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J Biol Chem, Vol. 274, Issue 26, 18218-18230, June 25, 1999
From We report the design, construction, and use of
the first very large non-immunized phage antibody library in Fab
format, which allows the rapid isolation and affinity analysis of
antigen-specific human antibody fragments. Individually cloned heavy
and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of
3.7 × 1010 independent Fab clones. The
performance of the library was determined by the successful selection
of on average 14 different Fabs against 6 antigens tested. These
include tetanus toxoid, the hapten phenyl-oxazolone, the breast
cancer-associated MUC1 antigen, and three highly related glycoprotein
hormones: human chorionic gonadotropin, human luteinizing hormone, and
human follicle-stimulating hormone. In the latter category, a panel of
either homone-specific or cross-reactive antibodies were identified.
The design of the library permits the monitoring of selections with
polyclonal phage preparations and to carry out large scale screening of
antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies
with off-rates in the order of 10 Display on filamentous phage in combination with selection forms a
powerful tool for the identification of peptide- or protein-based drugs
(1, 2). Of these, antibodies are especially of interest, due to their
capacity to recognize a variety of targets with high specificity and
affinity. In particular, the use of partially or completely human
antibodies, which elicit no or minimal immune response when
administered to patients, is yielding an increasing list of
FDA-approved protein-based drugs (3). Phage display technology enables
the generation of large repertoires of human antibodies (4-7), while
the biopanning procedure permits the selection of individual antibodies
with a desired specificity.
Key to the success of the technology were two critical observations:
(i) the expression of functional antibody fragments by secretion into
the periplasm of Escherichia coli (8, 9), and (ii) the rapid
access to variable region gene pools by the polymerase chain reaction
(10-12). For the construction of antibody libraries, V-genes are
amplified from B cell cDNA and heavy and light chain genes are
randomly combined and cloned to encode a combinatorial library of
single-chain Fv (scFv)1 or
Fab antibody fragments (4, 13-15). The natural primary (unselected) antibody repertoire within B cells contains a large array of antibodies that recognize a variety of antigens; this array can be cloned as a
"naïve" repertoire of rearranged genes, by harvesting the V-genes from the IgM mRNA of B cells of unimmunized human donors, isolated from peripheral blood lymphocytes (4), from bone marrow or
tonsils (7), or from similar animal sources (16). This procedure
provides access to antibodies that have not yet encountered antigen,
although the frequency of those genuine "germline" antibodies will
depend heavily on the source of B cells (17). A single naïve
library, if sufficiently large and diverse, can indeed be used to
generate antibodies to a large panel of antigens, including self,
non-immunogenic and relatively toxic antigens (4, 6). In a different
approach, antibodies may be built artificially, by in vitro
assembly of V-gene segments and D/J segments, yielding "synthetic"
antibodies (5). A major drawback of these procedures is that from the
initial naïve and synthetic libraries, only moderate affinity
antibodies were isolated (4, 18). Over the last few years, more
efficient techniques have been developed to build larger libraries of
antibody fragments, using sophisticated in vivo
recombination methods (6) or brute force cloning procedures (7, 19).
Such large libraries have yielded a greater number of human antibodies
per antigen tested, with on average much higher affinity (up to
subnanomolar). However, technical restrictions on the size of libraries
that may be obtained or handled in selection, the loss of library
diversity upon library amplification, and the relatively long
downstream analysis path of the selected antibodies, i.e.
large scale affinity analysis, have limited the spread of these
libraries as generic tools in antibody generation.
We describe here the generation of a very large antibody library based
on the display of Fab fragments on phage. The choice for the Fab format
was based on the notion that the monomeric appearance of the Fab should
permit the rapid screening of large numbers of clones on kinetics of
binding (off-rate) with crude protein fractions. Most large libraries
made to date use the single-chain format (scFv) for display on phage
(7, 19), but these fragments have the tendency to form dimers and
higher order multimers in a clone-dependent and relatively
unpredictable way (20-22). Multimeric antibody molecules bind more
strongly to immobilized antigen than monomeric fragments because of
their greater avidity, and therefore have higher "apparent"
affinities (23). This explains why an accurate determination of the
affinity is not easily possible with mixtures of mono- and multimeric
scFv fragments. As a consequence, the affinity assay used for ranking
individual clones (such as BIAcore analysis) often necessitates
time-consuming purification to obtain the monomeric fraction of the
selected antibody fragments (19, 24). An additional argument for the
choice of the Fab format is to avoid possible problems with avidity of
the displayed antibody fragment on the phage itself. The tendency of
Fabs to be expressed at lower levels than scFv fragments and the lack of multimerization will lead to a lower display frequency and lower
fraction of avid phage. The effect of multimerization of scFv on phage
with respect to the selection of fragments with very low affinities has
indeed been observed previously (6). Therefore, compared with scFv
libraries, selections with Fab phage may be more governed by affinity
rather than avidity, even when performing selections by panning on
immobilized antigen (24) or with soluble multivalent antigen (25).
This report describes the strategy for the construction of a very large
antibody library. An efficient cloning method, in which restriction
fragments instead of PCR products were used, made it possible to
generate a repertoire with as many as 37 billion different Fab clones.
The performance of the library was analyzed by the selection with an
extended panel of antigens including three closely related glycoprotein
hormones, yielding a diverse set of specific antibody fragments for
each antigen. Without using sophisticated selection protocols,
hormone-specific as well as cross-reactive Fabs were retrieved against
the highly homologous glycohormones, demonstrating that the library is
a rich source of antibody specificities. The affinities of the
anti-glycohormone antibodies varied between 2.7 and 38 nM.
Finally, the Fab format indeed permitted the rapid screening and a
reliable ranking of individual clones on off-rate using crude antibody fractions.
RNA Isolation--
As source of lymphoid tissues, we used
peripheral blood lymphocytes from 4 healthy donors and part of a
tumor-free spleen removed from a patient with gastric carcinoma. B
lymphocytes were isolated from 2 liters of blood on a Ficoll-Pacque
gradient. For RNA isolation, the cell pellet was immediately dissolved
in 50 ml of 8 M guanidinium thiocyanate, 0.1 M
2-mercaptoethanol (26). Chromosomal DNA was sheared to completion by
passing through a narrow syringe (1.2/0.5-mm gauge), and insoluble
debris was removed by low speed centrifugation (15 min at 2,934 × g at room temperature). RNA was pelleted by centrifugation
through a CsCl block gradient (12 ml of supernatant on a layer of 3.5 ml of 5.7 M CsCl, 0.1 M EDTA; in total four
tubes) during 20 h at 125,000 × g at 20 °C in
a SW41 rotor (Beckman). The yield of total RNA was approximately 600 µg. RNA was stored at
From the spleen, 2 g of tissue was used for homogenization with a
Polytron homogenizer in 20 ml of 8 M guanidinium
thiocyanate, 0.1 M 2-mercaptoethanol. The total volume was
increased to 80 ml with guanidinium thiocyanate buffer, and after
passage through a narrow syringe for shearing and removal of debris,
RNA was pelleted as described before, except for 15 h at
85,000 × g at 20 °C in a SW28.1 rotor (12 ml of
supernatant on 3.5 ml of 5.7 M CsCl, 0.1 M EDTA
in five SW28.1 tubes). From 2 g of tissue, 3 mg of total RNA was extracted.
Amplification of Variable Region Genes--
Random primed
cDNA was prepared with 250 µg of PBL RNA, while in a separate
reaction 300 µg of spleen RNA was used as template. RNA was
heat-denatured for 5 min at 65 °C in the presence of 20 µg of
random primer (Promega); subsequently, buffer and dithiothreitol were
added according to the supplier's instructions (Life Technologies, Inc.), as well as 250 µM dNTP (Amersham Pharmacia
Biotech), 800 units of RNasin (40 units/µl; Promega), and 2,000 units
of Moloney murine leukemia virus reverse transcriptase (200 units/µl;
Life Technologies, Inc.) in a total volume of 500 µl. After 2 h
at 42 °C, the incubation was stopped by a phenol/chloroform
extraction; cDNA was precipitated and dissolved in 85 µl of water.
