From the Institute for Biological Sciences, National
Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada, the
§ Biotechnology Research Institute, National Research
Council of Canada, Montréal Québec H4P 2R2, Canada, and
¶ Viventia Biotech Inc., Winnipeg, Manitoba R3T 3Z1, Canada
Received for publication, January 26, 2001, and in revised form, April 25, 2001
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ABSTRACT |
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We have constructed a human VH
library based on a camelized VH sequence. The library was
constructed with complete randomization of 19 of the 23 CDR3 residues
and was panned against two monoclonal antibody targets to generate
VH sequences for determination of the antigen contact
residue positions. Furthermore, the feasibility and desirability of
introducing a disulfide bridge between CDR1 and CDR3 was investigated.
Sequences derived from the library showed a bias toward the use of
C-terminal CDR3 residues as antigen contact residues. Mass
spectrometric analyses indicated that CDR1-CDR3 disulfide formation was
universal. However, surface plasmon resonance and NMR data showed that
the CDR3 constraint imposed by the disulfide bridge was not always
desirable. Very high yields of soluble protein products and lack of
protein aggregation, as demonstrated by the quality of the
1H-15N HSQC spectra, indicated that the
VH sequence for library construction was a good choice.
These results should be useful in the design of VH
libraries with optimal features.
Heavy chain antibodies, found in camelids (1, 2), lack light
chains and as a result have variable domains that reflect the absence
of a VL partner. Single domain antibodies
(dAbs)1 derived from the
variable domains (VHHs) of these antibodies are highly
soluble and the structural basis of solubility has been partially
elucidated. First, conserved human/murine interface residues are
generally replaced in heavy chain antibodies by residues that increase
the hydrophilicity of the VL interface either by non-polar
to polar substitutions or, in a more subtle way, by inducing local
conformational changes (3, 4). This explanation is supported by
experiments in which an insoluble human VH was made soluble
by introducing these substitutions (5). Second, in the solved
structures of two camel dAbs, the CDR3s fold back on the VH
surface, masking a significant surface area of the VL interface (3-6).
Two other features of VHHs are noteworthy. One is the
frequent occurrence of cysteine residues in CDR1 and CDR3 (7-10).
While the location of the CDR1 cysteine is typically fixed, that of the
CDR3 cysteine varies. These two residues form a disulfide linkage
between CDR1 and CDR3 (3, 11). In the crystal structure of a
dAb-lysozyme complex, the disulfide linkage imparts rigidity on the
CDR3 loop that extends out of the combining site and penetrates deep
into the active site of lysozyme (3). A second feature is the longer
average length of the VHH CDR3, relative to human or
murine VHs (8). A longer CDR3 increases the antigen-binding surface and partially compensates for the absence of the
antigen-binding surface provided by the VL in conventional
antibodies (3).
As antigen-binding fragments, dAbs are an attractive alternative to
scFvs because of their much smaller size and the fact that they have
affinities comparable to those of scFvs (4, 9, 12-15). Smaller size is
an advantage in applications requiring tissue penetration and rapid
blood clearance. Smaller molecules also offer a tremendous advantage in
terms of structural studies (5, 16, 17).
Phage antibody library construction is much simpler and more efficient
with dAbs as compared with Fabs or scFvs. Randomization can be
introduced at a much higher percentage of CDR positions without
exceeding practical library size. The problem of shuffling original
VL-VH pairings is also avoided. Camelid phage
dAb libraries constructed from the VHH repertoire of camels
immunized with target antigens have performed well (6, 9, 14). However,
in addition to the obvious problems of this approach, the non-human
nature of products from these libraries limits their usefulness.
Synthetic dAb libraries (13, 15), particularly those based on a human VH framework, alleviate these problems. Here we describe
the construction of camelized human dAb library that is based on the
VH of the human monoclonal antibody BT32/A6 (18). In prior
experimental studies with this
VH,2 the length
of its CDR3 and its solubility properties were found to be remarkable,
features which were later determined to be characteristic of camelid
heavy chain antibodies (1). To generate the library, the CDR3 was
randomized and cysteine residues were introduced at key positions with
the expectation that the residues would form the CDR1-CDR3 disulfide
bridge found in the camel antibody cAb-Lys3 (3). The library was
evaluated by panning against two monoclonal antibody targets.
