Protein Engineering vol.10 no.11 pp.

1311–1318, 1997

Novel selection method for engineered antibodies using the mechanism of Fv fragment stabilization in the presence of antigen

Kouhei Tsumoto1, Yoshiyuki Nishimiya1, Nobuhiro Kasai1, Hiroshi Ueda2, Teruyuki Nagamune2, Kyoko Ogasahara3, Katsuhide Yutani3, Kenji Tokuhisa4, Masaaki Matsushima4 and Izumi Kumagai1,5

of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-77, 2Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bunkyo-ku 113, 3Institute for Protein Research, Osaka University, Suita, Osaka 565, and 4Rational Drug Design Laboratories, Matsukawa, Fukushima 960-12, Japan whom correspondence should be addressed


Although the heavy and light chain domains of some antibody variable region fragments (Fvs) readily dissociate under physiological conditions, the Fvs are stable in the presence of antigen. This ‘antigen-driven Fv stabilization mechanism’ was applied to the selection of clones with specificity toward target antigens. The results can be summarized as follows. (i) Some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of anti-hen egg white lysozyme (HEL) monoclonal antibody HyHEL10 heavy chain variable region (VH) were randomized. (ii) The randomized VH fragments of HyHEL10 were displayed on a filamentous bacteriophage and mixed with the target antigen, before being applied to a light chain variable region (VL) which was immobilized on microtiter plates and subjected to selection by panning. (iii) After four rounds of panning, four clones that showed significant binding to human lysozyme (hL), which HyHEL10 recognized poorly, were selected from the HCDR2 library. (iv) The soluble Fv fragments selected were expressed in Escherichia coli, purified, and subjected to an inhibition assay of lysozyme enzymatic activities and an isothermal titration calorimetry. These Fv fragments had increased affinity toward hL, and thermodynamic analysis suggested that the reduced entropy loss due to binding by the replacement of residues in HCDR2 resulted in the higher hL binding activity. Keywords: antibody engineering/phage display/complementarity determining regions/Fv fragment/isothermal titration calorimetry

Introduction One of the major goals of antibody engineering is the development of a procedure to graft specific function into an antibody and to enhance the affinity toward the target antigen (Winter and Milstein, 1991). Antibodies may be regarded as a protein engineering system perfected by nature for the generation of a virtually unlimited repertoire of complementary molecular surfaces (Mariuzza and Poljak, 1993), and the structural variation of the antigen binding site is mainly confined to the complementarity determining regions (CDRs). The antibody binding site is formed by a small number of protein segments of variable structure (the CDRs) grafted onto a scaffolding of
© Oxford University Press

essentially invariant architecture (framework regions) (Padlan, 1994), and this characteristic has been critical to the success of artificial functional conversion of antibodies (Verhoeyen et al., 1988). The display of antibody fragments on the surface of filamentous bacteriophages by fusing them to a minor coat protein of the phage (pIII) (Smith, 1985; Scott and Smith 1990), and subsequent selection of the phage with antigen (by panning) has provided a powerful means of making antibodies of predefined binding specificity from V gene repertoires (Winter et al., 1994). Many approaches have been tried for obtaining a novel antibody (Marks et al., 1991; Kruif et al., 1995) or a functionally improved antibody (Hawkins et al., 1992; Riechman and Weill, 1993; Deng et al., 1995; Yang et al., 1995). In addition, the chain shuffling method (Kang et al., 1991; Marks et al., 1992) has been proposed; a new library is constructed and is subjected to selection by fixing one variable chain obtained from the naive library and shuffling the other chain from V genes repertoires. Most Fv fragments of immunoglobulins readily dissociate under physiological conditions. To overcome the instability of Fv fragments, linking domains with peptide linkers (single chain Fv, scFv) (Glockshuber et al., 1990) or disulfide bond (disulfide stabilized Fv, dsFv) (Brinkmann et al., 1993) has been attempted; however, Fv fragments are usually stable in the presence of their antigens (Glockshuber et al., 1990, Ueda et al., 1996). On the basis of the antigen driven stabilization