Oligonucleotides used for PCR amplification of human heavy and light
chain V-regions are described in Table I. IgM-derived heavy chain
variable regions were obtained by a primary PCR with an IgM constant
region primer. All primary PCRs were carried out with separate BACK
primers and combined FOR primers, to maintain maximal diversity. The
PCR products were reamplified with a combination of JHFOR primers,
annealing to the 3' end of VH, and Sfi-tagged VHBACK
primers, annealing to the 5' end, and subsequently cloned as VH
fragments. The light chain V-genes of the
PCR was performed in a volume of 50 µl using AmpliTaq polymerase
(Cetus) and 500 pM of each primer for 28 cycles (1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C); nine separate
IgM-derived VH amplifications were generated with 2 µl of
random-primed cDNA (equivalent to 6 µg of PBL RNA or to 7 µg of
spleen RNA) as template for each reaction. For the light chain
families, 6 different V Construction of the Primary and Secondary Repertoires--
For
the construction of the primary heavy chain and the two primary light
chain repertoires, the PCR products, appended with restriction sites,
were gel-purified prior to digestion and the different VH, V
The Fab library was obtained by cloning of VH fragments, digested from
plasmid DNA prepared from the heavy chain repertoires, into the plasmid
collection containing the light chain repertoires. Plasmid DNA,
isolated from at least 3 × 109 bacteria of the VH
library, was digested with SfiI and BstEII for
cloning in the vector that already contained Selection of the Library--
The rescue of phagemid particles
with helper phage M13-KO7 was performed according to (4) on a 10-liter
scale, using representative numbers of bacteria from the library for
inoculation, to ensure the presence of at least 10 bacteria from each
clone in the start inoculum. For selections, 1013
colony-forming units were used with antigens immobilized in immunotubes (Maxisorp tubes, Nunc) (4) or with soluble biotinylated antigens (28).
The amount of the immobilized antigens tetanus toxoid and the hapten
phenyl-oxazolone (conjugated to BSA in a ratio of 17 to 1) was reduced
10-fold during subsequent selection rounds, starting at 100 µg/ml at
round 1. Capture with biotinylated antigen in solution was used for a
100-mer peptide encoding five copies of the tandem repeat of MUC1 (29),
or with human chorionic gonadotropin (hCG), human luteinizing hormone
(hLH), human follicle-stimulating hormone (hFSH) and its chimeric
derivative (hFSH-CTP, containing the carboxyl-terminal peptide from the
hCG Screening and Sequencing of Clones--
Soluble Fab was produced
from individual clones as described before (4). Culture supernatants
were tested in ELISA with directly coated antigen or indirectly
captured biotinylated antigen via immobilized biotinylated
BSA-streptavidin. Tetanus toxoid and phOx-BSA were coated at 10 µg/ml
in 0.1 M NaHCO3, pH 9.6, for 16 h at
4 °C. For coating of hCG and hFSH-CTP, a concentration of 4 µg/ml
in 50 mM NaHCO3, pH 9.6, was used. For capture
of biotinylated antigens, biotinylated BSA was coated at 2 µg/ml in
PBS during 1 h at 37 °C. After 3 washes with PBS, 0.1% (v/v)
Tween 20, plates were incubated during 1 h with streptavidin (10 µg/ml in PBS/0.5% gelatin) (30). Following washing as above,
biotinylated antigen was added for an overnight incubation at 4 °C
at a concentration of 0.5 µg/ml for MUC-1 peptide, 3 µg/ml for hLH,
and 0.6 µg/ml for hFSH (binding to hCG was tested with directly
coated antigen). The plates were blocked during 30 min at room
temperature with 2% (w/v) semi-skim milk powder (Marvel) in PBS. The
culture supernatant was diluted 1- or 5-fold in 2% (w/v) Marvel/PBS
and incubated 2 h; bound Fab was detected with anti-myc
antibody 9E10 (5 µg/ml) recognizing the myc-peptide tag at
the carboxyl terminus of the heavy Fd chain, and rabbit anti-mouse-HRP
conjugate (Dako) (4). Following the last incubation, staining was
performed with tetramethylbenzidine and H2O2 as
substrate and stopped by adding 0.5 volume of 2 N H2SO4; the optical density was measured at 450 nm. Clones giving a positive signal in ELISA (over 2 times the
background), were analyzed by BstNI fingerprinting of the
PCR products obtained by amplification with the oligonucleotide primers
M13-reverse and geneIII-forward (4).
Large scale induction of soluble Fab fragments from individual clones
was performed on a 50-ml scale in 2× TY containing 100 µg/ml
ampicillin and 2% glucose. After growth at 37 °C to an
OD600 of 0.9, the cells were pelleted (10 min at 2,934 × g) and resuspended in 2× TY with ampicillin and 1 mM isopropyl-1-thio-
For sequencing, plasmid DNA was prepared from 50-ml cultures grown at
30 °C in LB-medium, containing 100 µg/ml ampicillin and 2%
glucose, using the Qiagen Midi-kit (Qiagen). Sequencing was performed
with the thermocycling kit (Amersham Pharmacia Biotech) with
CY5-labeled primers CH1FOR (5'-GTC CTT GAC CAG GCA GCC CAG GGC-3') and
M13REV (5'-CAG GAA ACA GCT ATG AC-3'); samples were run on the
ALF-Express (Amersham Pharmacia Biotech). V-gene sequences were aligned
to V-BASE or the Sanger
Center.2
Determination of Fine Specificities of the Anti-hormone Fabs by
Western Blot and Surface Plasmon Resonance--
An hCG preparation
purified from urine and immuno-affinity-purified recombinant hLH, hFSH,
and hFSH-CTP produced in CHO cells (30, 31) were used for Western blot
studies as described elsewhere (32). Between 0.5 and 1 µg of each
hormone was loaded per lane; proteins were diluted in non-reducing
sample buffer and boiled during 5 min or directly applied on gel
without heat treatment; proteins were transferred to blotting membrane
by electrotransfer. Blots were subsequently incubated for 16 h at
room temperature with a 10-fold diluted periplasmic fraction in PBS,
4% Marvel. Bound Fab was detected with anti-myc antibody
9E10 (5 µg/ml) and 4,000-fold diluted anti-mouse alkaline
phosphatase-conjugate (Promega), using the substrates
5-bromo-1-chloro-3-indolyl phosphate and nitro blue tetrazolium (Roche
Molecular Biochemicals) for visualization.
The specificity of the Fabs was further characterized by surface
plasmon resonance (BIAcore 2000, Biacore). Recombinant hLH, hFSH, and
the urinary hCG were immobilized on the flow cells of a CM chip using
the NHS/EDC kit (Biacore AB, Uppsala, Sweden), yielding a surface of
1906 RU for hLH, 1529 RU for hFSH, and 1375 RU for hCG. Periplasmic
fractions were diluted 3-fold in Hepes-buffered saline (10 mM Hepes, 3.4 mM EDTA, 150 mM NaCl,
0.05% (v/v) surfactant P20, pH 7.4) and analyzed using a flow rate of
10 µl/min.
Purification of Soluble Fab Fragments--
Fabs were obtained by
refolding of the total bacterial proteins from a 50-ml culture (33).
Briefly, the pelleted cells from a 50-ml induced bacterial culture were
resuspended in 8 ml of 8 M urea (in PBS). After sonication,
the mixture was rotated head over head for 30 min and insoluble
material was removed by centrifugation for 30 min at 13,000 × g. The supernatant was dialyzed against PBS with four buffer
changes. Insoluble proteins were removed by centrifugation and the
flow-through fraction, obtained by filtration through a 0.2-µm
membrane, was immediately loaded on an hCG column (bed volume 0.3 ml).