Library Construction and Panning--
Wild type dAb (Fig.
1) was constructed from BT32/A6 (18). Two
camelized versions, BT32/A6.ERG and BT32/A6.ERI (Table
I), were constructed by standard
protocols (19). BT32/A6.ERG was used as the template in PCR to amplify
a shorter fragment using primers
5'-TGTTCAGCTAGCGGATTCACCTTCAGTAGCTATTGTATGCACTGGGTCCGC-3' (A6VH.33C) containing the NheI site (underlined) and
5'-TGCTGCACAGTAATACACAGCCGT-3'. At the protein level this introduces
Cys and two Ala residues at positions 33, 93, and 94, respectively. In
camelid VHHs, positions 93 and 94 are predominantly
occupied by Ala residues and Cys is frequently found at position 33 (8, 10). The mutated fragment was used as the template in a second PCR
using the primers A6VH.33C and
5'-GCCCCAGATATCAAA(A/CNN)9GCA(A/CNN)10TGCTGCACAGTAATA-3'. The second primer results in the randomization of the first 19 residues
in CDR3, with the exception of 100e where a Cys is introduced to
facilitate the formation of intramolecular disulfide linkage between
33Cys and 100eCys in CDR1 and CDR3, respectively. The amplified fragments were used as templates in a third round of PCR
employing primers A6VH.33C and
5'-TGAGGAGACGGTGACCGTTGTCCCTTGG-CCCCAGATATCAAA-3' to
incorporate a 3' end BstEII site (underlined). The amplified fragments were purified, digested with NheI and
BstEII, and ligated to
NheI/BstEII-treated pSJF6-BT32/A6VH
phagemid.3 The product was
desalted using spin columns and used to transform Escherichia
coli strain XL1-Blue. Growth of the library was performed as
described by Harrison et al. (20). To sub-clone the library into a phage vector, library phagemid DNA template (180 pmol) and two
primers which were complimentary to the 5' and 3' ends of the dAb
genes, and incorporated flanking ApaLI and NotI
restriction sites, were used to PCR amplify the dAb genes. The products
were purified, cut with ApaLI and NotI, purified,
and ligated to the ApaLI/NotI-digested phage
vector fd-tetGIIID (21). Following this, 1.5 µg of the desalted
ligated product was mixed with 40 µl of competent E. coli
strain TG1 and the cells were transformed by electroporation. Using
standard methods, the sizes of the phagemid and phage libraries were
determined to be 2.1 × 107 and 6.6 × 107, respectively. Phage were produced and purified as
described previously (20). One clone, R3A10 (Table I), was selected
from the phage library because of its higher yield of soluble product. Two mutated versions of R3A10, R3A10.G47I and
R3A10.G47I/Cys
The library was panned against 3B1, a scFv specific for a bacterial
polysaccharide (22), and M2 anti-FLAG IgG (Sigma). Immuno MaxiSorpTM
(Nunc) microtiter plate wells were coated overnight by adding 150 µl
of 100 µg/ml antigen in PBS. Plates were rinsed three times with PBS,
blocked with 400 µl of PBS, 2% (w/v) skim milk (2% MPBS) at
37 °C for 2 h and rinsed as above. Phage (1012
transducing units in 2% MPBS) were added and incubated at room temperature for 1.5 h after which unbound phage were removed. The
wells were rinsed 10 times with PBS, 0.1% (v/v) Tween 20 and then 10 times with PBS. Bound phage were eluted by adding 200 µl of freshly
prepared 100 mM triethylamine and neutralized with 100 µl
of 1 M Tris-HCl, pH 7.4. Exponentially growing TG1 cultures (10 ml) were infected with 150 µl of eluted phage at 37 °C for 30 min. Serial dilutions of infected cells were used to determine the
titers of eluted phage as described above. The remainders of the
infected cells were pelleted, re-suspended in 900 µl of 2 × YT
(19), mixed in 300-µl aliquots with 3 ml of 0.7% agarose in
LB (19) at 50 °C and the phage propagated on plates overnight at 37 °C. Phage were purified, the titers were determined, and a
total of 1011 transducing units of phage were used for
further rounds of selection.