of Fv, we have recently proposed the novel ‘open sandwich’ method (Ueda et al., 1996). This method is an alternative sandwich method for determining antigen concentration. Its sensitivity is relatively high, and the background is significantly lower than for the usual sandwich method. Thus, ‘open sandwich’ is expected to be an alternative and powerful method for the functional conversion and improvement of antibodies by chain shuffling. We have focused on the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody, HyHEL10. The interaction is structurally well studied (Padlan et al., 1989) and therefore is most suitable for the precise analysis of antigen–antibody interactions. Previously we reported a thermodynamical analysis that combined bacterial expression system and site-directed mutagenesis (Tsumoto et al., 1994a,b, 1995, 1996). For convenient analysis of antigen–antibody interactions, a novel phage display system has been developed (Maenaka et al., 1996), and using this system, the HyHEL10 single chain variable region (scFv) fragment and the variable region of the heavy chain (VH) can be stably displayed on a filamentous phage. Although HyHEL10 recognizes several avian lysozymes (Smith-Gill et al., 1984a,b), the Fv fragment poorly recognizes human lysozyme (hL). As shown in Figure 1, the most significant difference in amino acid residues between the HEL epitope region recognized by HyHEL10 and the corresponding one of hL lies in the loop at site 98–102 of HEL, and the local 1311

L and H in the bottom column represent the sites recognized by the light and heavy chains respectively. 1312 . (A) Only residues recognized by HyHEL10 are shown.. the correct numbering of Trp63 to Pro102 in hL is Trp64 to Pro103. Comparison of the hL and HEL local structures recognized by HyHEL10. HLZ appeared in the figure represents hL. 1. and blue lines and zones those of HyHEL10 Fv. and the main chain and side chain of hL are shown by white lines and zones.K. The drawing is from the Protein Data Bank (3HFM). (C) HEL of (B) is replaced with hL. (B) X-Ray crystallographic analysis of the HyHEL10 Fv fragment-HEL complex (Padlan et al. HEWL appeared in the figure represents HEL. Numbering of amino acid residues is based on that of HEL. Fig. Red lines and zones represent the main chain and side chain of HEL. Since one residue is inserted between the 46th and 47th positions in hL.Tsumoto et al. 1989).

1989) supplemented with 200 µg/ml ampicillin.5 U Taq DNA polymerase (Perkin Elmer) in the manufacturer’s recommended buffer. HyHEL10. After removing the solution. Construction of HyHEL10 randomized VH fragments The HCDR2 of HyHEL10 was randomized by PCR. which had been mixed with hL or HEL and prediluted with 1 volume of binding buffer 30 min before. The phage was prepared by centrifugation and PEG precipitation and was subjected to the next selection stage. To graft specificity toward hL into the Fv fragment of HyHEL10. 100 µl of 10 µg/ml biotinylated VL was added to the wells. followed by ligation with the vector. Prior to use for PCR. clones on plates were cultured and phage VH was prepared according to the method described previously (Maenaka et al. K and H represent the mixtures of AGCT. and mixed to a final concentration of 25 pmol/ml (N. (Tokyo). Horseradish peroxidase (HRP)-conjugated anti-M13 mouse antibody was from Pharmacia Biotec Inc. The PCR reaction was carried out in 100 µl of 250 nM dNTP. From 200 ml of culture. The PCR amplified product was subjected to agarose gel electrophoresis.. Detection of binding activity of phage VH by enzyme-linked immunosorbent assay (ELISA) ELISA was performed in principle according to the manual of RPAS detection module (Pharmacia). until the early stationary phase was reached. The transformant E. After 1 h. 2. M13KO7. 1991. displayed on a filamentous bacteriophage and subjected to the selection of desired clones. and reinfected into early log-phase E. After discarding the buffer. and digested with XhoI and SacII. infected with helper phage. Absorbance was measured at 410 nm using a microplate reader (Bio-Rad. Phage VH was recovered by PEG precipitation (Maenaka et al. The plate was washed six times with PBST.. 200 µl SuperBlock (Piearce) was added to each well and incubated for 1 h at room temperature. 1996). with 25 pmol of each primer. 