The column material was prepared by coupling 8.4 mg of protein to
1 g of Tresyl-Sepharose according to the supplier's instructions
(Pierce). The column (1 ml column material) was washed with 10 volumes
of 100 mM Tris, 500 mM NaCl, pH 7.5; then
subsequently with 10 volumes of 100 mM Tris, 500 mM NaCl, pH 9.5; and 2 volumes of 0.9% NaCl. Bound Fab was
eluted in a batchwise fashion with 2 volumes of 0.1 M
triethylamine. After a 10-min incubation, the effluent was collected
and immediately neutralized with 0.5 volume of 1 M Tris, pH
7.5. The Fab fraction was dialyzed against PBS using a Microcon 30 spin
dialysis filter (Amicon). Finally, a gel filtration analysis was
carried out on a Superdex 75HR column (Amersham Pharmacia Biotech). The
yield was determined by measuring the optical density at 280 nm (using a molar extinction coefficient of 13 for Fabs).
Determination of On- and Off-rate Using Surface Plasmon Resonance
with Crude Fab Preparations--
The kinetics of binding were analyzed
by surface plasmon resonance on three different hCG surfaces (303, 615, and 767 RU immobilized, with 4955 RU of BSA on a separate flow cell as
a negative control). Obviously, ranking of the off-rates of the
individual clones needs to be done by analysis with the BIAevaluation
software. Fab present in crude periplasmic extracts was quantified on a
high density surface of purified anti-human Fab polyclonal antibody
(Pierce) as described (34). Anti-hCG Fabs controls were purified by
affinity chromatography on hCG columns as described above and used to
calibrate the system.
Design of the Non-immunized Phage Antibody Library--
We
considered a number of variables to address in the construction of a
novel, very large phage antibody library: (i) the primer design was
optimized for amplification of variable gene pools to maintain maximum
diversity; (ii) a highly efficient two-step cloning method was
developed to obtain a very large non-immunized library; (iii) an
antibody format and compatible cloning vector were chosen, which should
permit the rapid downstream analysis of selected clones.
In order to achieve access to as many different human heavy and light
chain V-region gene segments as possible, a new set of oligonucleotide
primers was developed (Table I), the
design of which was based on the most recent sequence information
provided by the V-base (see "Materials and Methods"). The primers
should allow efficient amplification of all commonly used V-gene
segments. Further, to obtain large sized libraries (over
1010 in diversity), we used a two-step cloning procedure;
heavy and light chain variable genes were first separately cloned as
digested PCR products, and then combined by restriction fragment
cloning to form a large library of Fab fragments. This cloning
procedure should be a more efficient route for library construction
than the relatively inefficient direct cloning of digested PCR
products, while avoiding the DNA instability often associated with
in vivo recombination systems (35).
As choice of antibody format, we preferred the Fab above the scFv
format, because this would allow us to develop rapid high through-put
affinity-screening assays for crude antibody preparations. Many scFv
fragments indeed form higher molecular weight species including dimers
(20, 21) and trimers (36), which complicate both selection and
characterization. We chose the Fab display format, in which the heavy
chain is linked to the phage coat protein pIII, and also carries a tag
for detection and purification (see below). The light chain is
expressed as separate fragment, secreted into the periplasm, where it
can pair with the heavy chain (37).
To incorporate all these improvements, a new phagemid vector, pCES1
(Fig. 1), was constructed, which allows
the stepwise cloning of antibody fragments in Fab format. In this
vector system, the variable heavy chain region genes are cloned as
VH-gene fragments; the vector supplies all Fabs with a human gamma-1
CH1 gene. The Fd fragment is fused to two tags for purification and
detection (a histidine tail for immobilized metal affinity
chromatography (38) and a c-myc-derived tag (39)), followed
by an amber stop codon (37) and the minor coat protein III of
filamentous phage fd. The antibody light chain is cloned as full VLCL
fragment, for directed secretion and assembly with the VHCH1 on the
phage particle.
Library Construction--
The Fab library was constructed in two
steps. In the first step, variable region gene pools were amplified
from approximately 4 × 108 B cells from the PBLs of
four healthy donors, and, as a source of possibly more heavily mutated
IgM antibodies, from a segment of a (tumor-free) spleen removed from a
patient with gastric carcinoma, containing approximately 1.5 × 108 B cells (40). Only IgM-derived VH segments were
amplified by using an amplification with an oligonucleotide primer
located in the first constant domain of this isotype. These products
were cloned into phagemid vector pCES1 for VL, and in pUC119-CES1 for VH (cloning was more efficiently in the smaller sized vector, in which
gene III was deleted). The PBL- and spleen-derived VH, V Quality Control of the Library by Selection with a Panel of
Antigens--
We evaluated the library by selection with different
antigens, the screening data of which are summarized in Table
III. First, the results from three model
antigens, the protein tetanus toxoid, the hapten 2-phenyloxazol-5-one
(phOx) (41), and the peptide MUC1, are discussed. Three rounds of
biopanning on tetanus toxoid yielded a diverse set of ELISA-positive
Fabs; in a series of 47 tetanus toxoid binding Fabs, at least 21 were
different with regard to BstNI fingerprint. Similarly, an
extensive panel of phOx-specific Fabs was retrieved after three rounds
of panning; at least 24 different clones were identified in a series of
50 ELISA-positive clones. Solution capture with biotinylated MUC1
peptide resulted in the selection of 14 different antibody fragments
out of 37 ELISA-positive clones selected after 3 rounds (Table
III).
Rapid Dissociation Rate Determination--
With such large panels
of antibodies isolated, it is crucial to have methods available to
readily determine the kinetic parameters of each individual
antibody-antigen interaction. Such an assay should be robust and
ideally employ non-purified antibody fragments. We tested whether it
would be feasible to use periplasmic fractions prepared from small
scale cultures for a rapid and accurate determination of the off-rate
of the antibodies using surface plasmon resonance. An example of an
overlay plot with the sensorgrams from a series of tetanus
toxoid-specific Fabs is shown in Fig. 2.
The plot of ln(R0/R)
versus time (Fig. 2, lower graphs)
reveals a linear relation with slope kd
(off-rate), thereby confirming a monophasic dissociation, which can be
expected for a truly monomeric Fab fragment binding to a low density
antigen surface. At the beginning of the dissociation phase, the
relation is not linear due to a difference in composition of the
BIAcore running buffer (Hepes-buffered saline) and the buffer solution
of the Fab samples (phosphate-buffered saline); this may be avoided by
pre-dialysis. Using this off-rate screening assay, we determined the
off-rates for the best tetanus toxoid- and MUC1-specific Fabs to be in
the order of 10 Selection of Fab Antibodies against Related Glycoprotein
Hormones--
As a more stringent test panel of antigens to assay the
performance of the library, we chose to derive antibodies to three structurally related glycoproteins: hCG, hLH, and hFSH (reviewed in
Ref. 42). These hormones are heterodimers sharing an identical
Selections were thus performed on biotinylated urinary hCG, recombinant
hLH, hFSH, and hFSH-CTP (the latter is a chimeric molecule containing
the carboxyl-terminal peptide of Specificity Analysis of the Selected Monoclonal Fabs--
ELISA of
monoclonal phage antibodies revealed that three rounds of selection
with hCG indeed resulted in the isolation of a high percentage (74%)
of clones positive for the gonadotropin. 27% of these clones were
hLH-cross-reactive; none were reactive against streptavidin.
