antibody VH⁄ VL interaction strength and antigen-binding affinity Kenji Masuda1, Kenzo Sakamoto3, Miki Kojima1,2,3, Takahide Aburatani3, Takuya Ueda1,2 and Hiroshi Ueda1,2,3,4 1 Departme
Trang 1antibody VH⁄ VL interaction strength and antigen-binding affinity
Kenji Masuda1, Kenzo Sakamoto3, Miki Kojima1,2,3, Takahide Aburatani3, Takuya Ueda1,2
and Hiroshi Ueda1,2,3,4
1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
2 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
3 Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo, Japan
4 PRESTO, JST, Kawaguchi, Saitama, Japan
Antibody plays a pivotal role in the humoral immune
response, primarily through the binding of its variable
region to its specific antigen with high affinity In
par-ticular, the antibody variable region (Fv) and its
deriv-atives are receiving increasing attention in many areas,
including diagnostics and therapy, primarily because of
their relative ease of production by many systems, including microbial culture However, because of their heterodimeric domain structure and weak heavy chain variable region fragment (VH)⁄ light chain variable region fragment (VL) interaction, Fv and⁄ or single-chain Fv (scFv) often show problematic physicochemical
Keywords
antibody variable region; antigen–antibody
interaction; combinatorial mutagenesis;
immunoassay; phage display
Correspondence
H Ueda, Department of Chemistry and
Biotechnology, School of Engineering,
University of Tokyo, Tokyo 113–8656, Japan
Fax: +81 3 5841 7362
Tel: +81 3 5841 7362
E-mail: hueda@chembio.t.u-tokyo.ac.jp
(Received 17 January 2006, revised 14
March 2006, accepted 16 March 2006)
doi:10.1111/j.1742-4658.2006.05232.x
While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region frag-ment (VH)⁄ antibody light chain variable region fragment (VL) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak VH⁄ VL interaction strength, in the absence of antigen To investigate the possible relationship between antigen-binding affinity and VH⁄ VL inter-action strength, a novel phage display system that can switch two display modes was employed We focused on the two framework region 2 regions
of the HyHEL-10 VH and VL, facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger VH⁄ VL inter-action The phagemid library, encoding VH gene 7 and VL amber codon gene 9, was used to transform TG-1 (sup+), and the phages displaying functional variable regions were selected The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant contain-ing VH-displaying phages and soluble VL fragment was used to evaluate the VH⁄ VLinteraction strength The results clearly showed the existence of
a key framework region 2 residue (H39) that strongly affects VH⁄ VL inter-action strength, and a marked positive correlation between the antigen-binding affinity and the VH⁄ VL interaction, especially in the presence of a set of particular VL residues The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region
Abbreviations
FR2, framework region 2; Fv, antibody variable region; HEL, hen egg lysozyme; OS, open sandwich; scFv, single-chain Fv; spFv, split antibody variable region; V H , antibody heavy chain variable region fragment; V L , antibody light chain variable region fragment.
Trang 2behavior, even if parental antibody retains superb
sta-bility and affinity For example, many Fv dissociate
into the two domains at low protein concentrations
and are too unstable for many applications at
physio-logical temperature [1] Also, scFv are prone to
spon-taneous dimerization and aggregation, as a result of
their weak VH⁄ VLinteraction, as well as their exposed
interconstant domain surface [2–4] Moreover, some
Fv lose their affinity by tethering with the interdomain
linker, probably because of local or global
conforma-tional change [5]
On the other hand, previously we found that the Fv
domain of anti-hen egg lysozyme (HEL) IgG
(HyHEL-10) has fairly strong antigen-binding affinity (Ka¼
2.5· 108Æm)1) [6] yet very weak VH⁄ VL interaction
strength (Ka<105Æm)1) in the absence of antigen [7] The
stability of the Fv–HEL complex was later shown to be
maintained by many water-mediated hydrogen bonds at
the imperfect VHand VLinterface [8] In contrast, in the
case of another anti-HEL IgG – D1.3 – the VH⁄ VL
interaction is very strong (Ka¼ 1010Æm)1) even in the
absence of antigen [9] Clearly, there should be
struc-tural differences between these two Fvs that make their
heterodimeric interaction strength quite different
How-ever, there has been no attempt to clarify experimentally
the determinant of VH⁄ VL interaction strength or its
relationship with antigen-binding affinity Previously,
the effect of mutations of the VH⁄ VLinterface residues
on antigen binding has been studied using Fab
frag-ments [10,11] However, while the primary effect of such
mutations should be altered VH⁄ VLinteraction strength,
in these studies the effect was unclear because of the
covalently and noncovalently interacting C-terminal
constant domains While Fv and its derivatives are
widely used, to date no attempt has been made to clarify
the effect of the interface mutations on the