10 ng pTZPsFv2 as a template and 0. Streptavidin was purchased from Sigma.coli strain BL21(DE3) harboring a plasmid clone was grown at 28°C in 2 YT (Sambrook et al. M13KO7 (Promega). The Escherichia coli strain JM109 was transformed with pTZPsVH2-ran. 1994). After three washes with PBST. 100 µl of 5000 times diluted horseradish peroxidase (HRP)-anti M13 (Pharmacia) was added and incubated for 1 h. pTZPsVH2 at the XhoI/SacII site using T4 DNA ligase (Boehringer). One hundred microliters of 10 µg/ml HyHEL10 VL fragment in PBS was applied to each well of the microtiter plates and incubated for 1 h at room temperature.2 -azinobis(3ethlbenzthiazoline-sulforic acid (ABTS) was from Wako Junyaku Inc. GT and ACT respectively).5) containing 500 mM NaCl. especially to HTyr53. After removing the solution from the wells. two fractions (bacterial supernatant and periplasmic fractions) were separated as follows (Tsumoto 1313 . The plasmid. recovered from the gel with Geneclean II (Bio101). phage VH premixed with soluble hL at room temperature for 30 min was applied to the VL-immobilized microtiter plate wells and incubated at room temperature for 1 h.. pTZPsFv2. Toyobo or Boehringer. using 630 nm as the control. Ser56 and Tyr58 in the CDR2 of the VH (HCDR2). DNA oligonucleotides for randomization of HyHEL10 VH and polymerase chain reaction (PCR) were synthesized using a 380A DNA synthesizer (Perkin Elmer). which is the modified phagemid vector of pLFv constructed for the phage display of HyHEL10 scFv (Maenaka et al. The randomized VH fragment was purified using Streptavidin Beads (Promega). Song et al. 55°C for 1.5 min and 72°C for 2 min) using a thermal cycler MP3000 (Takara). Increased affinity toward hL into the antiHEL monoclonal antibody. To induce the expression of soluble scFv. Colonies were transferred into an LB culture containing 100 mg/l ampicillin. and dissolved in 500 µl phosphate-buffered saline (PBS) per 20 ml culture. The PCR reaction was cycled 35 times (94°C for 1 min. DNA sequencing Di-deoxy reactions for DNA sequencing were performed using an Auto read Sequencing Kit (Pharmacia) according to the recommendations of the manufacturer. The variable region of the light chain (VL) fragment of HyHEL10 was prepared and biotinylated as described previously (Ueda et al. After washing the wells with PBS containing 0. 1996).. the back primer HCDR2RANBACK (5 -CGCCTCGAGTACATGGGCTACGTCAGCNNKNNKGGCNNKACCTHCTACAACCCC-3 ) and forward primer HyHELFOR (5 ACCCGCGGAGACGGTGACGAGGGTGCC-3 ) were biotinylated using a 5 -biotinylation kit (Amersham). and was designated pTZPsVH2-ran. After four stages. All other reagents were of biochemical research grade. 1996). infected with helper phage.. the Fv fragment could be introduced by the method described here. The number of clones present on the plates were counted at each stage. isopropyl β-D-thiogalactopyranoside (IPTG) was added to 1 mM and the culture was grown overnight at 28°C. a His 6 peptide tag was added to the C-terminus of the VH chain. and eluted with 100 mM Glycine (pH 2. Eluate was rapidly neutralized with 100 mM Tris–HCl (pH 8.. was used as a template. HEL was obtained from Seikagaku-kogyo (Tokyo). and expressed as a scFv fragment (Tsumoto et al. After washing twice with PBST. 1996). Analysis of DNA sequences was performed using an ALF express auto-read sequencer (Pharmacia). and then incubated overnight at 37°C. (Osaka).0).05% Tween-20 (PBST).coli JM109. Biotin-NHS was from Boehringer. Materials and methods Materials All enzymes for genetic engineering were purchased from Takara shuzo. 1994a). 1000 µl of the culture was plated and incubated at 37°C overnight. In this paper. we report the novel method for engineering of an antibody using the mechanism of Fv stabilization in the presence of antigen. these residues in HCDR2 were randomized. 100 µl of VH phage. type 550). Panning One hundred microliters of 10 µg/ml streptavidin (Sigma) was added to each well of a microtiter plate (Falcon) and incubated overnight at 4°C.01% H2O2 was added and incubated for 1 h. Ser54.Novel selection method for antibody engineering antigenic structure has been found to be rigid in comparison of hL with HEL (McKenzie and White. Preliminary modeling indicated that Pro103 of hL interfered with the close fitting of HyHEL10.. and incubated overnight at 37°C. was added to each well and incubated for 1 h at room temperature. incubated for 1 h at 37°C. Expression of soluble proteins For more convenient preparation of the selected clones. 200 µl of 50 mM sodium succinate buffer containing 10 mg/ml ABTS and 0.