BstNI fingerprint analysis of the ELISA-positive clones
revealed a high degree of diversity (8 different patterns). From a
representative hCG-specific (coded CG#4F) and hLH-cross-reactive (CG#5C) clone, the specificity was tested in BIAcore using unpurified soluble Fab fragments (Fig. 4). Clone
CG#4F gave a high response on hCG, with no visible binding to either
hLH or hFSH-CTP. In contrast, clone CG#5C bound to hCG and hLH, but not
to hFSH-CTP. Western blots, with the different hormones in non-reduced
form, showed the specific recognition of the
Selection with the hormone hLH resulted in the isolation of
hLH-specific and hCG-cross-reactive clones. Examination of individual clones from selection round three in ELISA revealed a large fraction of
hLH-specific clones (69%), and a minor group of cross-reactive clones
(16%); no streptavidin-reactive clones were selected. Within the group
of specific clones, a large array of different species (>21) could be
discriminated by fingerprint analysis; however, all cross-reactive
species had a single pattern. The unique hLH specificity was confirmed
for representative clones LH#2H and LH#3G, shown in surface plasmon
resonance (shown for clone LH#3G in Fig. 4); and on Western blot
(illustrated for clone LH#3G in Fig. 5). This Fab only recognizes the
intact
When hFSH was used as antigen during selection, six different
antibodies were isolated from the library, with one type, represented by clone FS#8B, dominating the selected population. This Fab only recognized hFSH in BIAcore (Fig. 4), and, as Western blot analysis demonstrated, in particular its
Upon selection with FSH-CTP, seven different Phage-selected Antigen-specific Clones Are Intact Fab
Fragments--
There have been some reports on the isolation from scFv
or Fab libraries of antigen-specific single-domain or other artificial antibody fragments (47, 48). Therefore, we tested the integrity of the
selected Fabs. First, the nature of the Fab fragments in periplasmic
fractions was determined in Western blot. When incubated in
non-reducing sample buffer, two products were detected with the 9E10
antibody, which recognizes the myc-tag at the end of the CH1
domain (Fig. 6A); the major
product is the intact Fab molecule, in which an intermolecular
disulfide bridge covalently links heavy and light chain fragments; the
low molecular product is most likely derived from non disulfide bridge
linked heavy chains. Analysis with anti-light chain sera reveals a
similar pattern and shows that the clones use a nearly equal percentage of Use of Diverse Germline Sequences--
A panel of 14 antigen-specific Fabs was fully sequenced (Table
IV; 3 anti-MUC1 antibodies positive in
BIAcore on 100-mer peptide, and 11 anti-gonadotropin antibodies). The
heavy chain genes are derived from the four largest VH families (VH1,
VH3, VH4, and VH6); the VL genes belong to one of four V Measurement of Affinities with Purified Anti-hCG Fabs--
The
affinities and off-rates of affinity-purified hCG-reactive Fabs LH#1C,
SC#2B, LH#3F, and CG#5C were determined. The off-rates for most Fabs
were in the order of 10 This report describes the construction of a phage display library
from the in vivo rearranged V-gene repertoire of human
donors and its evaluation by selection with a panel of hapten and
protein antigens. The source of antibody producing B cells was twofold: peripheral blood lymphocytes, which are mainly IgM-positive cells, and
B cells from a human spleen. The theoretical diversity of a
combinatorial antibody library made from the PBLs of one donor is much
larger than what can be practically made or accessed (1014
combinations with 107 individual B cells). However, there
may be a strong bias in the diversity introduced because of the
donor's recent immune history and major differences in mRNA
contents and clonal outgrowth. Therefore, in previously constructed
very large non-immunized libraries, B cells from many different donors
were used (7). Most probably the repertoire will be limited in
diversity by using random priming because plasma cells (mostly of the
IgG type) will produce 10,000-fold more mRNA when compared with
non-activated B cells; a better source for non-immunized libraries are
IgM primed V-genes (4). We have successfully used B cells from only a
few donors (four for the PBLs and one spleen), but have aimed to access
a more diverse pool (spleen and PBL-IgM in this library,
versus tonsil and PBL random primed V-genes). In the PBL
pool of adults, over 60% of the B cells are unmutated IgM+IgD+
naïve B cells, while the remaining cells are memory cells that
have acquired somatic mutations (50). Since the latter class contains
more mRNA (17), most of the VH genes derived from cloning of this
pool are expected to carry mutations. Similarly the IgM pool in spleen
B cells will contain antibodies with mainly mildly mutated germline
genes. Indeed, many of the selected antibodies carry a low level of
mutations in the heavy chain genes. Some antibodies (i.e.
clone LH#3G) are nevertheless completely germline encoded and yet of
high affinity and specificity (similarly to what has been described for
some B cell-derived antibodies (51)). There may be sources of B cells that will yield a truly naïve V-gene repertoire (possibly, bone marrow-derived and/or IgM+IgD+CD27 We employed an efficient two-step cloning procedure with DNA fragments
digested from plasmid DNA instead of PCR fragments, to obtain the
largest non-immunized human Fab repertoire reported to date, with a
theoretical diversity of 37 billion different clones.
The choice of the Fab format was based on the possibility to develop
rapid affinity/kinetic screens. Most large libraries made to date use
the single-chain format for display on phage (7, 19). One report
described the use of a human non-immunized Fab library on phage (not
permitting immediate screening of selected soluble Fab fragments) (35).
scFv fragments have the tendency to form dimers and higher order
multimers in a clone-dependent and relatively unpredictable
way (20-22). As a consequence, the affinity assay used (such as
BIAcore analysis) often necessitates purification of the selected
antibody fragments. For example, ranking for off-rates using BIAcore is
not easily possible with unpurified scFv fragments; the monomeric
fraction of selected scFv clones first needs to be purified by affinity
chromatography and gel filtration (19, 24). Our data suggest that the
off-rate screening of individual Fab clones using non-purified
bacterial preparations yield data similar to the off-rates determined
with the purified Fab fragments. Therefore, provided sufficient Fab fragment is produced, the true monomeric appearance of Fabs allows a
rapid initial screen for off-rate. In combination with a concentration determination assay (which could also be carried out on BIAcore; Ref.
34), this should allow the rapid affinity determination of large series
of antigen-specific Fabs. The Fab format is therefore more amenable
then scFv to high throughput affinity screening, and should be the
preferred format when rapid affinity measurement is crucial
(e.g. during affinity maturation studies).
Most large libraries made to date use the single-chain format for
display on phage (7, 19), which does not easily allow the rapid
screening of large numbers of clones on kinetics of binding (off-rate)
with crude protein fractions. One report described a very large human
synthetic library with Fab fragments displayed on phage, which was
constructed with an in vivo recombination system to combine
separately cloned heavy (with completely synthetic CDR3 sequences) and
light chain repertoires (with few randomized CDR3 residues) (35).
Although the authors also used affinity-purified Fab fragments for
affinity measurements without further purification by gel filtration,
screening of individual clones had to be performed after recloning of
the selected Fabs for soluble expression. Clearly, this system does not
allow a rapid screening procedure, while the low percentage (28%) of
clones having both a heavy and a light chain after the recombination
event suggests instability of the library.