antigen-binding affinity To investigate systematically this
unsought relationship between the antigen-binding
affinity of Fv and the VH⁄ VLinteraction strength, in the
present study we employed a novel phage display system
that can switch two display modes suitable for
evaluat-ing either Fv-antigen or VH⁄ VLinteractions [12]
As a target for the analysis, we focused on the two
framework region 2 (FR2) regions of VHand VL, each
facing the domain interface of the anti-lysozyme IgG,
HyHEL-10, a weak VH⁄ VL binder FR2 regions have
been implicated to play an important role in VH⁄ VL
interaction [13] In particular, dromedary antibodies
without light chains show characteristic sequence
alter-ation in VH FR2 to increase hydrophilicity, and are
stable without pairing with VL [14] Here, we made a
combinatorial library in which each FR2 residue
enco-ded was mixed with that of D1.3, a strong VH⁄ VL
binder, to describe the relationship, and also to iden-tify key residues in determining the interdomain inter-action strength
Results
Construction of an FR2 combinatorial library
To investigate the possible relationship between anti-gen-binding affinity and VH⁄ VL interaction strength, a novel phage display system that can switch two display modes, named the split Fv (spFv) system, was employed The spFv system can either display VHand
VL fragments on the N termini of M13 phage coat proteins p9 and p7, respectively, when an amber sup-pressor strain is used for the phage production, or dis-play VHfragment on the phage p9, and simultaneously secrete a his-myc tagged VL fragment to the culture supernatant, when a nonsuppressing strain is used as a host (Fig 1) [12] The ability to switch the two display modes enables side-by-side evaluation of antigen-bind-ing ability and VH⁄ VL interaction strength of the tar-get Fv, and also rapid screening of Fv that is suitable for open sandwich (OS) immunoassay, an immuno-assay that utilizes antigen-dependent Fv stabilization [7] The potential advantage of the spFv system over the conventional display system of monodomains on phage p3 was that it allowed simultaneous mutagenesis
of VH and VL domains, and also rapid evaluation of antigen-binding affinity of phage-displayed Fv frag-ment
For the efficient library selection with the spFv sys-tem, first, the original phagemid pKS1 was modified to stabilize the inserted sequence by inserting two HP ter-minators [15] both upstream and downstream of the spFv coding region, to yield a new vector, pKST2 Based on this vector, a combinatorial library of HyHEL-10 spFv, where each FR2 residue encoded was mixed with a D1.3-type residue, was constructed HyHEL-10 Fv was chosen as a model, not only because it has very low VH–VLaffinity, yet retains high antigen-binding affinity, but also it was efficiently dis-played as spFv or as sole VHon phage, together with soluble VL Out of 16 heavy and 14 light chain FR2 residues, according to the Kabat database [16], 7 and 6 positions, respectively, are different between HyHEL-10 and D1.3 To make a combinatorial library, two oligo-nucleotides encoding degenerate codons coding for both types of amino acids for these positions were used to amplify the 5¢ half of VHand the 3¢ half of VL fragments with degenerated FR2 residues (Fig 2) Using a linker DNA fragment connecting these two,
an overlap-extension PCR was carried out to yield
Trang 3the insert DNA, with a total theoretical diversity of 2.6· 105 The restriction enzyme-digested insert was ligated with pKST2 digested with the same restriction enzymes, which was used to transform electrocompe-tent TG-1(sup+) cells This resulted in a library of colonies with an estimated size of 7· 107, which was considered large enough to cover the theoretical diver-sity The Fv-displaying phages were thus prepared from the harvested cells
Selection of specific antigen binders
To enrich phages displaying functional Fv, a round
of biopanning was performed on immobilized antigen HEL As many combinations of mutations on the conserved FR2 sequence may destabilize the Fv struc-ture, and it might be difficult to remove the resultant nonspecific binders by repeated pannings, we adopted
a combination of a round of biopanning and subse-quent screening of functional Fv by ELISA A mini-mum round of panning would allow selection of binders with a variety of affinity to antigen, which might otherwise be lost After the panning, 1536 colon-ies were picked, and the corresponding monoclonal phages were prepared on 96-well plates and screened
by phage ELISA on HEL-immobilized (specific) and nonimmobilized (blank) wells Among them, 72 clones showing sufficient affinity and specificity with an absorbance of > 0.3, and more than fivefold the blank absorbance, were re-examined for their antigen specif-icity The display efficiencies of VH and VL fragments
Fig 2 The framework region 2 (FR2) library The sequence for FR2
residues, different between HyHEL-10 and D1.3, were mixed to
encode both types of amino acids As a result of degenerated
codon usage, five out of 13 positions encoded two other codons.