and dialysed against the same buffer for two days. SDS–PAGE To examine the secretory expression level. 2.2 106 104 105 106 Input numberb 1.u.4 5.).5 1010 109 109 109 number of clones recovered 1. 0. Schematic representation of open sandwich selection method. Fig.6 2.u. 40 ml water was added to give an osmotic shock and the cells were left on ice for 30 min.4 106 104 103 103 Input number 1.p.5 M sucrose.2 4. 0. 1994b). by panning with hL (10 µg/mL) are shown.4 2. by panning without hL are shown. number of phage is shown colony forming unit (c. Purification of soluble proteins Each fraction prepared was salted out with ammonium sulfate at 80% saturation. After the removal of the culture supernatant by centrifugation. previously equilib- .2 6.6 2.K.m. number of c.f. for 15 min. Binding of phage antibodies using ‘open sandwich selection’ methoda Backgroundc Rounds of panning 1 2 3 4 aExperimental bThe cThe Against hLd Number of clones recoveredb 1. The precipitates formed during dialysis were removed by centrifugation at 10 000 r.5)..4 1010 109 109 109 procedures are described in Materials and methods.083% sodium 1314 deoxycholate (DOC).f.f.u.2 M NaCl. Table 1.8 5.4 2. then loaded onto a Ni-NTA column (Qiagen).m. each fraction mentioned in the previous section was taken for protein analysis.1 mM EDTA and incubated for 5 min at room temperature. The protein precipitates were dissolved in 50 mM phosphate buffer (pH 7.8 3. Then. The supernatant was further concentrated by ultrafiltration. The cells were spun down and the supernatant was saved as the periplasmic fraction. the cell pellet was resuspended in 10 ml of 20 mM Tris–HCl (pH 7. and subjected to protein analysis by SDS–PAGE in the buffer system described by Laemmli (1970). Detail procedures are described in Materials and methods.Tsumoto et al.4 2.2) containing 0.p. and the precipitates were collected by centrifugation at 15 000 r. et al. dThe number of c. The total protein in the supernatant was precipitated with 6% trichloroacetic acid (TCA) and 0. for 10 min.2 6. The procedure for estimating values is described in Materials and methods.2 4.

0 1. wild-type HyHEL10 scFv.2). lane 2.5 µM lysozyme and incubated at 25°C for 1 h in 30 µl of 50 mM phosphate buffer (pH 7.05 0. fraction of wash with PBS.006 1. and the amplified fragments were ligated into the phagemid vector. are given in the Materials and methods. we chose the randomized site (53. Binding activities of HyHEL10 mutant Fv fragment toward HEL or hLa Mutants Wild-type 23 26 Wild-type 23 26 Lysozyme HEL HEL HEL hL hL hL Inhibition activity (mol/mol)b 0. lane 3. 1972) and that of hL or mutant HyHEL10 Fv fragments was estimated using quantitative amino acid analysis.10 0.4 104 (20 20 20 3) kinds of mutant. rated with the same buffer. respectively). SDS–PAGE analysis of expressed and purified scFv clone selected. hL.10 0. Mutation was introduced by a general PCR method (Figure 2).50 at 410 nm obtained in ELISA are shown. 66..4 0. The case of purification of clone 23 was shown. lanes 6–11. 45.15 0. The eluate was rapidly subjected to dialysis against PBS.2 105 identical transformants were obtained. 116. 30. and constructed VH-CDR2 libraries which consisted of only 2.2 adjusted with NaOH) containing 340 µg of Micrococcus lysodeikticus cells. Lane 1. hen egg-white lysozyme. 97. respectively.2) containing 200 mM NaCl in a calorimeter cell was titrated with a 75 µM solution of the mutant Fv fragments in the same buffer at 25°C. The Fv fragment at various concentrations was mixed with 1. Each mixture was then added to 970 µl of 50 mM Na2HPO4 (pH 6.05 0.0 5. The column was washed with the same buffer. human lysozyme. Table 3. N. indicating that full libraries for randomization of selected sites could be 1315 Fig. lane 4.06 0.0 100 500 100 300 Relative to wild typec 1.30 0. 54.05 0. Ser56 and Tyr58 in HCDR2. not determined..Novel selection method for antibody engineering Table 2.. preliminary modeling indicated that Pro103 of hL interfered with the close binding of HyHEL10. especially to HTyr53. 1995). 50. variable region of the heavy chain. cThe molar ratio relative to the wild type is shown. ratio of Fv fragment to HEL which showed the 50% reduced enzymatic activity were given. eluted with PBS containing imidazole of 1. 