As was postulated and observed by Griffiths and colleagues (35), the
size of the antibody library dictates the probability of the selection
of high affinity antibodies to the antigen. Comparison of the first
non-immunized scFv repertoire containing 2.9 × 107
clones (4), with recently constructed scFv repertoires of approximately
1010 clones (7, 19), confirms this postulation; increasing
the library size 500-fold resulted in approximately 100-fold higher affinities. This increase is caused by lowering the off-rates from
10 The affinities of the selected antibody fragments are, however, very
dependent on the antigen used for selection. Sheets and colleagues
reported an affinity varying between 26 and 71 nM for the
selected scFv fragments specific for the anti-Clostridia
botulinum neurotoxin type A fragments, whereas for antibodies to
the extracellular domain of human ErbB-2, Kd values
between 0.22 and 4.03 nM were found (19). The affinities of
the gonadotropin-specific Fabs selected from our library varied between
2.7 and 38 nM, which is comparable to the protein binding
scFv fragments from the non-immunized library made by Vaughan et
al. (7) and Sheets et al. (19). It also approaches the
values of the best antibodies in their kind.6
The size of the library is not only important for affinity, it also
determines the success rate of selection of antibodies against a large
set of different antigens. In this respect the library performs very
well; over 24 antibodies to the hapten phOx, and on average 13 antibodies against the other antigens were selected. Furthermore, the
specificities of the antibodies obtained by selections on the
gonadotropins are unique; due to the high degree of homology between
hLH and hCG, it has been very difficult to isolate hCG-specific monoclonal antibodies with the hybridoma technology, whereas there are
very few hLH-specific antibodies (32, 42). Using a straightforward selection procedure, taking no precaution to avoid the selection of
cross-reactive Fabs, we have readily isolated fragments with all
possible specificities: Fabs specific for any of the three hormones
hCG, hLH, and hFSH, and cross-reactive Fabs recognizing the common
In the limited set of 14 clones that were sequenced, we identified
antibodies with variable region genes from all large V-gene families,
including VH1/3/4, V This new phage library will be a valuable source of antibodies to
essentially any target. To date, we have been able to select specific
antibodies to over 20 antigens tested. The antibodies may be used as
research reagents or as a starting point for the development of
therapeutic antibodies. As the list of sequenced genomes and
disease-related gene products is expanding rapidly, there will be a
growing need for an in vitro and eventually automated method
for antibody isolation. As antibodies have been and will be ideal
probes for investigating the nature, localization, and purification of
novel gene products, this library is envisaged to play an important
role in target validation and target discovery in the area of
functional genomics.
We thank colleagues at the Department of
Pathology, in particular Dr. R. Hoet and C. Petrarca for
discussions, and collaborators (Dr. E. Krambovitis) for materials.
*
This work was supported by European Community Biotechnology
Program 5.1 Grant BIO4CT950252 (to P. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Functional Biomolecules, Unilever
Research Laboratorium Vlaardingen, AC Vlaardingen, The Netherlands.
2
V-BASE is available via the World Wide Web
(Medical Research Council Center for Protein Engineering, 1997;
http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html); Sanger Center
is also available via the World Wide Web (Sanger Center Germline Query,
1997; http://www.sanger.ac.uk/Data Search/gq-search.html).
3
H. J. de Haard and B. Kazemier, unpublished results.
4
P. Henderikx, K. E. Tengbjerg, R. Hoet, C. Petrarca, E. van der Linden, A. de Bruïne, J. Zeuthen, J. W. Arends, and H. R. Hoogenboom, manuscript in preparation.
5
R. C. Roovers, E. van der Linden, A. de
Bruïne, J. W. Arends, D. C. Boerman, and H. R. Hoogenboom,
manuscript in preparation.
6
H. J. de Haard and B. Kazemier, unpublished results.
The abbreviations used are:
scFv, single-chain
Fv fragment;
PCR, polymerase chain reaction;
PBL, peripheral blood
lymphocyte;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
ELISA, enzyme-linked immunosorbent assay;
RU, resonance units;
hCG, human chorionic gonadotropin;
hLH, human luteinizing hormone;
hFSH, human follicle-stimulating hormone;
CTP, carboxyl-terminal
peptide.
A Large Non-immunized Human Fab Fragment Phage Library That
Permits Rapid Isolation and Kinetic Analysis of High Affinity
Antibodies*
§,,,¶
Target Quest B.V. and the ¶ Department of
Pathology, Maastricht University and University Hospital Maastricht,
P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 to 104
s1 and affinities up to 2.7 nM were
recovered. The kinetics of these phage antibodies are of the same order
of magnitude as antibodies associated with a secondary immune response.
This new phage antibody library is set to become a valuable source of
antibodies to many different targets, and to play a vital role in
target discovery and validation in the area of functional genomics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C in ethanol.
and 
families were
obtained by PCR with a set of CKFOR or CFOR primer annealing to the
3' end of the constant domain and BACK primers, priming at the 5' end
of the V-regions. The DNA segments were reamplified with primers tagged
with restriction sites and cloned as VC and VC fragments.
C products and 11 VC products (C2
and C7 primers combined in each reaction) were obtained. All
products were purified from agarose gel with the QIAex-II extraction
kit (Qiagen). As input for reamplification to introduce restriction
sites, 100-200 ng of purified DNA fragment was used as template in a
100-µl reaction volume. The large amount of input, ensuring the
maintenance of variability, was checked by analysis of 4 µl of the
"unamplified" PCR mixture on agarose gel.
, and
V families combined into three groups. The VC and VC
fragments were digested with ApaLI and AscI, and
cloned into the phagemid vector pCES1 (Fig. 1). The VH fragments, 1.5 µg in total, were digested with SfiI and BstEII
and ligated in a 100-200-µl reaction mixture with 9 units of
T4-DNA ligase at room temperature to 4 µg, gel-purified
vector pUC119-CES1 (similar to vector pCES1, but with the pIII gene
deleted). The desalted ligation mixture for light or heavy chain pools
was used for electroporation of the E. coli strain TG1, to
create the one-chain libraries.
and light chain
libraries. To retain clones with internal BstEII site in the
V (this site is relatively frequent in some germline V-segments (27), and also in the constant domain of one of the families), the
cloning of VHCH1 in the light chain repertoire containing vector
was also carried out using SfiI and NotI cloning
sites, to create a less restriction-biased V library.
-subunit fused to the -subunit of hFSH). Antigens were
biotinylated at a ratio of 10-20 molecules of NHS-Biotin (Pierce) per
molecule of antigen according to the supplier's recommendations.
Unless stated otherwise, the antigens were used for selection at
concentrations of 100, 30, and 10 nM during
rounds 1, 2, and 3 respectively. For hFSH-CTP, 50, 15, and 10 nM was used, respectively; for MUC1 peptide, 500, 100, 20, and 5 nM was used.
-D-galactopyranoside. Bacteria were harvested after 3.5 h of growth at 30 °C by
centrifugation (as before); periplasmic fractions were prepared by
resuspending the cell pellet in 1 ml of ice-cold PBS. After 2-16 h of
rotating head-over-head at 4 °C, the spheroplasts were removed by
two centrifugation steps; after spinning during 10 min at 3,400 × g, the supernatant was clarified by an additional
centrifugation step during 10 min at 13,000 × g in an
Eppendorf centrifuge. The periplasmic fraction obtained was directly
used for determination of fine specificities by surface plasmon
resonance or for Western blot studies (described below).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Oligonucleotide primers used for construction of the library
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Fig. 1.
Phagemid vector pCES1 for display of antibody
Fab fragments. Schematic representation (A) and
polylinker region (B) of pCES1. The polylinker region
comprises two signal sequences (S; pelB and the gene III
leader sequence), the C domain, ribosome binding site
(rbs), CH1 domain, hexahistidine tag (H6), and a
c-myc-derived sequence (tag). Variable domain genes can be
cloned as ApaLI-XhoI or
ApaLI-Asc fragments (for VL or VLCL,
respectively) and SfiI/PstI-BstEII or
SfiI-NotI fragments (for VH or VHCH1,
respectively). The amber stop codon (*) between the antibody genes and
bacteriophage gene III enables the production of soluble Fab fragments
in a non-suppressor strain of E. coli. Expression of the
bicistronic operon is under control of the LacZ promotor
(pLacZ).