Fig 1 Split antibody variable region (spFv) system (A) Structure of the spFv phagemid coding region The vector uses pIX and pVII
of M13 phage to display the antibody heavy chain variable region fragment (VH) and anti-body light chain variable region fragment (V L ) on the phage, respectively An amber codon for the switch of display ⁄ secretion of the VLfragment is marked by an arrowhead (B) Display of antibody variable region (Fv) with sup+strain (TG1) as a host Antigen-binding affinity of the Fv can be evaluated (C) Display of the VHand secretion of tagged V L with sup–strain (HB2151) as a host Measurement of the VH⁄ V L interaction
on the plate immobilized with anti-tag immu-noglobulin is possible.
Trang 4were confirmed by ELISA with immobilized anti-flag
and anti-myc IgG, respectively From these analyses,
64 clones were confirmed to show more than eightfold
specific absorbance than the blank, and for sufficient
display of the two fragments The phagemids for these
clones were extracted from the stock strain and their
nucleotide sequences were determined Because the
clones containing amber codons were not suitable for
subsequent analysis with the nonsuppressing strain, 36
clones without any amber codons in the FR2 region
were chosen and used for further analyses
Evaluation of relative antigen-binding affinity
The relative antigen-binding affinity of 36 clones was
evaluated by phage ELISA after setting the titer of
each clone to 2.5· 108, 1· 109, and 4· 109
colony-forming units (CFU)ÆmL)1 As the widest range of
dis-tribution in absorbance was observed at 1· 109
CFUÆmL)1, we decided to compare the
antigen-bind-ing affinity at this titer, and to use the ratio of specific
absorbance minus background absorbance at this titer
against that of wild-type as an index of the relative
affinity to antigen The ELISA results of representative
three clones are shown in Fig 3A Both clones with
higher and lower signals than the wild-type were
observed at similar frequencies The ELISA signals
and FR2 sequences of all the mutants and the
wild-type, sorted by this index, are summarized in Table 1
It is worth noting that while two clones with low
affin-ity (1D5 and 4F1) had W at H47, the other 34 clones
had Y at this position, with generally higher affinity It
is possible that weak binders with W at this position
were counterselected by the biopanning In addition,
among the 13 highest antigen binders, eight shared a
common four VL residues (L41G, L45R, L48V and
L49K), and the combination was not observed for
lower-affinity clones In addition, 10 out of 13 clones
shared three common residues (L41G, L45R and
L49K), which were not observed in weaker binders
Evaluation of the VH⁄ VLinteraction strength
To evaluate the VH⁄ VLinteraction strength of these 36
clones, phages were used to infect a nonsuppressing
strain, HB2151, to produce culture supernatant
con-taining VH-displaying phage and myc-tagged soluble
VLfragment The culture supernatant was then applied
to either microplate wells immobilized with anti-myc
IgG (specific) or nonimmobilized wells (blank), washed,
and probed with horseradish peroxidase (HRP)-labeled
anti-phage IgG The specific absorbance minus the
blank absorbance was taken as an index of VH⁄ VL
interaction strength (Table 1) Also, to evaluate the antigen dependency of the interaction, HEL at three concentrations was included in the culture supernatant before ELISA The results for the OS ELISA of the representative clones are shown in Fig 3B While some clones showed a similar, or even superior, antigen-dependent increase in absorbance, others showed a decreased or diminished antigen-dependency To evalu-ate the OS-fitness of the clone, the ratio of the specific absorbance in the presence of 10 lgÆmL)1HEL to that
in the absence of HEL was taken as an index
To analyze the effect of the type of each FR2 resi-due on each index, the Student’s t-test was performed (Supplementary material Table S1) According to the test, it was clear that the residue type of H39 domi-nantly affects both VH⁄ VLinteraction strength and the OS-fitness When H39 was Lys (K), as in HyHEL-10, the VH⁄ VL interaction in the absence of antigen was generally weak, while the OS-fitness was high On the
Fig 3 Representative clones obtained after panning (A) Phage ELISA at 1 · 10 9 colony-forming units (CFU) per mL with ⁄ without immobilized antigen (B) Open sandwich (OS) ELISA where the soluble variable region fragment (VL) was immobilized with anti-myc immunoglobulin Binding of the heavy chain variable region fragment (V H )-phage in the presence⁄ absence of hen egg lysozyme (HEL) was detected with horseradish peroxidase (HRP)-anti-M13.