30 clones on plates were randomly picked up and sequenced. The initial rate of the decrease in A540nm was monitored at 25°C. (Northampton. VH. 10.10 0. 3. The ligand solution was injected 16 times in portions of 7 µl during 15 s.15 0. Binding of selected clones to lysozymes Clone number ELISAa HEL 23 26 2 18 Wild Type aAbsorbance hL 0.7 1The aExperimental conditions bThe values of the molar phagemid of the selected clone was sequenced and only the deduced amino acid sequence is shown.20 0. MA). Amino acid sequences of selected clones deduced from DNA sequencing(1) Table 4. 21 and 14 kDa.5 (Imoto et al. Estimation of protein concentration The concentration of HEL was estimated using A280 of 26. 1989) from MicroCal Inc. Samples for PAGE were prepared as described in the Materials and methods and then followed by 12% SDS–PAGE and stained with Coomassie Brilliant Blue C-250.05 0. 2After four rounds of panning. Then. Measurement of inhibitory activity toward lysozymes Inhibition of lysozyme enzymatic activity was measured as described previously (Ueda et al.05 0.. Thermogram data were analysed using a computer program (Origin) supplied by MicroCal Inc. 100 and 1000 mM. Ser54. molecular size marker (from top to bottom: 200.06 0. 1993).25 0. .8 2. 1.05 0. the lysozyme at a concentration of 3 µM in 50 mM phosphate buffer (pH 7.. HEL. Numbers of clones having the same sequences are shown as Frequences. Uncertainties are the average of at least three experiments S. lane 5. The experimental conditions were the same as those reported previously (Tsumoto et al. 56 and 58). pass through fraction. ELISA procedures are described in the Material and methods section.75 0. The adsorbed protein was eluted with the same buffer containing 1~1000 mM imidazole. The shaded boxes indicate mutated residues in HCDR2. wild -type HyHEL10. 1989). WT. Results and discussion Construction of VH-CDR2 libraries and panning As mentioned above. total proteins in culture supernatant.D.0 0.15 0. 5.15 1.D.. (Wiseman et al. Isothermal titration calorimetry Thermodynamic parameters of the interactions between lysozymes and the mutant Fv fragments were determined by microtitration calorimetry using an OMEGA titration microcalorimeter (Wiseman et al.

. HEL.9 –2. Thermodynamic study of the interaction between selected mutants and lysozymes The interactions between mutant HyHEL10 scFv fragments and lysozymes were investigated by titration calorimetry. indicating that the negative entropy change (–T∆S) was decreased by the mutation. Tsumoto et al. 1996). only a poor yield of Fv fragments is obtained from affinity chromatography (Tsumoto et al. Phage VH was mixed with soluble hL.3 T∆∆S 0 –4. The Gibbs energy was calculated to be –37 kJ mol–1. binding enthalpy and entropy.3 –36. ∆H and T∆S are change in Gibbs energy.. The thermodynamic parameters of the interactions between selected clones and HEL or hL are shown in Table 5. Inhibition assay of lysozyme enzymatic activity To investigate the binding of selected clones to HEL and hL.5 –31.36 2.7 –24. The phage prepared was subjected to the next selection stage. indicating that the entropy change made a positive contribution to the interaction. Twenty-four clones were found to have the same sequence (Table 2. however. 1989. pKTN-NSX. ∆∆H and T∆∆S are the differences in each of the values from those of wild-type Fv.5 ∆∆G kJ mol–1 0 6. The negative enthalpy of the interaction between clone 23 scFv and HEL was observed to be slightly reduced in comparison with the case of wild-type scFv.0 –39. Open sandwich ELISA analysis using the phagedisplayed VH and soluble VL was performed (Ueda et al.. Although the negative enthalpy (–∆H) of the interaction between clone 23 scFv and hL was almost the same as that of wild-type Fv. and was purified with only one step of metalchelating chromatography (Figure 3).. several clones on plates were sequenced and it was confirmed that certain clones are not biased. the inhibition of clone 23 scFv toward hL was significantly increased in comparison with wild-type Fv.0 –56. stoichiometry.. The