Báo cáo khoa học: The role of interface framework residues in determining antibody VH ⁄ VL interaction strength and antigen-binding affinity pptx

The role of interface framework residues in determining 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 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo, Japan PRESTO, JST, Kawaguchi, Saitama, Japan 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 5841 7362 Tel: +81 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 fragment (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 interaction strength, a novel phage display system that can switch two display modes was employed We focused on the two framework region 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 residue was mixed with that of D1.3, which has a far stronger VH ⁄ VL interaction The phagemid library, encoding VH gene 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 containing VH-displaying phages and soluble VL fragment was used to evaluate the VH ⁄ VL interaction strength The results clearly showed the existence of a key framework region residue (H39) that strongly affects VH ⁄ VL interaction strength, and a marked positive correlation between the antigenbinding 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 wildtype variable region was also confirmed by a SPR biosensor as a severalfold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region 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 particular, the antibody variable region (Fv) and its derivatives 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 singlechain Fv (scFv) often show problematic physicochemical 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; VH, antibody heavy chain variable region fragment; VL, antibody light chain variable region fragment 2184 FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS VH ⁄ VL interaction and affinity K Masuda et al behavior, even if parental antibody retains superb stability and affinity For example, many Fv dissociate into the two domains at low protein concentrations and are too unstable for many applications at physiological temperature [1] Also, scFv are prone to spontaneous dimerization and aggregation, as a result of their weak VH ⁄ VL interaction, 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 conformational change [5] On the other hand, previously we found that the Fv domain of anti-hen egg lysozyme (HEL) IgG (HyHEL10) has fairly strong antigen-binding affinity (Ka ¼ 2.5 · 108Ỉm)1) [6] yet very weak VH ⁄ VL interaction strength (Ka 0.3, and more than fivefold the blank absorbance, were re-examined for their antigen specificity The display efficiencies of VH and VL fragments FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS VH ⁄ VL interaction and affinity K Masuda et al were 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, · 109, and · 109 colonyforming units (CFU)ỈmL)1 As the widest range of distribution in absorbance was observed at · 109 CFmL)1, we decided to compare the antigen-binding 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 wildtype, sorted by this index, are summarized in Table It is worth noting that while two clones with low affinity (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 ⁄ VL interaction strength To evaluate the VH ⁄ VL interaction strength of these 36 clones, phages were used to infect a nonsuppressing strain, HB2151, to produce culture supernatant containing VH-displaying phage and myc-tagged soluble VL fragment 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 Fig Representative clones obtained after panning (A) Phage ELISA at · 109 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 antimyc immunoglobulin Binding of the heavy chain variable region fragment (VH)-phage in the presence ⁄ absence of hen egg lysozyme (HEL) was detected with horseradish peroxidase (HRP)-anti-M13 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, antigendependent increase in absorbance, others showed a decreased or diminished antigen-dependency To evaluate the OS-fitness of the clone, the ratio of the specific absorbance in the presence of 10 lgỈmL)1 HEL to that in the absence of HEL was taken as an index To analyze the effect of the type of each FR2 residue 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 dominantly affects both VH ⁄ VL interaction 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 FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS 2187 VH ⁄ VL interaction and affinity K Masuda et al Table Result of phage ELISA at · 109 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 (VL) 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 (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 contrary, when H39 was Gln (Q), as in D1.3, the VH ⁄ VL interaction was generally strong, while the OS-fitness was close to Relationship between antigen binding and VH ⁄ VL interaction The relationship between the indexes of antigen-binding affinity and VH ⁄ VL interaction strength was plotted 2188 (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 antigenbinding affinity and strong VH ⁄ VL interaction In FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS VH ⁄ VL interaction and affinity K Masuda et al Table Kinetic parameters of the wild-type (WT) and H39KQ mutant in variable region (Fv) and single chain Fv (scFv) formats kon (104 ms)1) Fv WT H39KQ H39KQ ⁄ WT scFv WT H39KQ H39KQ ⁄ WT Fig Scattered plot of VH and VL interaction against relative affinity of the Fv to the antigen The plot is classified by the type of H39, as indicated Fig Scattered plot of OS-fitness against relative affinity of the Fv