, and V
libraries were cloned separately to maintain diversity, to yield
one-chain libraries in size typical for libraries made by cloning of
PCR fragments (4): 1.75 × 108 individual clones for
the heavy chain, 9.4 × 107 clones for V, and
5.2 × 107 clones for V. An overview is given in
Table II. In the second step, the heavy
chain fragments were digested from plasmid DNA isolated from the
primary VH repertoire, and cloned into the vector containing the light
chain repertoires (again separately for PBL- and spleen-derived
repertoires; Table II). The libraries were combined using this
efficient cloning procedure, to create a non-immunized Fab repertoire
with 3.7 × 1010 individual clones (4.3 × 1010 recombinant clones, 86% of which have a full-length
Fab insert), with 70% of clones harboring a light chain, 30% a
chain. All of 20 clones with full-length Fab insert tested scored
positive in dot-blot analysis with the 9E10 antibody indicating an
expression level of soluble Fab of at least 0.2 mg/liter.
Size and composition of the phage antibody libraries
Overview of results of selections with a diverse set of antigens
2 to 104 s1
(Fig. 2).
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Fig. 2.
Off-rate screening in BIAcore of selected
tetanus toxoid and MUC1 binding Fab fragments. Periplasmic
fractions from four anti-tetanus toxoid clones (A) and from
anti-MUC1 clones (B) were injected at t = 115 s on flow cells with immobilized tetanus toxoid and MUC1
peptide, respectively. At t = 175 s, the
dissociation phase started by washing with Hepes-buffered saline
buffer. Below the sensorgram, the derivative
ln(R0/R) of two representative clones
was plotted against the time (R0 taken from
t = 175 s), which should be linear for a truly
monophasic dissociation. Resulting dissociation rates are shown below
each series of sensorgrams.
-chain with 92 amino acid residues, but have -subunits of
different composition and length. The -chain of hCG contains 145 amino acid residues, and the one from hLH only 121 residues, the latter showing 85% homology to -hCG. The -chain of hFSH is only 111 amino acids and shares 36% of the residues with hCG. Antibodies that
specifically detect hCG have been used extensively in pregnancy tests
(42) and for cancer diagnosis (43, 44). A large set of antibodies to
these targets would extend the limited number of hormone-specific
antibodies (especially against hLH), obtained using the hybridoma
technology (42). The human origin of the antibodies might be beneficial
when using these for imaging or therapy of testicular and bladder
cancer (43, 44).
-hCG fused to the -chain of FSH;
Ref. 45). The highest degree of enrichment with respect to the increase
in the number of eluted phage particles in round 3 versus
round 1 was found for hCG (10,000-fold), followed by hFSH-CTP
(1,000-fold), hFSH (300-fold), and hLH (150-fold). Polyclonal phage of
selected populations were tested for binding using sensor chips
containing immobilized hormones (46). Polyclonal phage selected on hCG
showed binding after two and three rounds of selection to all three
proteins, i.e. hCG, hLH, and hFSH, with the strongest signal
visible for hCG (Fig. 3A);
after three selection rounds, approximately 400 RU of bound material is
visible at the start of the dissociation phase (the large peaks visible
during the first phase of association and dissociation are caused by refractive index changes due to buffer effects). Similar analysis of
the polyclonal phage populations selected for three rounds on hFSH
showed a dominance of hFSH-specific binding approx. 150 RU), while
selections on hFSH-CTP yielded binders to both hFSH and hCG (250 RU,
Fig. 3B). In this case, the control surface was coated with
streptavidin; no specific signals were obtained (<50 RU between the
signal before and after injection of the phage preparation). Selections
on hLH yielded antibodies reactive with hFSH and hCG (thus most likely
anti--chain antibodies; marked LH (1) in Fig.
3B). When hLH was used at lower concentrations (at 10 nM in round 1 and 3 nM during the subsequent
selection rounds), a signal was seen with strepta-vidin only
(marked LH(2) in Fig. 3B), due to the selection
of streptavidin-specific antibodies. Thus, this polyclonal phage
screening provides a rapid test to check the overall quality of the
clones in the selected repertoire, and may also be used to guide the
choice of the conditions for the next selection round (46).
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Fig. 3.
Monitoring of selections with polyclonal
phage using surface plasmon resonance. Polyclonal phage
populations from rounds 1, 2, and 3 (R1, R2, and
R3, respectively) of the selection with hCG, were analyzed
on flow cells with hCG, hLH, and hFSH; at t = 70 s, t = 400 s, and t = 690 s,
phage was injected, ending at t = 120 s,
t = 450 s, and t = 740 s,
respectively (A). Analysis of phage from round 3 selected
with the antigens hCG, hFSH-CTP, hFSH, and hLH (the latter was selected
with 100 nM hormone (coded LH(1)) or 10 nM (coded LH(2)) at round 1), using flow cells
with hCG and streptavidin (upper sensorgram) or
with hFSH-CTP, and hCG (lower sensorgram);
injection was started at t = 60 s,
t = 270 s, t = 500 s,
t = 720 s, and t = 950 s, and terminated at t = 120 s, t = 330 s, t = 560 s, t = 780 s, and t = 1010 s, respectively
(B).
-subunit of hCG by
clone CG#4F, while the cross-reactive clone CG#5C reacted with the
-subunit of both hCG and hLH (Fig.
5).
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Fig. 4.
Analysis of specificity of selected
anti-glycoprotein hormone Fabs using surface plasmon resonance.
Periplasmic fractions from clones CG#4F, CG#5C, LH#3G, LH#3F, FS#8B,
and SC#2B were injected at t = 120 s on flow cells
with immobilized hFSH-CTP, hLH, and hCG, and on an empty surface; at
t = 180 s, the injection phase was stopped and the
cells were washed with Hepes-buffered saline buffer.
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Fig. 5.
Specificities of the Fabs determined with
Western blot analysis. The glycoprotein hormones hCG, hFSH, and
hLH were diluted in non-reducing sample buffer, and boiled
( 
T+) or not heat-treated (T), loaded on a
15% SDS-PAGE, blotted onto nitrocellulose filter, and detected with
the indicated Fabs. The molecular forms of the hormones recognized by
the Fabs of clones CG#4F, CG#5C, LH#3G, FS#8B, and SC#2B are indicated
with arrows.
/-heterodimer of hLH. Two representative clones of a
pan-reactive antibody in ELISA, coded LH#1C and LH#3F, reacted in
BIAcore with hFSH-CTP, hCG, and hLH (shown for clone LH#3F in Fig. 4),
and in Western blot analysis with the -chains from all three
hormones (data not shown).
-unit (Fig. 5). Further, the specificity of an -chain binding clone, SC#2B, was confirmed in
BIAcore (Fig. 4) and Western blot (Fig. 5).
-chain-specific Fabs
were identified by fingerprint analysis, from which the clones coded
SC#2B, SC#2F, SC#2G, and SC#4G were examined in more detail. Immunoblot
analysis with the recombinant Fab as detecting antibody confirmed the
-chain specificity (blot incubated with clone SC#2B is shown in Fig.
5).
and chains (found in 6 and 7 clones, respectively, of a total of 13 tested) (Fig. 6, B and C). From the
densities of the blots, it would be unlikely that all of the light
chain would be complexed as Fab. Instead, it appears that in many cases
more light than heavy chain fragment is produced, which may be expected from the design of the bicistronic expression cassette. Upon
purification of functional antigen-binding fragments using denaturation
and refolding, followed by affinity chromatography, this excess of light chain disappears, as expected (shown with a Coomassie
blue-stained protein gel, for five clones in Fig.
7). Upon reduction, equal amounts of
heavy and light chain are seen, while under non-reducing conditions
(shown for one clone only), the main product is represented by the
disulfide linked Fab-molecule, with an equal amount of the (most
likely) non-covalently linked VHCH1 and VLCL products visible.
Production yields of selected hormone-specific Fabs varied between 160 µg and 1.43 mg of Fab/liter of culture, which was in the same range
as was found for the unselected Fabs (data not shown).
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Fig. 6.