Gibbs energy change (∆G –RT lnK) of binding was calculated from the binding constant. Gibbs energy (–∆G) for the interaction between scFv and hL was increased.4 0 1. Ward et al.3 T∆S –32. human lysozyme. On the other hand.K. Fv fragments are usually stable in the presence of their antigens (Glockshuber et al.. Ueda et al. followed by preparation of phagemid from the cells.83 9. Tsumoto et al. Clone 26 scFv fragment was also found to inhibit hL enzymatic activity (Table 4). two clones (23 and 26) were chosen for further examination. in the case of mutants with a low affinity constant toward the target antigen. indicating that the negative entropy of the interaction between the mutant and HEL was increased by mutation.5 ∆H –80. 1990.5 3. few deletions of randomized genes were observed in phages displaying only VH fragments (data not shown). the deletion of genes encoding antibody fragments of randomly mutated CDRs has often been observed in phage-displaying scFv fragments (Tsumoto.8 6. Table 5. in a 1:1 molar ratio. indicating that clone 23 scFv has enhanced affinity toward hL.. however.1 49. constructed. pKTN2 (Tsumoto et al.0 0 7.22 0.. designated as clone 26).. Here the VH-CDR2 libraries were subjected to the novel selection method (Figure 2). Expression and purification of mutant soluble proteins To characterize the selected clones. The inhibitory activity of clone 23 scFv toward HEL was only slightly reduced.39 ∆G 48. binding constant. applied to the HyHEL10 VL-immobilized microtiter plate and incubated at room temperature. 1993). ∆G. The genes encoding these mutants were inserted into the expression vector. The inhibitory activities of the selected clones toward both HEL and hL were measured (Table 4). Abbreviations used: n.2 ∆∆H 0 2. several clones seemed to be enriched (Table 1). Colonies on the culture plates were randomly picked. Ka. As described above. the HyHEL10 Fv fragment completely inhibited the activity of its antigen. 1994b). eluted and reinfected into early logphase E. Gibbs energy was also slightly reduced. As discussed below.. respectively.K.3 28. The first step of the usual purification procedure for Fv fragments is affinity chromatography (Skerra and Pluckthun. Thermodynamic parameters of the interactions between HyHEL10 mutants and HEL or hLa Mutants Wild-type 23 26 Wild-type 23 26 aExperimental Lysozyme HEL HEL HEL hL hL hL Ka 106M–1 250 15. The vector was constructed for expression of the gene by fusing six histidines to its Cterminus.3 –42.5 1.4 0 –5. 1996). and Nishimiya.coli. designated as clone 23). which was the modified vector of high expression for the HyHEL10 Fv fragment.2 30. 1990. Metal chelating chromatography would overcome the difficulty in this purification step. hen egg-white lysozyme. and four clones were identical (Table 2. and the DNA sequences of the clones were determined.3 13. and the entropy change (T∆S –∆G ∆H ) upon the association could also be estimated.Tsumoto et al.. these clones had enhanced binding activity toward hL in comparison with wild-type Fv. unpublished results). HEL.7 –30. hL. After four rounds of panning. ∆∆G. As shown in Table 3. Soluble scFv fragments of these clones were highly 1316 expressed in E. 1995). We applied this mechanism to the selection of desired clones.6 –78.0 –16.3 conditions are given Material and methods. scFv fragments were used for precise analysis because of their convenient and efficient purification. 1996). The enthalpy change (∆H) and binding constant (K) upon antigen– antibody interaction were directly obtainable from the experimental titration curve (Wiseman et al. While the negative value of the enthalpy change of the interaction between clone 26 scFv and hL was 28 kJ mol–1 less than for the wild-type. As described previously (Ueda et al. On the other hand. 1988.2 –58. 1994b). which was almost the same as the case of wild-type scFv.9 –34. the entropy change made a . The plate was extensively washed.coli cells.9 –36.0 –34.Y. inhibition of the enzymatic activity of HEL and hL by these clones was tested. Before subjection to panning. Thirty clones were randomly picked up. and that of clone 26 scFv was significantly reduced.2 36.