to the antigen classified as in Fig addition to Gln at H39, these clones shared four common residues (L41G, L45R, L48V and L49K) in VL, similar to other high-affinity clones The relationship between the indexes of antigen-binding 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 expression levels of the VH ⁄ VL fragments 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 koff (10)5 s)1) Ka (108 0.35 ± 0.08 8.75 ± 1.39 25.3 ± 4.0 1.02 ± 0.01 7.93 ± 2.69 7.75 ± 2.63 3.83 ± 0.83 12.4 ± 5.6 3.67 ± 1.66 8.31 ± 0.20 7.10 ± 0.44 0.85 ± 0.05 8.61 ± 3.96 12.0 ± 4.35 1.40 ± 0.51 11.2 ± 5.1 6.47 ± 2.53 0.58 ± 0.23 M )1 ) 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 dissociation 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 0.58fold 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-binding 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 ⁄ VL interaction 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 described 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 generally 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 originating from H39 lysine are observed in the crystal FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS 2189 VH ⁄ VL interaction and affinity K Masuda et al A VL Arg38 Phe87 Gln37 Tyr94 Gln38 Phe40 Lys39 Lys39 VH B VL Tyr87 Gln37 Tyr94 Arg38 Gln38 Lys39 Gln39 VH Pro40 Fig 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 structure 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 glutamine 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 sufficiently 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 distribution 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 interaction strengths, further study is needed to analyze the distribution of natural repertoire The five strong antigen binders shared a common VL FR2 sequence in addition to H39Q In this group of Fvs, a weak positive correlation between antigen-binding affinity and VH ⁄ VL interaction strength, in other words, an apparent positive correlation of the stability of the Fv–antigen complex and that of Fv in the absence of antigen, was observed Because these four VL FR2 residues seem to enhance the antigen-binding affinity, irrespective of 2190 H39 type, these VL are optimized for high-affinity antigen binding through the mutation at remote sites When both VH and VL fragments are optimized for antigen binding, this ‘increasing the affinity by increasing the VH ⁄ VL interaction’ might represent a mechanism of increasing Fv affinity This is also supported by the kinetic study of purified H39KQ mutant Fv fragment, where enhancement in VH ⁄ VL interaction significantly improved the antigen-binding affinity of Fv, similarly to the level of scFv (Table 2) Some single VH domains of anti-protein IgG, including 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 hypothesis 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 (Stratagene, La Jolla, CA, USA) for general cloning, TG1 [supE, hsdD5, thi, D (lac-proAB), ⁄ F¢ traD36, proAB+, lacIq, lacZDM15] (Amersham Bioscience, Tokyo, Japan) and HB2151 [ara, D (lac-proAB), thi ⁄ F¢ proAB+, lacIq, lacZDM15] (Amersham Bioscience) for phage display Restriction and modification 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 described previously [12] To add an SfiI cloning site upstream FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS VH ⁄ VL interaction and affinity K Masuda et al of NcoI at the 5¢ end of the VH sequence, the NcoI–EcoRI fragment of pKS1(HyHEL-10) was transferred to pCantab5E (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 proteins 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¢-phosphorylated oligonucleotides – tHP1 (5¢-AGCGGTACCCGATA AAAGCGGCTTCCTGAC-3¢), tHP2 (5¢-AGGAGGCCG TTTTGTTTTGCAGCCCACCTC-3¢), tHP3 (5¢-GCTG AGGTGGGCTGCAAAACAAAACGGCCT-3¢) and tHP4 (5¢-CCTGTCAGGAAGCCGCTTTTATCGGGTACC-3¢) – were annealed and ligated to SapI-digested pKS2(HyHEL10) 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 encoded was mixed with that of D1.3) was constructed by overlap extension PCR as follows First, a DNA fragment encoding the N-terminal 55 residues (H1–H55) of HyHEL10 VH, whose FR2 residues were designed to be either HyHEL-10 or D1.3 type (VHFR2), was amplified with primers MH2BackSfi (5¢-GTCCTCGCAACTGCGGCCC AGCCGGCCATGGCCSARGTNMAGCTGSAGSAGTC WGG-3¢) and H10VHframe2 (5¢-ACCACTGTAGCTT 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 H10VLframe2 (5¢-GGCAACAACCTACACTGGTATCA ACAAAAAYMGSRCRAATCTCCTCRGCTCCTGRTCW AKTATGCTTCCCAGTCCATCTCT-3¢) and g7EcoFor (5¢-AGTGAATTCTCATCTTTGACCCCCAGCGATTAT ACCAA-3¢) As a linker to connect these two, a DNA encoding H49 to L39 with intervening sequences (linkerFR2) was also amplified with primers H10linkRV (5¢-GGGTACGTAAGCTACAGTG-3¢) and H10linkFR (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 (94 °C for min, followed by seven cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for min), followed by a normal cycling (30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for min) with primers MH2BackSfi and g7EcoFR, and Ex Taq DNA polymerase (Takara-Bio) The amplified 0.9 kb fragment encoding both VH and 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 gỈL)1 tryptone, gỈL)1 yeast extract, gỈL)1 NaCl, 100 lgỈmL)1 ampicillin, 1% glucose, 15 gỈL)1 agar) Several colonies were selected for extraction of the phagemid to check the quality of the library Nucleotide sequencing was performed using a 3100 Genetic Analyzer (Applied Biosystems, Tokyo, Japan) and BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with primers M13RV (Takara-Bio) and OmpARV (5¢-ACAGCTATCGCGATTGCAGTG-3¢) Preparation