Title: Thermodynamic Consequences of Grafting Enhanced Affinity toward the Mutated Antigen onto an Antibody
Abstract: In order to address the mechanism of enhancement of the affinity of an antibody toward an antigen from a thermodynamic viewpoint, anti-hen lysozyme (HEL) antibody HyHEL-10, which also recognize the mutated antigen turkey lysozyme (TEL) with reduced affinity, was examined. Grafting high affinity toward TEL onto HyHEL-10 was performed by saturation mutagenesis into four residues (Tyr53, Ser54, Ser56, and Tyr58) in complementarity-determining region 2 of the heavy chain (CDR-H2) followed by selection with affinity for TEL. Several clones enriched have a Phe residue at site 58. Thermodynamic analyses showed that the clones selected had experienced a greater than 3-fold affinity increase toward TEL in comparison with wild-type Fv, originating from an increase in negative enthalpy change. Substitution of HyHEL-10 HTyr58 with Phe led to the increase in negative enthalpy change and to almost identical affinity for TEL in comparison with mutants selected, indicating that mutations at other sites decrease the entropy loss despite little contribution to the affinity for TEL. These results suggest that the affinity of an antibody toward the antigen is enhanced by the increase in enthalpy change by some limited mutation, and excess entropy loss due to the mutation is decreased by other energetically neutral mutations. In order to address the mechanism of enhancement of the affinity of an antibody toward an antigen from a thermodynamic viewpoint, anti-hen lysozyme (HEL) antibody HyHEL-10, which also recognize the mutated antigen turkey lysozyme (TEL) with reduced affinity, was examined. Grafting high affinity toward TEL onto HyHEL-10 was performed by saturation mutagenesis into four residues (Tyr53, Ser54, Ser56, and Tyr58) in complementarity-determining region 2 of the heavy chain (CDR-H2) followed by selection with affinity for TEL. Several clones enriched have a Phe residue at site 58. Thermodynamic analyses showed that the clones selected had experienced a greater than 3-fold affinity increase toward TEL in comparison with wild-type Fv, originating from an increase in negative enthalpy change. Substitution of HyHEL-10 HTyr58 with Phe led to the increase in negative enthalpy change and to almost identical affinity for TEL in comparison with mutants selected, indicating that mutations at other sites decrease the entropy loss despite little contribution to the affinity for TEL. These results suggest that the affinity of an antibody toward the antigen is enhanced by the increase in enthalpy change by some limited mutation, and excess entropy loss due to the mutation is decreased by other energetically neutral mutations. complementarity-determining region hen egg white lysozyme turkey egg white lysozyme variable region of immunoglobulin heavy chain variable region of immunoglobulin light chain fragment of immunoglobulin variable regions phosphate-buffered saline mutant HyHEL-10 Fv in which Tyr53-Ser54-Ser56-Tyr58of VH chain are substituted with Ser53-Phe54-Ser56-Phe58 enzyme-linked immunosorbent assay 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) polyacrylamide gel electrophoresis polymerase chain reaction Antibody-proteinaceous antigen interactions have been extensively studied as one of the most promising models of protein-protein interaction (1.Janin J. Chothia C. J. Biol. Chem. 1990; 265: 16027-16030Abstract Full Text PDF PubMed Google Scholar, 2.Davies D.R. Cohen G.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7-12Crossref PubMed Scopus (492) Google Scholar, 3.Mariuzza R.A. Poljak R.J. Curr. Opin. Immunol. 1993; 5: 50-55Crossref PubMed Scopus (81) Google Scholar, 4.Webster D.M. Henry A.H. Rees A.R. Curr. Opin. Struct. Biol. 1994; 4: 123-129Crossref Scopus (124) Google Scholar) and have provided considerable knowledge about the general rules of various biochemical reactions (5.Padlan E.A. Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (807) Google Scholar, 6.Padlan E.A. Adv. Protein Chem. 1996; 49: 57-133Crossref PubMed Google Scholar, 7.Colman P.M. Adv. Immunol. 1988; 43: 99-132Crossref PubMed Scopus (195) Google Scholar). The most striking feature of the antibody-proteinaceous antigen interaction is its strict specificity and high affinity (8.Braden B.C. Poljak R.J. FASEB J. 1995; 9: 9-16Crossref PubMed Scopus (184) Google Scholar). Specificity and affinity of an antibody toward a target proteinaceous antigen are usually determined by the structural complementarity of the antigen-binding site of an antibody, referred to as the complementarity-determining region, CDR1 (9.Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Daives D.R. Tulip W.R. Colman P.M. Spinelli S. Alzari P.M. Poljak R.J. Nature. 1989; 342: 877-883Crossref PubMed Scopus (1137) Google Scholar, 10.Chothia C. Lesk A.M. J. Mol. Biol. 1987; 196: 901-917Crossref PubMed Scopus (1237) Google Scholar). Examination of structurally well refined antibody-antigen interactions (11.Jin L. Fendly B.M. Wells J.A. J. Mol. Biol. 1992; 226: 851-865Crossref PubMed Scopus (207) Google Scholar, 12.Covell D.G. Wallqvist A. J. Mol. Biol. 1997; 269: 281-297Crossref PubMed Scopus (68) Google Scholar) by site-specific amino acid substitutions of antigen and antibody is essential to the further discussion of antibody specificity. Recent advances in phage-display technology will enable us to engineer an antibody molecule for the purposes of grafting the desired specificity and affinity (13.Winter G. Griffiths A.D. Hawkins R.E. Hoogenboom H.R. Annu. Rev. Immunol. 