J.I.. Tsumoto et al.J. 682–687. Gorick.W.H. the mutant Fv fragments selected in our experiment may stabilize the conformation complementary to local antigenic structure.T.M. (1991) J.T. Structural investigations of the interactions between lysozymes and selected clones are now under progress. Res.G.K. (iii) Since variable region fragments are not combined with linkers.G.K.A. unpublished)...D. 18571–18577. 87.. Commun. 86. Inaka. MacKenzie.H. Voak.V.R. 1996).E. Biochem.. Lowary. Cold Spring Harbor Laboratory Press.W. Fritsch. Chem. 88. Science.K. McKenzie. 32612–32616. 92..A. Watanabe.Y. Chem..K. causing decrease in entropic loss. Griffiths..T. (1993) Curr.A. and Weill. Ueda. 271. 201. Sadowska.S. 1996).. for Scientific Research (No. 7.L. USA. Ogasahara. Bye. Immunol. (1991) Adv. (1992) Bio/Technology. Cohen. (1993) Biochemistry.D..U. 31. Biol. Tsumoto. Watanabe. Young.. 1991. we think that this method will be a powerful tool for functional conversion of other Fv fragments. 384–393.J. Smith-Gill. 1995..A.. (1988) Science.R. Smith-Gill.Y.. Kruif. and Potter..N. displaying only one partner of Fv fragments may overcome the instability of randomized genes during the selection procedure.G.R. 9491–9494.T.C.N. Phillips.06454688).R. 226.S.K.C.B. Ueda..G... 169–217.D..E.I. indicating that only Fv fragments which have a high affinity toward target antigens can be stabilized if random mutagenesis is introduced into the CDRs of Fv fragments.A. Lavoie. and Winter.S.E. Recent report of the crystal structures of hapten-germline Fab and hapten-affinity maturated Fab complexes has proposed that affinity maturation stabilize the configuration with optimal hapten complementarity of the antibody (Wedemayer et al. Mandel. Finnern. Sci.S. adjusting the reaction pH and ion strength to lower the interchain association.K..K...Y... Ueda...J.B. 522–540.. the HyHEL10 Fv fragment is stabilized in the presence of its antigen (Ueda et al.08780641) from the Ministry of Education.K.. Lee.. Johnson. Brousseau..P.S. Mackenzie. Watanabe. Mainhart.K. and Bundle.Y..W.D.M.J. Immunol. Kang.E.R. 546–551.S. (1995) J. USA.05258207). 228. (1990) Science.D. J. (1994) J. This may originate mainly from two factors..M. Yutani.M. 227.J.A.P.J.J.Y... Natl Acad.. Energetic advantages by reducing negative entropic loss on solvent displacement and/ or conformational change may have an advantage over reducing enthalpy change by decrease in hydrogen bond formation and van der Waals interactions.P. Sci. 8848–8855. 197. Padlan. Jung. 2289. entropic loss is reduced).. 1038–1041... 270. Ogasahara.I. (1990) Proc. Biophys.. (1994) Mol. Schwarz et al. North. Biol. 32.G.B.. Biol.e.. Opin. and Winter. (1993) Proc.D. These results clearly indicate that the negative entropy changes of the interactions between selected clones and hL are decreased (i. Rudikoff. Chem. 244.F.D. and Poljak.R. Mariuzza. Biol. Boel. 29.S.. 386–390.. Ueda.R. Maenaka.J.A. Sports and Culture of Japan. (1984a) J. and by a Grants-in-aid for Scientific Research in Priority Areas (No. Ouwehand. (1996) J. Altman.Y. (1992) J. Smith-Gill. (1996) Biochem. however.S. 240. Res. J. and Davies. 1315–1317.E.G. this method is more effective than the usual panning procedures using single-chain Fv fragment from three points.P..R.G. Sigurskjold. (1990) Biochemistry. Gallo.K. (1984b) J. Hoogenboom.. Although only one chain can be engineered by this protocol. (1991) J.H. 581–597.. USA. 388–394. Smith. 21874–21879.R. ¨ Skerra. Mol. and Matsusima.. (ii) Background clones (i.A.. 889–896. and for the Encouragement of Young Scientists (No. 28777–28782. Biochem. Hirama. Sheriff. Sigurskjold. Goldbaum. Tsumoto. 1317 . Song. Furuta. 263. Marks.I.. Immunol..A.I. 779–783.C.D.. 680–684. the novel method will save laborious and time consuming screening steps in the usual panning procedures. Scott..R. and that decreasing conformational changes in HyHEL10 HCDR2 upon binding result in a slight increase in affinity toward hL of clone 26 (Tsumoto and Kumagai.K. non-specific binding clones) were highly reduced from the libraries.K. Natl Acad. Protein Chem.R.. (1985) Science. and Kumagai.C. 7538–7542.. Spolar. Yutani. 