Examination of the integrity of produced Fab
fragments on Western blot. The periplasmic fractions from the
indicated glycoprotein-specific Fab clones were boiled in non-reducing
sample buffer and loaded on 12.5% SDS-PAGE. The blots were incubated
with the anti-myc mAB 9E10 (A), anti-human polyclonal antibodies (upper panel of
B), or with anti-human kappa polyclonal antibodies
(C). Affinity-purified Fab was used as a control on the blot
incubated with anti-myc mAB (indicated with pur
Fab).
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Fig. 7.
SDS-PAGE of affinity-purified Fabs. hCG
binding Fabs produced by clones LH#1C, LH#3F, CG#5C, SC#2B, and SC#2F
were purified by affinity chromatography on hCG-Sepharose, and analyzed
on a Coomassie-stained 12.5% SDS-PAGE gel under reducing conditions,
and for Fab SC#2B also under non-reducing conditions. As positive
control, a Fab fragment made by proteolytic digestion of a human
monoclonal antibody was included (marked with +). B is a
4-fold dilution of A of the same sample.
families or
one of three V families. Chain promiscuity is seen for the
-chain-specific clone SC#4G, the /-LH-specific clones LH#2H
and LH#3G, and -FSH-specific clone FS#8B, which all used a highly
homologous V2 light chain gene segment (A19, previously coded DPK15)
combined with different heavy chain fragments. This promiscuity for A19
was previously found in antibodies derived from a synthetic Fab
repertoire (35). The three anti-MUC1 antibodies use heavy and light
chain genes derived from two different VH and V families; clone
MUC#9 uses a VH with a cross-over of two segments. It is remarkable
that both MUC#4 and MUC#9 VH genes use the same reading frame of the same D-segment (D6-13; with a stretch of 13 or 16 bp from this segment, respectively; Ref. 49), encoding an alanine-glycine stretch
(AAAG; Table IV). This may reflect a similar mode of binding to MUC1,
despite the use of a different light chain by these two clones.
V-gene segments and CDR3 sequences used by the selected Fabs
2 and 103
s1 (Table V). The off-rate
values obtained using crude periplasmic fractions were in good
agreement with the values found for the purified Fabs, validating the
utility of the off-rate screen with unpurified Fab fragments. The
affinities, 23 and 38 nM for the -subunit-specific antibody LH#1C and the -subunit
hCG/hLH-cross-reactive antibody CG#5C, respectively, are comparable to
the affinity of antibodies selected from a murine immune phage antibody
library3; the top affinity,
2.7 nM for the -chain-specific Fab SC#2B (Table IV),
approaches the values of the best anti-hCG monoclonal antibodies.3
Affinities of anti-hormone Fabs for hCG
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B cells), but it remains to be
seen if these V-gene sources will also yield better libraries. Indeed,
when comparing synthetic antibody libraries (35), which incorporate
germline encoded V-gene segments with non-immunized human V-gene
libraries such as the one presented here, it is difficult to pinpoint
any performance differences with regard to affinity and specificity of
selected antibodies.
1-102 s1 for fragments
selected from the smaller sized library to 103 to
104 s1 for those from the larger library.
This is in the same order of magnitude as we observe for the off-rates
of our selected antibody fragments. Since Fab fragments lack the
tendency to dimerize, Fab libraries could possibly display a lower
fraction of avid phage than equivalent scFv libraries library. This
does not appear to have reduced the number or average affinity of
selected antibodies. An indication that antibodies from this library
behave similarly or better with regards to affinity comes from a
comparison of selections of two different libraries on the same two
antigens under identical conditions. Antibodies to MUC1 selected from a large non-immunized scFv library (29) have faster off-rates then the
equivalent Fabs isolated from the library described in this study.
Further, they show a very distinct V-gene usage and have a different
fine specificity.4 Similarly,
when comparing the off-rates of phage antibodies against the
pancarcinoma marker epithelial glycoprotein-2, one of the Fabs selected
from the present library appears to have a 10-fold slower off-rate than
the best scFv (7).5
-chain or epitopes on the -chain shared by hCG and hLH. These
selections demonstrated that antibodies directed against different
epitopes within single antigen molecules can be retrieved from the library.
1/3, and V1/2; but less frequently used
segments of family VH6, V2/7, and V7 were also retrieved. Most
likely, the use of an extended set of variable region gene primers,
designed on the most recent sequence information of the germline
V-regions, and/or the separate PCRs, combined with partially separate
cloning, ensured access to a highly diverse sample of the human V-gene
repertoire. The average amino acid mutation frequency of the selected
human V-genes was calculated to be 4.0% for the VH segments (50 amino
acid mutations in 1267) and 7.3% for the VL (92 out of 1260). This
mutation frequency is the reverse of that reported for IgG+ B cells
(52) (10% for VH and 6% for VL). The higher mutation rate of the
VL-genes in the combinatorial repertoire may serve to contribute the
rather restricted natural light chain diversity. With respect to the VH
segments of the selected antibodies, the mutation frequency is
remarkably lower (mean 3.57 (±2.90) mutated residues per VH) than what
was found in the selected scFv fragments by Vaughan and colleagues (7) (mean 7.53 (±4.25) mutated residues). This is most likely caused by
the amplifications with VH-based primers for the construction of the
latter library, instead of the IgM primer used by us for the primary
PCRs. The light chain segments seem to have a similar mutation
frequency (Vaughan et al. reported 8.23 ± 5.20 residues, versus 6.57 ± 5.56 residues in the Fabs
reported here).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence and reprint requests should be
addressed. Tel.: 31-433874630; Fax: 31-433874609; E-mail:
hho{at}lpat.azm.nl.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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 R. To, T. Hirama, M. Arbabi-Ghahroudi, R. MacKenzie, P. Wang, P. Xu, F. Ni, and J. Tanha Isolation of Monomeric Human VHS by a Phage Selection J. Biol. Chem., December 16, 2005; 280(50): 41395 - 41403. [Abstract] [Full Text] [PDF]
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 H. J. W. De Haard, S. Bezemer, A. M. Ledeboer, W. H. Muller, P. J. Boender, S. Moineau, M.-C. Coppelmans, A. J. Verkleij, L. G. J. Frenken, and C. T. Verrips Llama Antibodies against a Lactococcal Protein Located at the Tip of the Phage Tail Prevent Phage Infection J. Bacteriol., July 1, 2005; 187(13): 4531 - 4541. [Abstract] [Full Text] [PDF]
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S. Schoonbroodt, N. Frans, M. DeSouza, R. Eren, S. Priel, N. Brosh, J. Ben-Porath, A. Zauberman, E. Ilan, S. Dagan, et al. Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library Nucleic Acids Res., May 19, 2005; 33(9): e81 - e81. [Abstract] [Full Text] [PDF]
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 N. Loizos, Y. Xu, J. Huber, M. Liu, D. Lu, B. Finnerty, R. Rolser, A. Malikzay, A. Persaud, E. Corcoran, et al. Targeting the platelet-derived growth factor receptor {alpha} with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: Implications as a potential therapeutic target Mol. Cancer Ther., March 1, 2005; 4(3): 369 - 379. [Abstract] [Full Text] [PDF]
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 L. Kausmally, K. Waalen, I. Lobersli, E. Hvattum, G. Berntsen, T. E. Michaelsen, and O. H. Brekke Neutralizing human antibodies to varicella-zoster virus (VZV) derived from a VZV patient recombinant antibody library J. Gen. Virol., December 1, 2004; 85(12): 3493 - 3500. [Abstract] [Full Text] [PDF]
