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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 9, Num. 2, 2006, pp. 171-175
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Electronic Journal of Biotechnology, Vol. 9, No. 2, April
15, 2006, pg. 171-175
TECHNICAL NOTE
Improved
affinity selection using phage display technology and off-rate based
selection
Bin Yuan1, Philip
Schulz2, Ruitian
Liu3, Michael
R. Sierks*4
1Department
of Chemical and Materials Engineering,
Arizona State University,
Tempe, AZ 85287,
USA,
Tel: 480 965 0826,
Fax: 480 965 0037,
E-mail: Bin.Yuan@asu.edu
2Department of Chemical and Materials
Engineering, Arizona State University,
Tempe, AZ 85287, USA,
Tel: 480 965 0826,
Fax: 480 965 0037,
E-mail: Philip.Schulz@asu.edu
3Department of Chemical and Materials Engineering,
Arizona State University,
Tempe, AZ 85287, USA
4Department of Chemical and Materials Engineering,
Arizona State University,
Tempe, AZ 85287, USA,
Tel: 480-965-2828,
Fax: 480 965 0037,
E-mail: sierks@asu.edu
*Corresponding author
Financial
support: This work was supported in part by grants from the NIH (AG17984),
American Health Assistance Foundation (A2001-043) and the Alzheimers Association
(IIRG-01-2753).
Received July 4,
2005 / Accepted October 28, 2005
Code Number: ej06020
Abstract
Flow
systems such as a BIAcore biosensor can be very efficient tools to isolate
high affinity antibody fragments from affinity matured phage display libraries.
Here we show that using flow based selection, we can readily isolate a
variant with a 35-fold higher affinity, especially with a 7 fold better
off-rate, compared to the parent clone after only a single round of selection
from a second generation affinity matured phage display library. The flow
system represents a fast method to isolate affinity improved antibody fragments
and can be particularly useful for isolating antibodies to antigens that
have poor
solubility, are toxic to the host cell, or prone to aggregation.
Keywords:
affinity maturation, β-amyloid (Aβ), dissociation rate, phage display.
Over 30%
of the biopharmaceuticals under development in clinical trials today are
recombinant antibodies (Hudson and Souriau, 2003). The powerful
capabilities of surface display technology to isolate high affinity antibody
fragments against a wide variety of target antigens has been frequently demonstrated
(Kretzschmar and von Ruden, 2002). Antibody fragments isolated
using immunotube based selection often have low affinities to the target
antigen (Knappik et al. 2000) Soluble selection using magnetic
beads and biotinylated antigen can lead to selection of higher affinity antibody
fragments (Hawkins et al. 1992), but the soluble selection
method may not be suitable for those antigens that have poor solubility or
are prone to aggregation. Soluble or immunotube based selection methods also
do not necessarily ensure that the selected antibody fragments will have
the lowest off-rates, an important characteristic for potential clinical
applications (Jirholt et al. 2001). Cell sorting methods
are effective selection tools for cell surface display libraries (Yeung
and Wittrup, 2002), however for antigens that are toxic, have limited
solubility or are prone to aggregation, phage display technology may be preferred.
An alternative selection method for use with phage display libraries is to
use a flow system such as a biosensorwhere selection is based on dissociation
rates rather than affinity constants, correlating higher affinities with
longer elution times (Malmborg et al. 1996).
We constructed
a second-generation phage display library by randomizing the light chain
CDR3 region of a parent scFv (H1) isolated against β-amyloid (Aβ),
a peptide that aggregates readily. We then compared two different methods
to select for scFvs having improved affinity to Aβ, a static immunotube
biopanning based selection, and a flow based system using a BIAcore X biosensor.
Using the static selection method, we obtained an scFv (H1-v2) with a four-fold
higher affinity than the parent antibody (H1), while when using the flow
based system; we obtained an scFv (H1-v3) having a 35-fold higher affinity.
Materials
and Methods
Aβ 1-40
was used as antigen and positive single clones were selected from the Nissim
library (Nissim et al. 1994) following standard biopanning
protocols (Vaughan et al. 1996). The clone, H1, which showed
the highest binding activity with Aβ 1-40 was isolated and used as a
parent clone for affinity maturation studies.