of phage library pKST2 vector digested with NcoI and NotI (2.13 pmol), and the split Fv fragment digested with the same (21.3 pmol), was ligated by adding 0.5 vol of Ligation High (Toyobo, Osaka, Japan) and incubated at 16 °C for h After ethanol precipitation, the pellet was dissolved in 20 lL of Milli-Q water The solution was divided into two, each was mixed with 100 lL of electrocompetent TG-1 cells, and electroporated with Easyject (EquiBio, Ashford, UK) with mm cuvettes Then, 900 lL of 2YT medium was added, and the solution was transferred to a microtube and incubated at 37 °C for 30 One microlitre 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 30 °C for 16 h The TG-1 colonies on the plate were harvested with mL of 2YT (16 gỈL)1 tryptone, 10 gỈL)1 yeast extract, gỈL)1 NaCl, 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 37 °C 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, incubated without shaking at 37 °C for 30 min, and centrifuged in a desktop centrifuge at 760 g for 15 at °C After removal of the supernatant, the pellet was resuspended in 50 mL of 2YT containing 100 lgỈmL)1 ampicillin and 50 lgỈmL)1 kanamycin (2YTAK) medium in a 500 mL baffled flask, and vigorously shaken at 30 °C, 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 incubated on ice for h After centrifugation (6500 g, °C, 30 min), the pellet was resuspended in mL of 10 mm Tris ⁄ HCl, mm EDTA, pH 8.0 (TE), centrifuged at 15 000 g, °C 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 FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS 2191 VH ⁄ VL interaction and affinity K Masuda et al well of 10 lgỈmL)1 HEL in 10 mm NaCl ⁄ Pi, blocked at room temperature for 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 30 °C for 90 After discarding the phage solution, 200 lL of PBST was added and incubated for This was repeated once, and twice with 200 lL NaCl ⁄ Pi Then, 100 lL of 0.2 m glycine-HCl (pH 2.2) containing mgỈmL)1 BSA was added and incubated for 10 min, to elute well-bound phages The eluate was recovered and neutralized with vol of m Tris base In the case of phage ELISA, the plates, after incubation with phage, were washed three times with PBST and incubated at room temperature for h with 100 lL per well of 5000-fold diluted HRP-conjugated mouse anti-M13 (Amersham Bioscience) in MPBS The plate was washed three times with PBST and developed with 100 lL per well of substrate solution (100 lgặmL)1 3,3Â,5,5Âtetramethylbenzidine; Sigma, Tokyo, Japan; 0.04 lLặmL)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 m sulfuric acid and the absorbance read at 450 nm, with the absorbance at 650 nm used as a control Measurement of VH ⁄ VL interaction strength HB2151 cells carrying each spFv-encoding phagemid were used to prepare culture supernatant containing VH-displaying phage and soluble VL The overnight culture was centrifuged at 6500 g for 30 and the supernatant was recovered and stored at °C To perform OS-ELISA, 100 mL per well of lgỈmL)1 anti-myc (9E10) IgG was coated overnight in NaCl ⁄ Pi After blocking at room temperature for h with MPBS, the plate was washed three times with PBST and incubated at room temperature for h with 100 lL per well of culture supernatant mixed with HEL, if necessary, which was mixed for 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 CCTCCACCGCTCGAGACGGTGACCG-3¢), ScFvH10 VLbk (5¢-CAGGCGGAGGTGGCTCTGGCGGTGGCGG ATCGACGGACATTGAGCTCAC-3¢) and SplitVLSeqFor (5¢-CCTCTTCTGAGATGAGTTTTTGTTCT-3¢) were used to amplify the fragments encoding linker-tagged VH and VL, respectively The gel-purified fragments were 2192 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 ATCCAGCTCCAA-3¢) After confirmation of the sequences, BL21(DE3, pLysS) was transformed with the plasmid, cultured stepwise into 100 mL of Luria–Bertani medium (LB; containing 100 lgỈmL)1 ampicillin and 34 lgỈmL)1 chloramphenicol) at 27 °C until the D600 reached 0.5, when the culture was induced with 0.1 mm isopropyl thio-b-d-galactoside After shaking for 16 h at 27 °C, 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 absorbance at 280 nm [25], and subjected to SPR kinetic analysis with Biacore 2000 at 25 °C 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 lLỈmin)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 References Glockshuber R, Schmidt T & Pluckthun A (1992) The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli Biochemistry 31, 1270–1279 FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS VH ⁄ VL interaction and affinity K Masuda et al Reiter Y, Brinkmann U, Kreitman RJ, Jung SH, Lee B & 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Supplementary material Table S1 Summary of the Student’s t-test for the effect of individual framework region (FR2) substitutions This material is available as part of the online article from http://www.blackwell-synergy.com The following supplementary material is available online: 2194 FEBS Journal 273 (2006) 2184–2194 ª 2006 The Authors Journal compilation ª 2006 FEBS ... observed in weaker binders Evaluation of the VH ⁄ VL interaction strength To evaluate the VH ⁄ VL interaction strength of these 36 clones, phages were used to infect a nonsuppressing strain, HB2151,... strong VH ⁄ VL binder, to describe the relationship, and also to identify key residues in determining the interdomain interaction strength Results Construction of an FR2 combinatorial library To investigate... evaluating either Fv-antigen or VH ⁄ VL interactions [12] As a target for the analysis, we focused on the two framework region (FR2) regions of VH and VL, each facing the domain interface of the anti-lysozyme