Trang 5contrary, when H39 was Gln (Q), as in D1.3, the
VH⁄ VL interaction was generally strong, while the
OS-fitness was close to 1
Relationship between antigen binding
and VH⁄ VLinteraction
The relationship between the indexes of
antigen-bind-ing affinity and VH⁄ VLinteraction strength was plotted
(Fig 4) When the plot was classified by the type of H39, a clear trend was observed in that the clones with
a stronger VH⁄ VL interaction had Gln at H39 (H39Q group), and those with a weaker interaction had Lys
at H39 (H39K group) While there appeared to be no strong correlation between the antigen-binding affinity and the VH⁄ VL interaction strength determined, five clones in the H39Q group showed both high antigen-binding affinity and strong VH⁄ VL interaction In
Table 1 Result of phage ELISA at 1 · 10 9 colony-forming units (CFU) per mL against hen egg lysozyme (HEL)-immobilized and blank wells, and antibody heavy chain variable region fragment (VH) ⁄ antibody light chain variable region fragment (V L ) interaction strength without HEL (amyc-blank) determined by the split variable region (split Fv) system The results are sorted by the HEL-blank value, and shown with partial framework region 2 (FR2) sequences HyHEL-10-type, D1.3-type and other residues are shown in roman, hatched and in italic, respectively Residues that have possible relationship with antigen binding affinity are shown in bold Values for the wild-type HyHEL-10 are underlined.
Trang 6addition to Gln at H39, these clones shared four
com-mon residues (L41G, L45R, L48V and L49K) in VL,
similar to other high-affinity clones
The relationship between the indexes of
antigen-bind-ing affinity and the OS-fitness was also plotted (Fig 5)
In this plot, a clearer H39-dependency was observed,
possibly because the OS-fitness as an absorbance ratio
contained less experimental error owing to the
expres-sion levels of the VH⁄ VLfragments Apparently, all the
H39Q members show limited antigen-dependency in
VH⁄ VL interaction strength On the contrary, for the
clones in the H39K group, a clone with higher affinity,
as well as higher OS-fitness, was observed, while many
other types of mutants were also observed
SPR analysis of antigen-binding affinity
For the quantitative evaluation of the H39 mutation,
kinetic analysis for antigen–Fv interaction of the
wild-type and H39KQ mutant of purified Fv and scFv proteins was performed using an SPR biosensor As shown in Table 2, in the concentration range of 50–100 nm where the wild-type VH and VL are fully dissociated [7], H39HQ mutant Fv showed 25-fold and approximately eightfold higher association and dissoci-ation rate constants, respectively, than the wild-type
Fv, which resulted in a 3.7-fold higher equilibrium association constant On the contrary, the scFv with the H39KQ mutation showed a similar or reduced association rate and a similar or higher dissociation rate than the wild-type scFv, which resulted in a 0.58-fold equilibrium association constant Apparently, the mutation to strengthen the VH⁄ VL interaction was almost as effective as tethering by the (G4S)3 linker used in scFv, but no synergistic effect in antigen-bind-ing affinity was observed
Discussion
In the present study, we showed a functional analysis
of FR2 residues for the antigen-binding affinity as well
as VH⁄ VLinteraction based on the selected clones from
a combinatorial library Through the construction of a sufficient size of combinatorial library and subsequent analysis, it became clear that a residue near the bottom
of the FR2 loop determines VH⁄ VL interaction strength, as well as its dependency on antigen binding The importance of H39 in Fv stability has been des-cribed for the Fv of M29 antibody [17] Although the
Fv was designed based on HyHEL-10, it is not clear whether or not H39 is dominantly tuning the VH⁄ VL interaction of other Fvs, including HyHEL-10 In addition, the effect of the H39 mutation on antigen binding has not yet been analyzed The reason for gen-erally weak, and stronger, VH⁄ VL interaction of H39K and H39Q group Fvs, respectively, may be ascribed
to their ability to form interchain hydrogen bonds (Fig 6) While no interchain hydrogen bonds origin-ating from H39 lysine are observed in the crystal
Fig 4 Scattered plot of VHand VLinteraction against relative
affin-ity of the Fv to the antigen The plot is classified by the type of
H39, as indicated.