The second
generation library was constructed by randomizing the CDR3 light chain region
of the parent H1 scFv using a two-step PCR protocol. The scFv gene of H1
was amplified first with the primers: LMB3 (5-CAG GAA ACA GCT ATG AC-3) and CDR3-6-VL-FOR (5-CTT GGT CCC TCC
GCC GAA TAC CAC NNN NNN NNN NNN NNN NNN AGA GGA GTT ACA GTA ATA GTC AGC CTC-3)
where N represents a random nucleotide, and then amplified again with the
primers: LMB3 (5-CAG GAA ACA GCT ATG AC-3)
and JL-NOT-FOR (5-ATT GCT TTT CCT TTT TGC GGC CGC GCC TAG GAC GGT CAG CTT
GGT CCC TCC GCC-3). The PCR product obtained after the
two PCR steps was digested with Nco I and Not I, and ligated
into plasmid pHEN2 (Griffiths et al. 1994) and then transformed
into E. coli TG1 cells. Biopanning using immobilized antigen on immunotubes
was performed essentially as described (Vaughan et al. 1996),
except that 1 µg/mL Aβ was immobilized on the surface of the immunotubes
and the second-generation library was panned for three rounds. Individual
clones were randomly selected as described (Vaughan et al.
1996), and clones providing the strongest ELISA signals were selected
for further characterization.
BIAcore
based selection was performed as follows: Aβ was diluted to 200 µg/mL
in 10 mM Sodium acetate buffer (pH 4.8)
and immobilized onto a C1 chip (BIAcore) following the amine binding protocol
provided by the manufacturer. C1 chip was used here because the short dextran
linkers on the surface can facilitate access of filamentous phage particles
to the immobilized antigen (Malmborg et al. 1996). Immobilization
of Aβ resulted in a 300 RU increase in the observed signal. For selection,
40 µL of second-generation library stocks containing 1.3 x 1013 cfu/mL
were passed over the C1 sensor chip at a flow rate of 1 µL/min. After association
of the phage to the chip surface, unbound phage were washed off the chip
using the rinse command. Dissociation of bound phage from the chip surface
was performed using a 1 µL/min continuous flow of running buffer (HBS-EP,
BIAcore) for 7 hrs. Regeneration of the chip surface was accomplished with
10 µL 0.1 M glycine-NaOH, pH 12. We collected
10 µL aliquots at 3, 4, 5, 6, and 7 hrs during the dissociation phase and
also during the regeneration step. The collected samples were immediately
used to infect E. coli HB2151. Individual clones were selected by
ELISA as described (Vaughan et al. 1996) for further characterization.
Individual
clones from immunotube and BIAcore were cultured and induced with 1 mM IPTG. The cultures were centrifuged
and filtered through a 0.2 µm membrane. The resulting samples containing
soluble scFv were used for BIAcore studies. Two clones, H1-v2, which represented
the highest ELISA signal obtained from the immunotube based selection, and,
H1-v3, which represented the highest ELISA signal obtained from the BIAcore
based selection, were purified using a Protein A column as described and
used for further characterization. The off-rates of soluble scFv samples
obtained from crude supernatant and the on- and off-rates of purified scFv
samples were evaluated using a BIAcore X biosensor. Briefly, Aβ was
immobilized on a CM5 sensor chip by amine binding yielding a final increase
of 150 RU. Samples containing scFv were serially diluted in running buffer
(HBS-EP, BIAcore) and 40 µL of the diluted samples were injected at a flow
rate of 10 µL/min. For the purified scFv samples (H1-v2 and H1-v3), both
association rate constants (ka) and dissociation rate constants
(kd) and the affinity constants (dissociation constants, KD= kd/ka)
were calculated assuming a single binding site model.
Results
and Discussion
The
parent scFv, H1 (KD = 2.61 x 10-6 M toward Aβ 1-40)
was obtained after four rounds of biopanning using the Nissim phage display
antibody library (Nissim et al. 1994). The second generation
library has a theoretical diversity of 206 (6.4 x 107)
and contained approximately 9.7 x 108 clones indicating a diversity
of at least 106 different clones. After four rounds of selection
by static panning, 50% of the antibody clones recovered showed positive ELISA
signals (a positive signal was defined as ELISA reading at least two times
higher than the background value) (Figure 1). We selected
the nine different clones with the strongest ELISA signals, determined their
amino acid sequences by DNA sequencing, and determined the off rates (Table
1). The clone H9 gave the highest ELISA signal, so we selected this clone
for further studies, renaming it H1-v2. Flow based selection was performed
on the BIAcore X. After only a single round of selection, 90% of the clones
tested from the 6 and 7 hrs and regeneration step aliquots gave positive
ELISA signals (defined as above) (Figure 1b). We again
selected the nine different clones with the highest ELISA signals, determined
their amino acid sequence, and off rates (Table 1).
The clone D9 had the highest ELISA signal, so we selected this clone for
further studies, and renamed it H1-v3.