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 Y. Li, H. Li, M.-N. Wang, D. Lu, R. Bassi, Y. Wu, H. Zhang, P. Balderes, D. L. Ludwig, B. Pytowski, et al. Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody Blood, August 15, 2004; 104(4): 1137 - 1144. [Abstract] [Full Text] [PDF]
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 R. W. Siegel, N. Velappan, P. Pavlik, L. Chasteen, and A. Bradbury Recombinatorial Cloning Using Heterologous Lox Sites Genome Res., June 1, 2004; 14(6): 1119 - 1129. [Abstract] [Full Text] [PDF]
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 K. Persaud, J.-C. Tille, M. Liu, Z. Zhu, X. Jimenez, D. S. Pereira, H.-Q. Miao, L. A. Brennan, L. Witte, M. S. Pepper, et al. Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C J. Cell Sci., June 1, 2004; 117(13): 2745 - 2756. [Abstract] [Full Text] [PDF]
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D. Lu, H. Zhang, D. Ludwig, A. Persaud, X. Jimenez, D. Burtrum, P. Balderes, M. Liu, P. Bohlen, L. Witte, et al. Simultaneous Blockade of Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor Signaling Pathways in Cancer Cells with a Fully Human Recombinant Bispecific Antibody J. Biol. Chem., January 23, 2004; 279(4): 2856 - 2865. [Abstract] [Full Text] [PDF]
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 B. Liu, F. Conrad, M. R. Cooperberg, D. B. Kirpotin, and J. D. Marks Mapping Tumor Epitope Space by Direct Selection of Single-Chain Fv Antibody Libraries on Prostate Cancer Cells Cancer Res., January 15, 2004; 64(2): 704 - 710. [Abstract] [Full Text] [PDF]
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D. Burtrum, Z. Zhu, D. Lu, D. M. Anderson, M. Prewett, D. S. Pereira, R. Bassi, R. Abdullah, A. T. Hooper, H. Koo, et al. A Fully Human Monoclonal Antibody to the Insulin-Like Growth Factor I Receptor Blocks Ligand-Dependent Signaling and Inhibits Human Tumor Growth in Vivo Cancer Res., December 15, 2003; 63(24): 8912 - 8921. [Abstract] [Full Text] [PDF]
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D. Lu, J. Shen, M. D. Vil, H. Zhang, X. Jimenez, P. Bohlen, L. Witte, and Z. Zhu Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity J. Biol. Chem., October 31, 2003; 278(44): 43496 - 43507. [Abstract] [Full Text] [PDF]
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R. Rauchenberger, E. Borges, E. Thomassen-Wolf, E. Rom, R. Adar, Y. Yaniv, M. Malka, I. Chumakov, S. Kotzer, D. Resnitzky, et al. Human Combinatorial Fab Library Yielding Specific and Functional Antibodies against the Human Fibroblast Growth Factor Receptor 3 J. Biol. Chem., October 3, 2003; 278(40): 38194 - 38205. [Abstract] [Full Text] [PDF]
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W. E. Biddison, R. V. Turner, S. J. Gagnon, A. Lev, C. J. Cohen, and Y. Reiter Tax and M1 Peptide/HLA-A2-Specific Fabs and T Cell Receptors Recognize Nonidentical Structural Features on Peptide/HLA-A2 Complexes J. Immunol., September 15, 2003; 171(6): 3064 - 3074. [Abstract] [Full Text] [PDF]
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G. Denkberg, A. Lev, L. Eisenbach, I. Benhar, and Y. Reiter Selective Targeting of Melanoma and APCs Using a Recombinant Antibody with TCR-Like Specificity Directed Toward a Melanoma Differentiation Antigen J. Immunol., September 1, 2003; 171(5): 2197 - 2207. [Abstract] [Full Text] [PDF]
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C. J. Cohen, O. Sarig, Y. Yamano, U. Tomaru, S. Jacobson, and Y. Reiter Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies J. Immunol., April 15, 2003; 170(8): 4349 - 4361. [Abstract] [Full Text] [PDF]
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 J. Hill, C. Copse, S. Leary, A. J. Stagg, E. D. Williamson, and R. W. Titball Synergistic Protection of Mice against Plague with Monoclonal Antibodies Specific for the F1 and V Antigens of Yersinia pestis Infect. Immun., April 1, 2003; 71(4): 2234 - 2238. [Abstract] [Full Text]
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C. J. Cohen, N. Hoffmann, M. Farago, H. R. Hoogenboom, L. Eisenbach, and Y. Reiter Direct Detection and Quantitation of a Distinct T-Cell Epitope Derived from Tumor-specific Epithelial Cell-associated Mucin Using Human Recombinant Antibodies Endowed with the Antigen-specific, Major Histocompatibility Complex-restricted Specificity of T Cells Cancer Res., October 15, 2002; 62(20): 5835 - 5844. [Abstract] [Full Text] [PDF]
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G. Denkberg, E. Klechevsky, and Y. Reiter Modification of a Tumor-Derived Peptide at an HLA-A2 Anchor Residue Can Alter the Conformation of the MHC-Peptide Complex: Probing with TCR-Like Recombinant Antibodies J. Immunol., October 15, 2002; 169(8): 4399 - 4407. [Abstract] [Full Text] [PDF]
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 C. Gao, S. Mao, G. Kaufmann, P. Wirsching, R. A. Lerner, and K. D. Janda A method for the generation of combinatorial antibody libraries using pIX phage display PNAS, October 1, 2002; 99(20): 12612 - 12616. [Abstract] [Full Text] [PDF]
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V. A. Somers, R. J. Brandwijk, B. Joosten, P. T. Moerkerk, J.-W. Arends, P. Menheere, W. O. Pieterse, A. Claessen, R. J. Scheper, H. R. Hoogenboom, et al. A Panel of Candidate Tumor Antigens in Colorectal Cancer Revealed by the Serological Selection of a Phage Displayed cDNA Expression Library J. Immunol., September 1, 2002; 169(5): 2772 - 2780. [Abstract] [Full Text] [PDF]
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P. Chames, R. A. Willemsen, G. Rojas, D. Dieckmann, L. Rem, G. Schuler, R. L. Bolhuis, and H. R. Hoogenboom TCR-Like Human Antibodies Expressed on Human CTLs Mediate Antibody Affinity-Dependent Cytolytic Activity J. Immunol., July 15, 2002; 169(2): 1110 - 1118. [Abstract] [Full Text] [PDF]
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G. Denkberg, C. J. Cohen, A. Lev, P. Chames, H. R. Hoogenboom, and Y. Reiter Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity PNAS, July 9, 2002; 99(14): 9421 - 9426. [Abstract] [Full Text] [PDF]
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A. Lev, G. Denkberg, C. J. Cohen, M. Tzukerman, K. L. Skorecki, P. Chames, H. R. Hoogenboom, and Y. Reiter Isolation and Characterization of Human Recombinant Antibodies Endowed with the Antigen-specific, Major Histocompatibility Complex-restricted Specificity of T Cells Directed toward the Widely Expressed Tumor T-cell Epitopes of the Telomerase Catalytic Subunit Cancer Res., June 1, 2002; 62(11): 3184 - 3194. [Abstract] [Full Text] [PDF]
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 P. Henderikx, N. Coolen-van Neer, A. Jacobs, E. van der Linden, J.-W. Arends, J. Mullberg, and H. R. Hoogenboom A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized Am. J. Pathol., May 1, 2002; 160(5): 1597 - 1608. [Abstract] [Full Text] [PDF]
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D. S. Wilson, A. D. Keefe, and J. W. Szostak The use of mRNA display to select high-affinity protein-binding peptides PNAS, March 7, 2001; (2001) 61028198. [Abstract] [Full Text]
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P. Chames, S. E. Hufton, P. G. Coulie, B. Uchanska-Ziegler, and H. R. Hoogenboom Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library PNAS, July 5, 2000; 97(14): 7969 - 7974. [Abstract] [Full Text] [PDF]
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D. S. Wilson, A. D. Keefe, and J. W. Szostak The use of mRNA display to select high-affinity protein-binding peptides PNAS, March 27, 2001; 98(7): 3750 - 3755. [Abstract] [Full Text] [PDF]
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