Fig 5 Scattered plot of OS-fitness against relative affinity of the
Fv to the antigen classified as in Fig 4.
Table 2 Kinetic parameters of the wild-type (WT) and H39KQ mutant in variable region (Fv) and single chain Fv (scFv) formats.
kon(10 4 ms)1) koff(10)5s)1) Ka(10 8
M )1)
Fv
H39KQ 8.75 ± 1.39 7.93 ± 2.69 12.4 ± 5.6 H39KQ ⁄ WT 25.3 ± 4.0 7.75 ± 2.63 3.67 ± 1.66 scFv
H39KQ 7.10 ± 0.44 12.0 ± 4.35 6.47 ± 2.53 H39KQ ⁄ WT 0.85 ± 0.05 1.40 ± 0.51 0.58 ± 0.23
Trang 7structure of HyHEL-10, two interchain hydrogen
bonds are formed between two glutamines of H39 and
L38 in the structure of D1.3 In addition, an additional
interchain hydrogen bond (H39Q–L87Y) is possible in
the latter structure
Both H39 and the corresponding VL residue, L38,
are nearly conserved on the genome, and 93% are
glu-tamine in 5355 expressed VH and VL sequences [18]
Probably, a major part of natural Fvs are stabilized by
the hydrogen bonds between them During B-cell
development, clones expressing both chains with
suffi-ciently strong interchain (H–L) interaction are believed
to be selected [19] However, as a result of covalent
linkage of the variable domains through the C-terminal
constant domains, not much is known about the
distri-bution of the VH–VL interaction strength in natural
B-cell repertoire Although our model study suggests
expression of clones with a variety of VH–VL
interac-tion strengths, further study is needed to analyze the
distribution of natural repertoire The five strong
anti-gen binders shared a common VL FR2 sequence in
addition to H39Q In this group of Fvs, a weak
posit-ive correlation between antigen-binding affinity and
VH⁄ VLinteraction strength, in other words, an
appar-ent positive correlation of the stability of the
Fv–anti-gen complex and that of Fv in the absence of antiFv–anti-gen,
was observed Because these four VLFR2 residues seem
to enhance the antigen-binding affinity, irrespective of
H39 type, these VLare optimized for high-affinity anti-gen binding through the mutation at remote sites When both VH and VL fragments are optimized for antigen binding, this ‘increasing the affinity by increas-ing the VH⁄ VL interaction’ might represent a mechan-ism of increasing Fv affinity This is also supported by the kinetic study of purified H39KQ mutant Fv frag-ment, where enhancement in VH⁄ VLinteraction signifi-cantly improved the antigen-binding affinity of Fv, similarly to the level of scFv (Table 2)
Some single VHdomains of anti-protein IgG, inclu-ding HyHEL-10 and D1.3, were known to retain the specificity and affinity to antigen in itself [7,20] At least for these antibodies, the role of the VL domain might be to increase the affinity by supporting the VH domain The phenomenon may also be observed in the chain shuffling experiments As antigen-induced
VH⁄ VL rearrangement is also observed for several Fab fragments [21–23], further verification of the hypothe-sis is needed However, this will not be difficult if the spFv system is utilized
In the present study, we demonstrated that library screening is indeed possible using the spFv system The results presented suggest that the VH⁄ VL interaction strength can be effectively engineered by substituting H39 (or L38) This, in turn, suggests that antibodies previously considered unsuitable to OS-immunoassay can be converted to suitable antibodies by a point mutation (H Ueda, unpublished results) Screening of the mutants with minimal reduction in antigen-binding affinity may also be possible by applying this FR2 engineering approach
Experimental procedures
Materials
The Escherichia coli strains used were XL-10 Gold (Strata-gene, La Jolla, CA, USA) for general cloning, TG1 [supE, hsdD5, thi, D (lac-proAB),⁄ F ¢ traD36, proAB+, lacIq,
HB2151 [ara, D (lac-proAB), thi⁄ F ¢ proAB+
, lacIq, lac-ZDM15] (Amersham Bioscience) for phage display Restric-tion and modificaRestric-tion enzymes were from Takara-Bio (Otsu, Japan), or New England Biolabs (Ipswich, MA, USA) Oligonucleotides were from Espec Oligos (Tsukuba, Japan)
Construction of the spFv phagemid
The spFv expression vector for anti-hen egg lysozyme HyHEL-10 Fv, pKS1(HyHEL-10), was constructed as des-cribed previously [12] To add an SfiI cloning site upstream
A
B
V L
Gln37
Phe87
Gln38
Lys39
Tyr94
Arg38
Lys39
Phe40
VH
V L
Gln37
Tyr87
Gln38
Lys39
Tyr94
Arg38
Gln39
Pro40
VH
Fig 6 3D structures of HyHEL-10 (A) and D1.3 (B) around H39.