Table 1. Characters
of the selected clones from static selection and flow based selection.
|
Static
Selection
|
CDR3
(L)
Sequence
|
OD1
Mutant/
parent
|
Dissoc.
Rate
(kd,
s-1)
|
Affinity
constant (KD, M)
|
Flow
based
Selection
|
CDR3
(L)
Sequence
|
OD1
Mutant/
parent
|
Dissoc.
Rate
(kd,
s-1)
|
Affinity
constant (KD, M)
|
H1
(parent)
|
NSRDSSGNHVV
|
1
|
4.02e-3
|
2.61e-6
|
H1
(parent)
|
NSRDSSGNHVV
|
1
|
4.02e-3
|
2.61e-6
|
A1
|
NSSNRPTQYVV
|
1.4
|
3.79e-3
|
N.A.
|
A9
|
NSSTPTQKHVV
|
3.3
|
1.23e-3
|
N.A.
|
A7
|
NSSRDQEGTVV
|
1.8
|
5.55e-3
|
N.A.
|
A11
|
NSSPQNKTLVV
|
2.5
|
2.54e-3
|
N.A.
|
C12
|
NSSKNDSVLVV
|
1.6
|
1.92e-2
|
N.A.
|
D7
|
NSSDQNITSVV
|
3.6
|
8.65e-4
|
N.A.
|
D5
|
NSSHVILNRVV
|
1.4
|
7.15e-3
|
N.A.
|
D9
(H1v3)
|
NSSTRHNPTVV
|
4.1
|
6.05e-4
|
7.28e-8
|
D7
|
NSSDSKNRPVV
|
2.0
|
6.54e-3
|
N.A.
|
D12
|
NSSQRHLPNVV
|
3.9
|
7.94e-4
|
N.A.
|
E1
|
NSSQRDSLKVV
|
1.7
|
3.29e-3
|
N.A.
|
C1
|
NSSIPRKLIVV
|
2.4
|
1.55e-3
|
N.A.
|
F11
|
NSSKTSNRDVV
|
1.5
|
8.25e-3
|
N.A.
|
C11
|
NSSDNGSKHVV
|
2.9
|
6.72e-4
|
N.A.
|
H5
|
NSSCNQDSLVV
|
1.8
|
5.36e-3
|
N.A.
|
C12
|
NSSHLHNHPVV
|
3.2
|
3.22e-3
|
N.A.
|
H9
(H1v2)
|
NSSYCVRTLVV
|
2.2
|
7.45e-3
|
6.53e-7
|
F9
|
NSSKTLNVDVV
|
3.6
|
1.74e-3
|
N.A.
|
2Average
|
|
|
7.40e-3
|
|
|
|
|
1.47e-3
|
|
1OD
values represent the ratio of Mutant/parent readings, where each
individual reading is obtained by calculating OD450-OD650.
2Average
value is the average of the nine clones isolated for each selection
method.
|
The average
dissociation rate for the nine different clones selected using the flow-based
method (1.47 x 10-3) was five-fold better than the average rate
for the nine clones selected by conventional immunotube panning (7.40 x 10-3)
(Table 1). The clone selected from flow based panning,
H1-v3, had the lowest dissociation rate from that group, however H1v2 did
not have the best dissociation rate from the static selection group.
The H1,
H1-v2 and H1-v3 scFvs were purified and analyzed by BIAcore. The association
rates (ka), dissociation rates (kd),
and dissociation constants (KD) were obtained (Table
1). The dissociation constants (KD) of H1-v2 (6.53
x 10-7 M) and H1-v3 (7.28 x 10-8 M) are four-fold and
35-fold better than the value obtained for the parental H1 clone (2.61 x
10-6 M) respectively, clearly demonstrating the value of a flow-based
selection method.
Another
advantage of the flow based method is that the total number of recovered
phage drops dramatically in those samples that contain the scFvs with the
slowest off rates, facilitating identification of strong binding scFvs when
limited antigen is available (Figure 1). An additional
powerful feature of flow-based system is that selection can be performed
very quickly, in only a few hours.
Here we
demonstrate that human based scFv fragments that can bind Aβ can be
isolated from a synthetic antibody library and that the affinity of these
scFvs can be greatly improved (35-fold) after only a single round of affinity
maturation, improving the off-rate over seven-fold. Further improvements
in antibody affinity and off-rate can be obtained by generating additional
antibody libraries by randomization of other CDR regions in the heavy and
light chains (Yang et al. 1995), leading to antibody fragments
that can be candidate therapeutics for treating Alzheimers Disease (Lombardo
et al. 2003).
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