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NATURE BIOTECHN O LO GY VOLUME 23 NUMBER 9 SEPTEMBER 2005 1105

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]).

Published online 7 September 2005; doi:10.1038/nbt1126

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1106 VOLUME 23 NUMBER 9 SEPTEMBER 2005 NATURE BIOTECHNO LO GY

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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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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