2nd Edn. Bonnert. and clones which have significantly enhanced binding activity toward hL were enriched during selection.M.T.L. USA.M. and Narang. (1995) Proc... the method reported here can diminish the effect of introducing linkers on affinity (Chaudhary et al... Marks. and Winter. Maenaka.J. (1991) Eur.A. Tsumoto.J. Application of ‘open sandwich selection’ method for functional conversion of Fv fragments As described above. 41..B.B.. and Kumagai. 222. Biophy. Tello.T.L.. 239–246.. Immunol. Jones..A and Poljak. Deng.T.R.E.K. (i) Few deletions of randomized genes were observed in phages displaying only VH fragments.Y. and Kumagai. Glockshuber. McCafferty.H. Acknowledgements We thank Dr Katsumi Maenaka (Oxford University) and Dr Yuichiro Nakaoki (University of Tokyo) for constructing the phage display system for the HyHEL10 VH fragment. 1997). Young.K. and it seems that the hydrophobic effect of the Trp residue and the burial of the electrostatic charge of Arg are important for the enhanced affinity toward hL of the clone 23. Nakaoki.. Maenaka. and Pluckthun. 963–967. and Burton.T.. 11120–11123.U.K. This work was also supported in part by Takeda Science Foundation.. (1970) Nature.E. Reiter. 90.C. Sci.e..T. USA. Watanabe. Here it was shown that stabilization is applicable to the functional alteration of Fv fragments. The deletion of genes encoding antibody fragments of randomly mutated CDRs has often been observed in phage-displaying scFv fragments and therefore. Chem. Mariuzza....F.H. 133.R. (1994b) J. Yutani... Padlan.D. Jr (1994) Science. Bundle. Russell.N. Thus.M.F.K.. solvent displacement (Sigurskjold et al.. 665–868. Yutani.Novel selection method for antibody engineering positive contribution to the interaction between clone 26 scFv and HEL.M.K.D.R. (1989) Proc. Commun.R. Imoto.N.. 4992–4996..S..R..S. Bundle. Mol. (1995) Eur. 173–315. Open sandwich selection may not be applicable to Fv fragments with strong interchain associations.F. Tsumoto. (1991) Proc.R. Watanabe. Natl. Mol. and Narang. Silverton. 1994. 1991) and conformational change (Spolar and Record.D..A. 50–55..J.J. and Record. Sci.S. Since this method is based on a weak VH–VL interaction and a lot of Fv fragments have weak interchain associations..H. 5938–5942.K.S.K. Natl Acad.N. Biol.M.. and Kumagai.K. and While. and Pastan.H. Tsumoto et al. or further engineering of the framework region seem to be effective (Ueda et al. Hughes-Jones. Mol.R.. 97–105. 1994a).D. 218. and Rupley. References Anand.M. Hawkins. and Logtenberg. and Pluckthun.K.A. 132. Ogasahara..J.B.I.. (1989) Molecular Cloning: A Laboratory Manual. 248. Thorpe.T. 249. Schwarz. (1972) The Enzymes..J.K.I.C. Biol. Lavoie.T. 266. 269. Tsumoto.. Chaudhary...J.C. Acad. Malia..A. Fitzgerald. Brinkmann. Natl Acad.J. Biol.. Pftizinger.S. This work was supported in part by the Original Industrial Technology R&D Promotion Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. (1995) J. 5. resulting in enhanced affinity toward hL.M..K. and Maniatis. 10. Sci.. (1994a) Biochem. Biol. and Smith..K. Riechmann.. Laemmli. Ogasahara. Sambrook. 777–782. Vol.I. Ueda.D.coli (Anand et al.N. and Kumagai...S... Since the selection method reported here can be regarded as an in vitro mimicry of affinity maturation. and Mainhart. 1996). 1990) and expression level in E.K.A. and Pastan. 1362–1367.

1714–1718. Gussow.G..E.. 179. (1994) Annu. and Milstein.K.G. Wedemayer.L. Ueda. Watanabe. (1995) J. Rev. revised July 22. Wiseman.. 1997 1318 .G. and Winter..A.P. and Barbas III.D. and Mahoney.T. Ueda.A... Brandts..T.. Briones. (1993) Gene. 1997.H. Jones.F. and Winter.G.D. Suzuki. and Kumagai. accepted July 23.D.T. Nishimura.L.C. Yang. 1665–1669. 254. Griffiths..K.K. 433–455. Williston.D. Pinz-Sweeney. 544–546.R. Burton. 349..P.. Received June 9.. 129–134. (1989) Anal. 14.J..I. (1991) Nature.C.A.. Wang. and Lin. 392–403..C. Biochem.Tsumoto et al.. Kubota.R.G.S...H.E.W.C.I.P. Biol. Winter.J.K. 131–137.A... (1988) Science.F. 239.P.P. Winter. Ward.T. 293–299.. 12. Nagamune..P. Verhoeyen. and Stevens. and Hoogenboom. Schueler. 129. Tsumoto..S..G. Schultz..M..E. (1996) Nature Biotech. Tsumoto. 341. Mol. Hawkins. (1997) Science.H. 276.. Milstein.K.R.C.A.W.G. Patten. 1534–1536. Kumagai. Griffiths.Y. Winter. Immunol. (1990) Nature..H.R.-N. Green..K. 1997.