503
Phage display of combinatorial antibody libraries
Christoph
Rader and Carlos F Barbas Ill*
Abbreviations
CDR
complementarity-determining region
HA
hemagglutinin peptide
scFr
single-chain variable region fragment
encapsulated
genotype via the phage surface forms the
basis of the technique
(reviewed
in [3-S]). Typically,
the antibody fragments are displayed on the surface of
phage as either Fab fragments [6], single-chain
variable
region fragments (scFvs), or dimeric scFvs, also known
as diabodies, which differ from scFvs in the reduced
length of the linker peptide used and their preference to
associate as dimers. Library construction
is facilitated by
the ready availability of phagemid vectors, which allow
for construction
and display of libraries of all of these
types of antibody fragments using a single rare cutting
restriction enzyme, S’Z. Selection of interesting members
from the library based on the displayed antibodies’ binding
specificity and affinity is generally performed over several
rounds of selection and amplification in a process known
as panning. Antibodies have been sleeted from immune
humans [7-16,17*,18,19*,‘20**,21~], macaques (the variable
region genes of macaques one highly homologous to those
of humans, making cloning from immune macaques an
attractive approach to the development
of human antibodies [Z]), mice [23**,24,25’,26’,27’*,28*,29”],
rabbits [30],
and chickens [31*], as well as from large naive libraries
[32-34,3Soo]. This review focuses on the most interesting
studies using this technique published in 1996.
Introduction
Selection
Monoclonal antibodies were isolated from the first combinatorial antibody library expressed by phage in 1989 [l].
Driven by the success of this initial report, incorporation
of the filamentous
phage display method of Smith into
general practice was rapid [Z]. It should be noted that at
the beginning there was much skepticism concerning both
the likelihood of finding high affinity antibodies within
a random combinatorial
library of antibody
fragments
and the ability to display a 50 kDa heterodimeric
Fab
protein on the surface of filamentous
phage. Some of
the most skeptical voices of this time are now among
the technique’s most prolific practitioners. There are now
more than 300 published examples in the literature for this
technique,
which forms the basis of an annual practical
course at the Cold Spring Harbor Laboratory.
While in the past the selection of phage displayed antibody libraries was mainly restricted to panning on purified
antigens immobilized
on artificial surfaces, an increasing
number of antibodies have now been selected by panning
on cell surface expressed
antigens
[9,11,18,23**]. Cell
panning has been used when the antigen was not available
in purified form or when it has yet to be characterized at
all, in other words in the search for new cell markers. For
example, the majority of antigens expressed on the surface
of human red blood cells are defined only serologically
and are not available in purified form. Cell panning of
combinatorial
antibody libraries derived from auto- and
alloimunized
individuals
has been used to select for
these antigens [18]. Moreover, cell panning has become
important for the selection of antibodies with therapeutic
potential. In contrast to panning on purified immobilized
antigen, cell panning selects for antibodies that are more
likely to bind to epitopes accessible in &XX In addition,
as a result of homophilic or heterophilic
interactions
of
the antigen on the cell surface, selection is focused on
biologically relevant epitopes. This has been suggested
for the HIV-l envelope glycoprotein
gp120. Oligomeric
gp120, as presented
on the HIV-l virion or an HIV-l
infected cell, was found to select HIV-l neutralizing
antibodies
from a phage library much more efficiently
than monomeric gpl20 [36*]. In fact, Parren et al. [36*]
suggested that the ability of antigen preparations to select
The selection of antibodies
displayed
an important methodology
diagnostic,
from combinatorial
on the surface of filamentous
for the generation
and therapeutic
natural immune responses.
molecules
libraries
phage has become
of reagent,
and for the study of
Using this technique,
genes have been cloned from multiple species
directly from large man-made
repertoires
genes. Recent studies demonstrate
for the in vitro evolution of antibodies
antibody
or expressed
of antibody-encoding
that the technique
allows
to create molecules
whose affinity for antigen exceeds that observed
in nature.