Possible hydrogen bonds, calculated by SWISSPDB VIEWER [26], are
shown as dotted lines.
Trang 8of NcoI at the 5¢ end of the VH sequence, the NcoI–EcoRI
fragment of pKS1(HyHEL-10) was transferred to
pCan-tab5E (Amersham Bioscience), denoted pKS2(HyHEL-10)
To avoid instability of the spFv fragment, possibly as a
result of basal expression of VH-p9 and VL-p7 fusion
pro-teins before induction, two glutamine permease terminators
(tHP) [15] were incorporated upstream and downstream of
the spFv coding sequence To insert the terminator into the
SapI site upstream of the lac promoter, four
5¢-phosphoryl-ated oligonucleotides – tHP1 (5¢-AGCGGTACCCGATA
AAAGCGGCTTCCTGAC-3¢), tHP2 (5¢-AGGAGGCCG
AGGTGGGCTGCAAAACAAAACGGCCT-3¢) and tHP4
(5¢-CCTGTCAGGAAGCCGCTTTTATCGGGTACC-3¢) –
were annealed and ligated to SapI-digested
pKS2(HyHEL-10) To insert tHP downstream of the ORFs, tHP4 and
tHP7 (5¢-AATTGGTACCCGATAAAAGCGGCTTCCTG
AC-3¢), as well as tHP2 and tHP8 (5¢-AATTGAGG
TGGGCTGCAAAACAAAACGGCCT-3¢), were annealed
and ligated to the EcoRI-digested plasmid, described above,
resulting in pKST2(HyHEL-10)
Construction of the FR2 library
A combinatorial library (in which each FR2 residue
enco-ded was mixed with that of D1.3) was constructed by
over-lap extension PCR as follows First, a DNA fragment
encoding the N-terminal 55 residues (H1–H55) of
HyHEL-10 VH, whose FR2 residues were designed to be either
HyHEL-10 or D1.3 type (VHFR2), was amplified with
primers MH2BackSfi (5¢-GTCCTCGCAACTGCGGCCC
AGCCGGCCATGGCCSARGTNMAGCTGSAGSAGTC
ACGTACCCCAWSYACTCCAGACSKTTACCTGGARR
TTKACGAAYCCAGCTCCAATAATCACTGGT-3¢) with
pKST2(HyHEL-10) as a template Also, the fragment
encoding L30-L107 of HyHEL-10 with diversified FR2
residues (VLFR2) was similarly amplified with primers
ACAAAAAYMGSRCRAATCTCCTCRGCTCCTGRTCW
(5¢-AGTGAATTCTCATCTTTGACCCCCAGCGATTAT
ACCAA-3¢) As a linker to connect these two, a DNA
encoding H49 to L39 with intervening sequences
(5¢-GATACCAGTGTAGGTTG-3¢) The three fragments
(VH FR2, VL FR2, and linker FR2) were assembled by
splice overlap extension (SOE)-PCR as follows: a thermal
cycling without primer (94C for 5 min, followed by seven
cycles of 94C for 30 s, 55 C for 30 s, and 72 C for
1 min), followed by a normal cycling (30 cycles of 94C
for 30 s, 55C for 30 s, and 72 C for 1 min) with primers
MH2BackSfi and g7EcoFR, and Ex Taq DNA polymerase
(Takara-Bio)
The amplified 0.9 kb fragment encoding both VHand VL (split Fv fragment) was recovered from a 1.5% agarose gel, digested with NcoI and NotI, repurified on the gel and ligated with pKST2 digested with the same enzymes The ligation mix was electroporated to E coli TG-1, and plated
on a YTAG agar plate (8 gÆL)1 tryptone, 5 gÆL)1 yeast extract, 5 gÆL)1 NaCl, 100 lgÆmL)1ampicillin, 1% glucose,
15 gÆL)1agar) Several colonies were selected for extraction
of the phagemid to check the quality of the library Nucleo-tide sequencing was performed using a 3100 Genetic Analyzer (Applied Biosystems, Tokyo, Japan) and BigDye Terminator Cycle Sequencing Kit (Applied Biosystems)
(5¢-ACAGCTATCGCGATTGCAGTG-3¢)
Preparation of phage library pKST2 vector digested with NcoI and NotI (2.13 pmol), and the split Fv fragment diges-ted with the same (21.3 pmol), was ligadiges-ted by adding 0.5 vol