Clones selected for expression were transferred to an expression vector
that added C-terminal c-Myc and His5 tags and were grown as described previously (23). The dAbs were purified from periplasmic fractions (24) by immobilized metal affinity chromatography (25) except that the starting buffer was 10 mM HEPES, 10 mM imidazole, 500 mM NaCl, pH 7.0. To detect
the presence of dimer/multimer dAb in protein preparations, gel
filtration chromatography was performed using Superdex 75 (Amersham
Phamacia Biotech) (26).
Alkylation Reactions--
Cold acetone (5 × volume) was
added to 200 µg of dAb solution and the contents were mixed and then
centrifuged in a microcentrifuge at maximum speed at 4 °C for
10 min. The pellet was dissolved in 500 µl of 6 M
guanidine hydrochloride and 55 µl of 1 M Tris buffer, pH
8.0, were added. Subsequently, a 25 M excess of DTT, relative to Cys residues, was added and the mixture was incubated at
room temperature for 30 min. A 2.2 M excess, relative to
DTT, of freshly made iodoacetic acid was added and the reaction was incubated as described above. The alkylated product was then
concentrated in 50 µl of distilled water using an Ultrafree-MC 10,000 NMWL filter unit (Millipore). Control experiments were identical except that DTT was replaced with water. The molecular sizes of native and
iodoacetate-treated dAbs were determined by infusion-electrospray ionization mass spectrometry (positive ion mode) using Quatro triple
quadrupole mass spectrometer (Micromass).
Binding Studies--
Following panning, phage clones were
screened by standard enzyme-linked immunosorbent assay
procedures using a horseradish peroxidase/anti-M13 monoclonal antibody
conjugate (Amersham Pharmacia Biotech) as the detection reagent.
Surface plasmon resonance was performed using a BIACORE Upgrade
(Biacore AB) (27). Approximately 14,000 resonance units of anti-FLAG M2
IgG or control IgG were immobilized on research grade CM5 sensor chips
by amine coupling. Single-domain antibodies were passed over the sensor
chips surfaces in 10 mM HEPES buffer, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% P-20 (Biacore AB)
at 25 °C and at a flow rate of 5 µl/min. To assess the effect of
the CDR1-CDR3 disulfide bridge on dAb binding to M2, dAbs were
incubated with DTT prior to injection and the above buffer was
supplemented with an appropriate amount of DTT. The influence of the
disulfide bridge on binding was also investigated by construction and
characterization of a mutant, M2R2-1.Cys NMR Sample Preparation--
Isotopically labeled proteins were
prepared from cells grown in 15N- or
15N/13C-enriched Bioexpress medium (Cambridge
Isotopes Laboratory). Six-ml amounts of LB (19) containing 100 µg/ml
ampicillin were inoculated with single colonies and incubated at
37 °C and 260 rpm until an A600 of ~5 was
reached. The cells were centrifuged and re-suspended in 3 ml of sterile
PBS. Aliquots were transferred to 25 ml of Bioexpress, 100 µg/ml
ampicillin in 125-ml Erlenmeyer flasks, to give an
A600 = 0.06, and incubated at 37 °C at 200 rpm for 9-10 h. The dAbs were purified as described above and dialyzed
extensively in 10 mM sodium phosphate, 150 mM
NaCl, 0.5 mM EDTA, at either pH 5.5 or 6.8. NMR samples
were prepared by concentrating the protein to ~1 mM using
a YM10 membrane (Amicon).
NMR Spectroscopy--
NMR experiments were performed at 298 and/or 308 K on a Bruker Avance-800 spectrometer equipped with pulse
field gradient accessories. Two-dimensional
1H-15N HSQC spectra (28) were acquired using
solvent suppression via the WATERGATE method implemented through the
3-9-19 pulse train (29, 30). Triple-resonance experiments (Ref. 31 and references therein) included HNC Panning--
The library was panned, in both formats, against 3B1
scFv. With the phagemid format, panning failed to enrich for binders and PCR analysis of clones selected at different stages of the panning
process revealed almost universal deletion of the dAb inserts. This is
probably the result of monovalent display since multivalent display
with the phage vector format gave seven different dAbs that bound to
3B1 (Table II). As shown in Fig.