Addresses
The Skaggs institute for Chemical Biology and the Department of
Molecular Biology, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, California 92037, USA
*e-mail: carlos@scripps.edu
Current Opinion in Biotechnology 1997, 8:503-508
http://biomednet.com/elecref/0958186900800503
0 Current Biology Ltd ISSN 0958-l 669
The basic principles
of the filamentous
phage display
technique
are illustrated
in Figure 1. A large library
of phage is prepared
following the cloning of genes
encoding
antibody
heavy and light chain fragments;
because heavy and light chains are recombined randomly,
we talk about combinatorial antibody libraries. Each phage
has the potential to display a unique antibody with a
specific antigen binding site on its surface, which is the
selectable phenotype
of interest. Within the phage coat
is the genotype
that encodes the displayed molecule.
This linkage
of displayed
antibody
phenotype
with
strategies:
cell panning and beyond
504
Protein engineering
Figure 1
(a)
V
V
Helper
phage
Male E. coli
Xba I Xho I
Sfi I
Sfi I
Gene
Ill fragment
trP
Sfi I
Fab or scFvsfi
Vectors
designed
for the expression
of antibody
fragments
I
0 1997 Current Opinion in Biotechnology
on the phage
surface
or in soluble
form. Typically,
phagemid
vectors
have
been utilized for the display of combinatorial
antibody libraries on the surface of filamentous
phage. Antibody fragments are fused to the
carboxy-terminal
domain of the minor coat protein of filamentous
phage, the gene Ill protein, which is displayed in low copy number at one
end of the phage. Whereas the amino-terminal
domain of the gene III protein binds to the F’ pili, allowing for infection of male E. coli, the
membrane-bound
carboxy-terminal
domain
is important
for phage
promoter drives the synthesis of a dicistronic
transcript.
translation of two separate polypeptide
chains encoded
a heavy chain
(VR-C&l)
fused
to the carboxy-terminal
assembly.
(a) In the Fab display version
of pComb3H
161 a single
IacZ
Two ribosome binding sites (Shine and Dalgarno sequences;
SD) give rise to the
sequentially.
One encodes a complete light chain (VL-CL), the other the Fd fragment
domain
of the gene Ill protein.
The gene Ill fragment
is followed
of
by the frp transcriptional
terminator. To generate a combinatorial
antibody library, light chain and Fd fragment encoding sequences
are amplified independently
and
combined randomly in pComb3H.
The Sacl and Xbal restriction enzyme sites are provided for cloning light chain encoding sequences.
The Xhol
and Spel sites are provided for cloning Fd fragment encoding sequences.
Alternatively,
the sequences
encoding light chain and Fd fragment can
be fused
by overlap
extension
PCR and cloned
directionally
using two asymmetric
sites of the rare eightbase
cutter
Sfi I. The leader peptides
ompA and pelB target both polypeptides
to the periplasm of E. coli, where the soluble light chain and the membrane-bound
Fd fragment
associate via a disulfide bond. The transformation
of male E. coli with Fab encoding pComb3H
and the subsequent
addition of helper phage
leads to the production
of phage that, as their phenotype,
display one copy of Fab fragment linked to the phage surface by the gene Ill protein
fragment and which contain the corresponding
single-stranded
phagemid as their genotype. (b) In the scFv display version of pComb3H
the
variable immunoglobulin
domains of light chain (Vt) and heavy chain (V,), are fused by a peptide linker (L). While a long, flexible linker allows
for the monovalent
scFv display, a shorter linker leads to the formation of scFv dimers (diabodies) with two antigen-binding
sites. (C) Digestion of
pComb3H,
encoding a selected Fab or scFv, with Spel and Nhel and self ligation removes the gene Ill fragment and allows for the production
of
soluble Fab or scFv. Alternatively,
Fab- or scfv-encoding
sequences
are generally subcloned
using the Sfil sites for high-level expression into the
compatible
expression vector pPhoA-HGHA
[6], which adds a histidine tag (H6) to the amino terminus and a hemagglutinin
decapeptide
(HA) to
the carboxyl
terminus
of the expressed
antibody
fragment
for detection
and purification.
Phage display of combinatorial antibody libraries
neutralizing antibodies from phage libraries could be used
as a measure of the quality of the antigen as a vaccine.