of Ligation High (Toyobo, Osaka, Japan) and incubated
at 16C for 2 h After ethanol precipitation, the pellet was dissolved in 20 lL of Milli-Q water The solution was divi-ded into two, each was mixed with 100 lL of electrocompe-tent TG-1 cells, and electroporated with Easyject (EquiBio, Ashford, UK) with 2 mm cuvettes Then, 900 lL of 2YT medium was added, and the solution was transferred to a microtube and incubated at 37C for 30 min One microli-tre was taken for colony counting after serial dilution, and the rest was plated onto a YTAG agar large square plate (Sumitomo Bakelite, Tokyo, Japan), and incubated at 30C for 16 h The TG-1 colonies on the plate were harvested with 5 mL of 2YT (16 gÆL)1 tryptone, 10 gÆL)1 yeast extract, 5 gÆL)1NaCl, pH 7.6), mixed and stored at)80 C after adding 0.5 vol of 50% glycerol, except for 50 lL, which was used to inoculate 100 mL of 2YTAG, and shaken at 37C until the attenuance (D) at 600 nm reached 0.5 Then, 10 mL of culture was added with helper phage, M13KO7, at a multiplicity of infection (m.o.i.) of 20, incu-bated without shaking at 37C for 30 min, and centrifuged
in a desktop centrifuge at 760 g for 15 min at 4C After removal of the supernatant, the pellet was resuspended in
50 mL of 2YT containing 100 lgÆmL)1 ampicillin and
50 lgÆmL)1kanamycin (2YTAK) medium in a 500 mL baf-fled flask, and vigorously shaken at 30C, 250 r.p.m for
20 h After incubation, the culture was centrifuged at 6500 g for 10 min, 0.2 vol of 20% polyethylene glycol 6000⁄ 2.5 m NaCl (PEG⁄ NaCl) was added to the supernatant and incu-bated on ice for 1 h After centrifugation (6500 g, 4C,
30 min), the pellet was resuspended in 1 mL of 10 mm Tris⁄ HCl, 1 mm EDTA, pH 8.0 (TE), centrifuged at
15 000 g, 4C for 20 min, and the supernatant was used as the phage library
Biopanning and phage ELISA
Thirty wells of Falcon 3912 microplate (Becton Dickinson, Oxnard, CA, USA) were coated overnight with 100 lL per
Trang 9well of 10 lgÆmL)1 HEL in 10 mm NaCl⁄ Pi, blocked at
room temperature for 2 h with NaCl⁄ Pi containing 2%
skim milk (2% MPBS), washed three times with NaCl⁄ Pi
containing 0.1% Tween-20 (PBST), the phage library (1011
CFU) in 1% MPBS was added to the well and incubated
at 30C for 90 min After discarding the phage solution,
200 lL of PBST was added and incubated for 1 min This
was repeated once, and twice with 200 lL NaCl⁄ Pi Then,
1 mgÆmL)1 BSA was added and incubated for 10 min, to
elute well-bound phages The eluate was recovered and
neutralized with 1 vol of 2 m Tris base
In the case of phage ELISA, the plates, after
incuba-tion with phage, were washed three times with PBST and
incubated at room temperature for 1 h with 100 lL per
well of 5000-fold diluted HRP-conjugated mouse anti-M13
(Amersham Bioscience) in MPBS The plate was washed
per well of substrate solution (100 lgÆmL)1
3,3¢,5,5¢-tetramethylbenzidine; Sigma, Tokyo, Japan; 0.04 lLÆmL)1
H2O2, in 100 mm NaOAc, pH 6.0) After incubation
for 5–30 min, the reaction was stopped with 50 lL
per well of 1 m sulfuric acid and the absorbance read
at 450 nm, with the absorbance at 650 nm used as a
control
Measurement of VH⁄ VLinteraction strength
HB2151 cells carrying each spFv-encoding phagemid were