2, these dAbs bound to the target
antigen, 3B1, in enzyme-linked immunosorbent assay experiments with no
detectable binding to control BSA. In each instance, the consensus
sequence was present at the extreme C-terminal end of CDR3 (Table
II).
In the phage vector format, the library was also panned against M2 IgG,
an antibody raised against the FLAG peptide DYKDDDDK (35) and shown to
recognize the consensus sequence XYKXXD (36). Twenty-four different dAbs with the FLAG consensus sequence were identified by sequencing of clones randomly selected after 3 rounds of
panning (Table III). No consensus
sequence other than XYKXXD could be identified.
Interestingly, like the 3B1 binders, all the FLAG consensus sequences
occurred in the C-terminal half of CDR3 and, with two exceptions,
occupied identical positions. To ascertain if this observation was
related to the presence of CDR1-CDR3 disulfide linkage, the reduced
version of the same library was also panned against M2 IgG. Panning,
including the washing steps, was performed in the presence 1 µM, 1 mM, 10 mM, and 100 mM DTT. In 13/13, 6/8, 8/8, and 10/10 instances for dAbs
obtained by panning in the presence of 1 µM, 1 mM, 10 mM, and 100 mM DTT,
respectively, the FLAG consensus sequence was located C terminus of
CDR3 (data not shown).
Size Exclusion Chromatography--
Size exclusion chromatography
of the non-camelized product revealed three components corresponding to
monomer, dimer, and higher oligomer on the basis of their elution
volumes. The monomer peak eluted unusually late suggesting that the dAb
interacted nonspecifically with the gel matrix. This is a property of
human and murine-derived dAbs that is not unusual and which has been documented previously (5, 12). Camelized BT32/A6 dAbs gave products
that were exclusively monomer and which eluted at the expected volume.
Verification of CDR1-CDR3 Disulfide Linkage
Formation--
Formation of the CDR1-CDR3 disulfide linkage was
verified by mass spectrometric analysis of alkylated dAbs isolated from
the library. The mass spectrum obtained for M2R2-5 after treatment with
iodoacetic acid had a major peak with a mass of 16,545.19 ± 2.4 Da (Fig. 3A) which corresponds
to the mass of the untreated protein. In contrast, the major peak
observed following reduction and alkylation had a mass of
16,783.29 ± 2.94 Da (Fig.
3B). The mass increase of
237.70 ± 2.94 Da indicates alkylation of 4 Cys residues.
Theoretically, alkylation of four cysteines should give a mass increase
of 236.16 Da. The 2 additional cysteine residues are the conserved ones
at positions 22 and 92. In Fig. 3A a minor peak
corresponding to the Mr of a dAb alkylated at
one position may have resulted from a side reaction involving
alkylation of another amino acid such as histidine. Identical
experiments were performed for eight additional anti-M2 dAbs as well as
R3A10.G47I and in all instances formation of the CDR1-CDR3 disulfide
bridge was confirmed. This demonstrates that disulfide linkage
formation is independent of the CDR3 sequence and is likely a function
of the overall fold of the protein.
Binding Kinetics--
The binding to M2 IgG of six of the dAbs
listed in Table III (M2R2-1, M2R2-2, M2R2-4, M2R2-10, M2R2-13, and
M2R3-4) was investigated by surface plasmon resonance. The binding data
fit poorly to a 1:1 interaction model in all instances, making the
derivation of kinetic and affinity constants impossible. However, when
binding studies were conducted in the presence of DTT it was observed that the amount of binding increased significantly, particularly for
M2R2-2. Furthermore, data collected in the presence of DTT fit much
better to a 1:1 interaction model. Similarly, the data for the binding
of a M2R2-1 mutant lacking the CDR1-CDR3 disulfide bridge to
immobilized M2 IgG fit reasonably well to the simple interaction model.