Taking a great step forward, Pasqualini
and Ruoslahti
[37**] developed a methodology
for the in vtio panning
of phage displayed peptide libraries in order to select
peptides that home to target organs. Phage were injected
in the tail vein of mice, allowed to circulate for a few
minutes, and were isolated and reamplified from brain and
kidney tissue after the mice had been snap frozen in liquid
nitrogen. After three rounds of in &JO panning, peptide
sequences that selectively target brain and kidney were
identified. Because the phage were allowed to circulate for
but a few minutes, it is unlikely that they left the blood
vessels; therefore, the peptides were assumed to bind cell
surface molecules selectively expressed on the endothelial
cells of brain and kidney [37”]. In principle,
in vjvo
panning should be feasible using phage displayed antibody
libraries too. Selective targeting of endothelial
markers
by peptides or antibodies promises applications in gene
and cancer therapy. Cell surface molecules selectively
expressed on mature or angiogenic endothelial cells that
form the blood vessels of tumors have become a promising
target for cancer therapy [38]. Only a few molecular
markers of tumor blood vessels are known, however,
making in viva panning of phage displayed peptide and
antibody libraries a promising too1 for their identification.
Peptides and antibodies selected by tumor targeting will
find many diagnostic and therapeutic applications.
Antibodies
to clinically relevant targets
Since their first application to cloning antibody segments
involved in the human immune response by the laboratories of Barbas, Burton, and Lerner, phage antibodies
have become an important resource for the development
of therapeutic
antibodies
and the study of the human
antibody repertoire as it relates to disease. Antibodies have
been selected to bind the classical therapeutic
targets,
tumor antigens [9,25’,26*,32] and viral antigens (reviewed
in [7]; [8,15,17’,21*]). Furthermore,
this methodology
has facilitated studies of human immune responses and
autoimmunity
[10,11,13,14,16,19*].
The first human IgE antibodies with known specificity
have now been isolated from phage displayed antibody
libraries [ZO”]. IgE Fabs specific for a grass pollen allergen
were selected by screening a phage displayed IgE Fab
library derived from peripheral blood lymphocytes
of an
allergic individual.
By interfering
with the binding of
IgE polyclonal antibodies to allergens, IgE Fab selected
from phage displayed
antibody
libraries may become
a therapeutic
tool for the treatment
of allergies [ZO**].
Another antibody directed to a clinically relevant target
that became accessible only through phage display is
one directed to the human platelet alloantigen
1 located
on integrin
aIIbB3 [12]. Human platelet alloantigen
1
can be encoded
by two alleles that differ in one
nucleotide.
Due to this difference, 98% of the Caucasian
Rader and Barbas
505
population have a leucine at position 33 of cxI&, while
2% have a proline at this position. Pro33 homozygotes
are at risk of producing Leu33-specific
antibodies
after
transfusion
or during pregnancy. Monoclonal antibodies
directed to native O(II& Leu33 would be an important
tool for phenotyping
and therapeutic
applications,
but
have not been obtained by hybridoma technology. Griffin
and Ouwehand
[12] have now selected an anti-o&B3
Leu33 human scFv by screening a phage displayed scFv
library derived from peripheral blood lymphocytes
of an
alloimmunized
pregnant
individual.
As shown by flow
cytometry, the isolated scFv selectively binds to platelets
expressing ~I&
LeU33.
A particularly interesting
report details the generation of
an antibody that selectively
binds to a peptide-MHC
complex [23”]. For this, a phage displayed Fab library was
generated from spleens of mice that had been immunized
with a peptide-MHC
complex consisting of an influenza
virus derived hemagglutinin
(HA) peptide and the mouse
MHC class I molecule Kk. To eliminate unspecific binders
efficiently the library was panned on cells that displayed
HA-Kk, alternating with panning on HA-Kk-coated microspheres. One of the selected Fabs, Fab13.4.1, bound to
HA-Kk with an affinity of about 50nM. Neither isolated
HA, isolated Kk, nor a complex between Kk and a different
peptide was bound by Fab13.4.1. Thus, Fab13.4.1 binds a
particular peptide-MHC
complex selectively, an activity
reserved to T-cell receptors in viva. Antibodies
specific
for peptide-MHC
complexes will be important diagnostic
and therapeutic
tools: the contents of cells containing
intracellular
parasites or aberrant proteins related to a
cancer can now be seen with a soluble antibody and,
if desired, the cell can be targeted for destruction
by
antibody effector functions or tethered toxins.