used to prepare culture supernatant containing VH
-display-ing phage and soluble VL The overnight culture was
centri-fuged at 6500 g for 30 min and the supernatant was
recovered and stored at 4C
To perform OS-ELISA, 100 mL per well of 1 lgÆmL)1
anti-myc (9E10) IgG was coated overnight in NaCl⁄ Pi
After blocking at room temperature for 2 h with MPBS,
the plate was washed three times with PBST and incubated
at room temperature for 1 h with 100 lL per well of
culture supernatant mixed with HEL, if necessary, which
was mixed for 1 h before and preincubated The plate was
washed six times with PBST and phages were detected as
described above Data are presented as an average of three
measurements
Preparation of Fv⁄ single chain Fv and kinetic
analysis with Biacore
To prepare soluble scFv fragment, primers M13RV and
ScFvH10VHF (5¢-CCAGAGCCACCTCCGCCTGAACCG
VLbk (5¢-CAGGCGGAGGTGGCTCTGGCGGTGGCGG
ATCGACGGACATTGAGCTCAC-3¢) and SplitVLSeqFor
used to amplify the fragments encoding linker-tagged VH
and VL, respectively The gel-purified fragments were
assembled by SOE PCR, digested with NcoI and NotI, and ligated with pET20b digested with the same To express Fv,
a fragment encoding a stop codon and Shine–Dalgarno sequence were inserted between BstEII and Sse8387I sites
of the above plasmid, which was amplified with primers VHBstBack (5¢-GGGACCACGGTCACCGTCTCGAGCT GAGCTGCTGACTAC-3¢), H10VkNotFor (5¢-AGCCGC GGCCGCGCTTATTTCCAGCGCGGTCCCCCCTCC-3¢) and pKST2(HyHEL-10) as a template, and digested by the same enzymes The H39KQ mutation was introduced by the QuikChange mutagenesis kit (Stratagene) using primers H39KQU (5¢-TTGGAGCTGGATACGGCAATTCCCAG GGA-3¢) and H39KQD (5¢-TCCCTGGGAATTGCCGT
sequences, BL21(DE3, pLysS) was transformed with the plasmid, cultured stepwise into 100 mL of Luria–Bertani medium (LB; containing 100 lgÆmL)1 ampicillin and
34 lgÆmL)1 chloramphenicol) at 27C until the D600 reached 0.5, when the culture was induced with 0.1 mm iso-propyl thio-b-d-galactoside After shaking for 16 h at
27C, the culture supernatant was precipitated with 65% saturated ammonium sulfate, and the scFv protein was purified from the precipitate using Talon affinity resin (BD Bioscience, Tokyo, Japan) according to the manufacturer’s protocol Alternatively, the protein was purified using a HEL affinity column made from HiTrap NHS-activated
HP column (Amersham Biosciences) essentially as described previously [24]
The purified protein was quantified based on the absorb-ance at 280 nm [25], and subjected to SPR kinetic analysis with Biacore 2000 at 25C with 300 resonance units of immobilized HEL on a CM5 sensorchip, and HBS-EP (Biacore, Tokyo, Japan) as a buffer at a flow rate of
20 lLÆmin)1 Three measurements were performed for each analyte concentration of 50 and 100 nm, and kinetic and equilibrium constants were calculated using biaevaluation 4.1 program (Biacore)
Acknowledgements
We are grateful to Drs Shinya Tsukiji and Teruyuki Nagamune for allowing the use of Biacore This work was supported by a Grant-in-Aid for Scientific Research (B14350430, B17360394) from JSPS, and a Grant-in-Aid for Exploratory Research (14655303) from MEXT, Japan
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