Global analysis of the data gave an association rate constant of 340 M NMR Studies--
Yields of at least 10 mg/liter of bacterial
culture of soluble product were obtained for native and camelized forms
of BT32/A6 dAb, facilitating labeling studies. The BT32/A6
VH fragment (BT32/A6, Table I) was reasonably soluble in
aqueous solution but exhibited broad NMR signals in both
one-dimensional proton and two-dimensional NOESY spectra (data not
shown), indicating partial aggregation. Various schemes of camelization
resulted in variant VH molecules with significantly
enhanced solubility and improved NMR spectral quality (Table I).
However, with the VH fragments studied here, including
BT32/A6.ERI with the Y47I substitution, the NMR data quality was
not improved by the presence of CHAPS detergent, which was found to be
essential for the improved solution properties of the human
VH fragment studied previously (10, 38, 39). On the other
hand, both the proton and HSQC spectra of R3A10.G47I responded to the
addition of DTT with a sharpening of some selected resonances (spectra
not shown). The localized spectral changes in the presence of DTT are
very likely induced by the selective reduction of the disulfide bond
between CDR1 and CDR3 (see next section).
In dramatic contrast, the R3A10.G47I/Cys Minimizing the size of antigen-binding proteins to a single
immunoglobulin domain has been one of the primary goals of antibody engineering over the past decade. However, low levels of soluble expression in Escherichia coli and solubility problems have
hampered development of such molecules. The discovery of camelid heavy chain antibodies (1) opened up new opportunities for development of
single domain antibodies, including the incorporation of features of
these antibodies into human VH frameworks. Camelization of human VHs is a promising technology for the generation of
small antigen-binding fragments that should be useful for therapeutic purposes in humans. However, while the camelized antibodies described in the literature (5, 13, 38, 39) have tremendously improved physical
properties relative to their non-camelized counterparts, these
properties are still less than ideal.
Davies and Reichmann (5) camelized a human VH by
introducing G44E, L45R, and W47G mutations. However, the yields in
E. coli of soluble camelized product were low (typically
less than 1 mg/l) and in order to obtain the yields and stability
required for NMR studies they opted for a W47I mutation instead of the W47G mutation (5, 13). This resulted in yields of up to 5 mg/liter,
which is an order of magnitude lower than the yields reported here for
camelized BT32/A6. A NMR structure of a human VH camelized
in this manner has been described (39) but, in order to reduce
aggregation and achieve sufficient solubility, the addition of CHAPS
detergent to the sample during NMR data collection was a requirement.
In contrast, we were able to collect high quality NMR data in the
absence of detergent, establishing that BT32/A6 VH is an
ideal template for the generation of camelized dAbs. The NMR spectra
and size exclusion chromatograms indicated that the camelized BT32/A6
VH molecules had properties that were indistinguishable
from those of dAbs derived from natural heavy chain antibodies.
In addition to the mutations introduced at positions 44, 45, and 47, it
is obvious that some features inherent in the wild type VH
contribute to the solubility of camelized BT32/A6 VH. Several observations support this. First, it has been observed that a
murine VH existed as a monomer at millimolar concentrations without the need for camelization (15) while camelization of a
different VH failed to eliminate aggregation (40). Second, x-ray crystallographic experiments have shown that, in addition to
residues 44, 45, and 47, several others at positions scattered throughout the VHH surface also contribute to solubility
(4). Third, long CDR3s like that present in BT32/A6 VH are
known to contribute to VHH solubility (3, 6).
As a template for camelized VH library construction, the
unusually long CDR3 of BT32/A6 offers the option of introducing a CDR1-CDR3 disulfide bridge and provides a larger antigen-binding surface, features which increase library diversity. Formation of the
disulfide bond was confirmed for several sequences and introduction of
the two cysteines did not have a negative impact on the yield of
soluble product. For anti-M2 dAbs the presence of the disulfide imposed
a constraint that prevented optimal interaction with M2. The SPR data
indicated that the binding of dAbs containing the introduced disulfide
bond to M2 was heterogeneous with reduction of the bond resulting in
binding data that fit well to a 1:1 interaction model. This was
consistent with NMR data showing that elimination of the R3A10
disulfide bridge changed CDR1 and CDR2 conformation and eliminated
structural heterogeneity without any change in the overall fold of the
protein. The structural heterogeneity of the disulfide form was
apparently not attributable to incomplete disulfide bond formation.
