally diverse antibody gene pools and for this reason display most diversity at the center of the binding site (depicted in green), with some level of somatic mutations throughout the V regions (green dots). Upper panel: for the construction of synthetic antibody libraries, synthetic diversity (in red) is introduced into (c) the CDR3 of a one V-gene segment99,153,(d) the CDR3 of multiple V-gene segments (indicated in pink)11,14 or (e) in several chosen CDR positions (indicated by red dots)82,138,139. Alternatively, in semi-synthetic libraries, natural and synthetic diversity is combined; for example, in (f) heavy-chain natural diversity in CDR3 (in green) is combined with synthetic hot-spot diversity for CDR1 and CDR2 (in red)15 or (g) natural CDRs (in green) are reshuffled into one or more chosen antibody frameworks86,154 (also see refs. 10,87). In the lower panel, during affinity maturation strategies, diversity is introduced into the V genes in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via an (h) high (red) or (i) low (pink) level of mutagenesis94 or (j) targeting isolated hot spots of somatic hypermutations or residues suspected of affecting affinity on experimental basis or structural reasons (red dots)97,155,156. Random mutations can be introduced throughout the whole V gene using (k) E. coli mutator strains, error-prone replication with DNA polymerases or RNA replicases (shown in pink)28,157, (l) the replacement of regions that are naturally diverse via DNA shuffling or similar techniques using natural partner repertoires (shown in light green)18,150 or a combination of both. Alternative techniques target hypervariable loops extending into framework-region and CDR4 residues (ref. 142) employ loop deletions and insertions in CDRs or use hybridization-based diversification158 (not shown). Some recent examples are listed in Table 3.

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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 cells124–126.

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 domains133 selected 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 or affinity optimization, combinations with fully automated two-hybrid systems may eventually yield a platform suitable for generating 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 developmentsLibrary 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 time, 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 panel of 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 librariesName Target Indication Company Clinical phase

Humira(adalimumab)

TNFα 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 β1 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 β 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

αGMCSFRα Granulocyte-macrophagecolony-stimulating factorreceptor α

Autoimmune diseases CaT/AMRAD Preclinical

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parameters; and fourth, 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 antigens. 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 novo binding-site design may also lead to libraries of conventional mAbs with a propensity either to bind such types of epitopes143 or 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 matured to 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.”

ACKNOWLEDGMENTSI thank many colleagues including Jane Osbourn and Lutz Jermutus, Clive Wood, Zhenping Zhu, Patrick Bauerle, Herren Wu, David Chen and Lex Bakker for sharing unpublished results and am grateful to Mark Alfenito for reviewing the manuscript.

COMPETING INTERESTS STATEMENTThe author declares that he has no competing financial interests.

Published online at http://www.nature.com/naturebiotechnology/

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