Directed evolution
of antibody affinity
It is generally assumed that the ability to create antibodies
of higher affinity will provide therapeutic and diagnostic
benefits. Phage display provides a format for the directed
molecular
evolution
of antibody
affinity (reviewed
in
[7]). While low affinity antibodies are readily improved
with respect to affinity, antibodies
that already possess
nanomolar binding of their antigen require more subtle
refinements
to improve their already high affinity [7].
Several strategies that have been recently explored are
saturation mutagenesis
of complementarity
determining
regions (CDR) (CDR walking mutagenesis) [6,39”,40,41],
chain shuffling [41-44], and the use of mutator strains
of Eschric/ria co/i [45*], which provide mutations in r&o
akin to error-prone PCR in vim. There appears to be
consensus
that the CDR walking mutagenesis
strategy
is the most reliable of affinity maturation methodologies
yet devised [6,39”]. This approach has been applied to
human anti-HIV [6] and anti-c-erbB-2
[39*.] antibodies to
create antibodies with monovalent picomolar affinities not
found in nature. In this approach, saturation mutagenesis
of a CDR is constrained
to libraries that examine all
506
Protein engineering
possible amino acids in the target CDR. Two strategies
are evident, either sequential or parallel optimization
of
CDR. In the sequential approach, the library of antibodies
with a single randomized
CDR is screened by several
rounds of panning
against the antigen. The selected
clone(s) are then used in the construction
of a second
library where a different
single CDR is randomized.
The resulting library is screened by additional
rounds
of panning, and the selected clone(s) are used in the
construction
of a third library and so on. Sequential
optimization takes into account that optimal binding may
result from the interdependence
of CDRs. In the parallel
approach, independent
libraries are constructed
where
each library represents the randomization of a given CDR.
Each library of antibodies is screened by several rounds
of panning against the antigen. Then, the individually
optimized CDRs are combined. If the free energy change
of individually optimized CDRs combined is nearly equal
to the sum of the free energy changes observed in the
single optimized CDRs, the free energy changes are said
to be additive. As additivity within the antibody binding
site is virtually impossible
to predict, sequential
CDR
optimization is preferred over parallel. To date the greatest
gains in affinity have resulted from the optimization
of
heavy chain CDR3 and light chain CDR3 regions, but
there are sure to be many exceptions.
An advantage
of this strategy over PCR or mutator strain induced
mutagenesis is that modifications to the parental antibody
are constrained to the hypervariable regions. It is expected
that changes in the primary sequence in these regions
are less likely to generate immunogenic
antibodies than
changes in the more sequence
constrained
framework
regions.
Acknowledgements
Conclusions
Methodologies
for the creation and selection of combinatorial antibody libraries on the surface of phage continue
to be improved and extended.
Antibodies
have been
selected to molecules
both large and small to study
protein recognition, immunity, and problems in basic and
applied sciences. While it was anticipated
that in viva
recombination
of heavy and light chain sequences based
on Cre/lox strategies [46] would allow for the creation
of libraries that exceed 1012 molecules, in practice this
approach has yet to demonstrate
its potential because of
severe problems with the genetic stability of the vectors.
These problems are not, however, insurmountable.
Other
interesting strategies are evolving that enable the cloning
of genes encoding both antibodies
and their antigens
using phage display. For example, Krebber et a/. [47]
investigated
a phage display system with the potential
to examine library versus library interactions.
It may be
possible in the future to clone a cDNA together with a
specific antibody which binds it. There is still hope that
phage display of combinatorial
antibody libraries “will be
the component of a machine that, before the turn of the
century, will evolve proteins on demand to meet new
challenges in biotechnology”
[4].
Christoph Rader was supported
by the Swiss National Science Foundation
and Carlos F Barbas III by grants from the National Institute of Health and
an Investigator Award from the Cancer Research Institute.
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with a synthetic peptide. An immunotoxin produced by fusing the selected
scFv to Pseudomonas
exotoxin A revealed good affinity, cytotoxicity, and
stability.
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26.
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