Construction and pooling of two libraries, one with and one without the
bridge, would appear to be desirable. The presence of the bridge would
probably be desirable in some instances since this is a common feature
of heavy chain antibodies and it introduces structural diversity into
the library.
Synthetic VH libraries described to date have been based on
randomization of CDR3s comprised of 12 amino acids or less (13, 15)
and, therefore, covered only the lower range of camelid CDR3 lengths.
Employing VHs with longer CDR3s, such as BT32/A6, may
provide the level of complexity required for isolation of lead
antibodies specific for a variety of antigens. Libraries containing
shorter and partially randomized CDR3s, such as the C-terminal
residues, could be constructed and pooled with the library described
here to further increase diversity. While exclusion of CDR1 and CDR2
randomization compromises library complexity, construction of primary
libraries based only on CDR3 randomization remains an attractive choice
because of design simplicity. In addition, limited randomization
minimizes the problem of generating theoretical library sizes that
exceed practical library sizes. Libraries constructed on a single
framework offer the advantage of relatively simple affinity maturation
of lead antibodies in that secondary CDR1 and CDR2 libraries can be
generated using single primer pairs.
Our data demonstrated that, for the antigens described here at least,
only a small region of the long CDR3 was available for antigen binding.
The structural basis for this observation could not be determined since
the CDR3 was unstructured, regardless of whether or not the CDR1-CDR3
disulfide bridge was present. It was somewhat surprising to find that
only the C-terminal portion of the CDR3 was involved in antigen binding
since the crystal structure of a camel anti-lysozyme dAb with a similar
CDR3 length showed that the N-terminal half of the CDR3 was involved in
binding (3). Based on this finding it had been suggested that more efficient camelized dAb libraries can be constructed in which the
N-terminal region of CDR3 is randomized leaving the C-terminal side
unchanged. Our finding clearly shows that using camelid dAbs as a guide
to construct camelized libraries can be misleading. It is therefore
wise, before committing to larger libraries, to construct smaller test
libraries in order to map the antigen binding contact area of CDR3.
Larger libraries then can be constructed by only randomizing those
amino acids in CDR3 which are involved in antigen recognition.
As more and more information accumulates on the properties of native
and camelized VHs, it is becoming clear that there are still unresolved issues surrounding factors that contribute to the
solubility and monomeric nature of isolated VHs. The work described here indicates that BT32/A6 VH is a useful
molecule for addressing these issues. Unmodified BT32/A6 VH
is unusually soluble and camelization converts the molecule to one with
excellent solution properties. More detailed structural analysis of the camelized VHs described here is underway and it is expected
that the resulting data will provide useful information for the design of human single domain antibodies that contain a minimum of non-human residues, thereby making them particularly valuable as therapeutic reagents.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Table I), were constructed by standard
protocols.
View larger version (31K):
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Fig. 1.
Sequence of BT32/A6 dAb. The locations
of the CDRs and the NheI and BstEII sites are
bold and underlined. The Kabat numbering system
(37) is used.
Variable residues and solution properties of BT32/A6 VHs
(Table I), that
lacks the bridge. Surfaces were regenerated with 10 mM HCl.
Sensorgram data were analyzed using the BIAevaluation 3.0 software
package (Biacore AB).
,
HNC
C
, and
C
C
(CO)NH for both R3A10.G47I and
M2R2-1.Cys
. The NMR data were processed using
NMRPipe/NMRDraw (32) and analyzed by the use of the NMRView software
program (33). Sequence-specific assignments of the backbone NH,
15N, and
13C
/13C
resonances were achieved only for the residues having strong 1H-15N HSQC cross-peaks through a combined
analysis of the HNC
, HNC
C
,
and C
C
(CO)NH spectra. Chemical-shift
values were referenced internally to the proton resonance of sodium
2,2-dimethyl-2-silapentane-5-sulfonate and indirectly for
15N and 13C assuming
15N/
1H = 0.101329118 and
13C/
1H = 0.251449530 (34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The CDR3 sequences of dAbs isolated by panning against 3B1 scFv
View larger version (18K):
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Fig. 2.
Specificity of the dAbs listed in Table II
for 3B1 scFv. Phage displayed dAbs were tested in an enzyme-linked
immunosorbent assay format using bovine serum albumin-coated wells and
the anti-M2 dAb M2R2 as controls.
The CDR3 sequences of dAbs isolated by panning against M2 IgG in the
absence of DTT
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Fig. 3.
Deconvoluted molecular mass profiles obtained
by mass spectrometry of M2R2-5 after treatment with iodoacetic acid
alone (A) and DTT (B) followed by
iodoacetic acid. The minor peak referred to in the text is
indicated by its molecular mass.
View larger version (17K):
[in a new window]
Fig. 4.
1H-15N NMR HSQC
spectra of R3A10.G47I/Cys (A) and
M2R2-1.Cys
(B),
both with CDR1-CDR3 disulfide bridge deletion (Table I). The
spectrum for R3A10.G47I/Cys
was acquired at pH 6.8 and a
temperature of 308 K and that for M2R2-1.Cys
at pH 6.8 and a temperature of 298 K. Cross-peaks from the CDR1 and CDR2 loops
are boxed and labeled by italicized
letters.
1 s
1 and a dissociation rate
constant of 3.4 × 10
4 s
1. From these
rate constants the dissociation constant of the interaction was
determined to be 1 × 10
6 M.
VH
fragment had significantly enhanced solubility and dramatically
improved spectral properties (Table I). Specifically, all the HSQC
peaks of R3A10.G47I influenced by the presence of DTT were well
resolved in the R3A10.G47I/Cys
spectrum; also HSQC peaks
were detected and assigned to residues such as Ser31,
Tyr32, and Ala33 from the CDR1 loop (Figs. 1
and 4A). In other words, removal of the CDR1-CDR3 disulfide
bridge in this fragment appears to eliminate the conformational
heterogeneity leading to resonance doubling or broadening experienced
by R3A10.G47I. Also, the absence of this disulfide bond does not
interfere with the formation of an intact tertiary structure as a
majority of the HSQC peaks remained at similar positions in both the
R3A10.G47I and R3A10.G47I/Cys
molecules. Fig.
4B shows the HSQC spectrum of M2R2-1.Cys
, a
VH fragment derived from the panel of binding hits after
panning the phage library using M2 antibody as the "antigen" (Table
III). Excluding those from the side chain amides, ~120 HSQC
cross-peaks were observed for both R3A10.G47I/Cys
and
M2R2-1.Cys
and most (>90%) of these HSQC peaks were
assigned, as shown in part in Fig. 4. Very importantly, both CDR1 and
CDR2 residues were at least partially assigned for
R3A10.G47I/Cys
and M2R2-1.Cys
while the
CDR3 residues in both molecules were still missing in the HSQC spectra.
Therefore, the engineered CDR1-CDR3 disulfide bond appears to affect
the conformational behavior of the CDR1 residues, but still leaves the
large CDR3 loop unstructured in solution and mostly undetectable in NMR
HSQC experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Doris Bilous for oligonucleotide synthesis, Joe Michniewicz for DNA sequencing, and Simon Foote for providing the expression and phagemid vectors. We thank Dr. Pierre Thibault and Don Krajcarski for assistance with the mass spectrometry and Drs. Darren Fast, Joycelyn Entwistle, Keith Lewis, and Martin Young for helpful discussions. We acknowledge the technical assistance of Ginette Dubuc.
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FOOTNOTES |
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* This is National Research Council of Canada publication 42431.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.
To whom correspondence should be addressed. Tel.:
613-990-0833; Fax: 613-941-1327; E-mail: roger.mackenzie@nrc.ca.
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M100770200
2 J. Tanha, S. Narang, H. Kaplan, M. Dan, and C. R. MacKenzie, unpublished results.
3 S. J. Foote, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: dAb, single domain antibody; CDR, complementarity determining region; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; Fab, antigen-binding fragment; HSQC, heteronuclear single-quantum coherence spectroscopy; IgG, immunoglobulin G; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PBS, phosphate-buffered saline; scFv, single chain variable fragment of an antibody; SPR, surface plasmon resonance; VH, antibody heavy chain variable domain; VHH, variable domain of a heavy chain antibody; VL, antibody light chain variable domain.
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