1994; 12: 433-455Crossref PubMed Scopus (1409) Google Scholar, 14.Schier R. Bye J. Apell G. McCall A. Adams G.P. Malmqvist M. Weiner L.M. Marks J.D. J. Mol. Biol. 1996; 255: 28-43Crossref PubMed Scopus (274) Google Scholar, 15.Yang W.P. Green K. Pinz-Sweeney S. Briones A.T. Burton D.R. Barbas III, C.F. J. Mol. Biol. 1995; 254: 392-403Crossref PubMed Scopus (309) Google Scholar). Thus, investigation of the interactions between mutated antigen and mutated antibody molecules with enhanced affinity for the mutated antigens would be particularly useful in expanding our understanding of the fine specificity and high affinity of an antibody toward an antigen. It is known that antibody molecules recognize mutant antigens with subtle structural changes, for example, in cases in which amino acid replacement has occurred in antibody binding regions (epitopes). Conformational changes in CDRs (16.Chitarra V. Alzari P.M. Bentley G.A. Bhat T.N. Eisele J.-L. Houdusse A. Lescar J. Souchon H. Poljak R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7711-7715Crossref PubMed Scopus (129) Google Scholar) or the acceptance of other binding orientations of mutated antigens (i.e. derivative of the cognate antigen of an antibody) (17.Arevalo J.H. Taussig M.J. Wilson I.A. Nature. 1993; 365: 859-863Crossref PubMed Scopus (137) Google Scholar, 18.Arevalo J.H. Hassig C.A. Stura E.A. Sims M.J. Taussig M.J. Wilson I.A. J. Mol. Biol. 1994; 241: 663-690Crossref PubMed Scopus (109) Google Scholar, 19.Lescar J. Pellegrini M. Souchon H. Tello D. Poljak R.J. Peterson N. Greene M. Alzari P.M. J. Biol. Chem. 1995; 270: 18067-18076Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) have been suggested as the driving force in the binding of an antibody to mutated antigens. However, no further approach for quantitative estimation of the contribution of each antibody residue to the recognition of mutated antigens has been addressed. We have focused on the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody, HyHEL-10. The interaction has been structurally well studied (20.Kondo H. Shiroishi M. Matsushima M. Tsumoto K. Kumagai I. J. Biol. Chem. 1999; 274: 27623-27631Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 21.Padlan E.A. Silverton E.W. Sheriff S. Cohen G.H. Smith-Gill S.J. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5938-5942Crossref PubMed Scopus (518) Google Scholar) and is therefore particularly suitable for the precise analysis of antigen-antibody interactions (22.Tsumoto K. Ueda Y. Maenaka K. Watanabe K. Ogasahara K. Yutani K. Kumagai I. J. Biol. Chem. 1994; 269: 28777-28782Abstract Full Text PDF PubMed Google Scholar, 23.Tsumoto K Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24.Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1996; 271: 32612-32616Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25.Rajpal A. Taylor M.G. Kirsch J.F. Protein Sci. 1998; 7: 1868-1874Crossref PubMed Scopus (33) Google Scholar, 26.Pons J. Rajpal A. Kirsch J.F. Protein Sci. 1999; 8: 958-968Crossref PubMed Scopus (99) Google Scholar). It has been shown that HyHEL-10 recognizes several avian lysozymes (27.Smith-Gill S.J. Lavoie T.B. Mainhart C.R. J. Immunol. 1984; 133: 384-393PubMed Google Scholar, 28.Lavoie T.B. Drohan W.N. Smith-Gill S.J. J. Immunol. 1992; 148: 503-513PubMed Google Scholar, 29.Kam-Morgan L.N.W. Smith-Gill S.J. Taylor M.G. Zhang L. Wilson A.C. Kirsch J.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3958-3962Crossref PubMed Scopus (64) Google Scholar) and can bind to turkey egg white lysozyme (TEL) with decreased affinity (27.Smith-Gill S.J. Lavoie T.B. Mainhart C.R. J. Immunol. 1984; 133: 384-393PubMed Google Scholar). X-ray crystallographic study of the HyHEL-10 Fv·HEL complex (20.Kondo H. Shiroishi M. Matsushima M. Tsumoto K. Kumagai I. J. Biol. Chem. 1999; 274: 27623-27631Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and free TEL (30.Harata K. Abe Y. Muraki M. Proteins Struct. Funct. Genet. 1998; 30: 232-243Crossref PubMed Scopus (36) Google Scholar) has suggested that one of the differences in antigenic structure is located in the loop region from Ile98 to Gly102, particularly the substitution of Asp101 (HEL) into Gly (TEL). This region is mainly recognized by complementarity-determining region 2 of the heavy chain (CDR-H2). Thus, some of the residues in CDR-H2 (Tyr53, Ser54, Ser56, and Tyr58) were subjected to saturation mutagenesis. VH libraries were phage-displayed and selected by affinity toward TEL using the mechanism of Fv fragment stabilization under coexistent antigens (31.Ueda H. Tsumoto K. Kubota K. Suzuki E. Nagamune T. Nishimura H. Schueler P.A. Winter G. Kumagai I. Mahoney W.C. Nat. Biotechnol. 1996; 14: 1714-1718Crossref PubMed Scopus (141) Google Scholar, 32.Jäger M. Plückthun A. J. Mol. Biol. 1999; 285: 2005-2019Crossref PubMed Scopus (43) Google Scholar). In this paper we report thermodynamic analyses of the interactions between mutant antibody molecules selected in vitro for a mutated antigen and the wild-type (HEL) and mutated (TEL) antigens. On the basis of the results obtained, the molecular mechanism of improvement of antibody affinity toward a mutated antigen is discussed from a thermodynamic viewpoint. All enzymes used in genetic engineering were purchased from Takara Shuzo (Shiga, Japan) or Toyobo (Osaka, Japan). Biotin-NHS was purchased from Roche Molecular Biochemicals. Streptavidin was purchased from Sigma. Streptavidin-conjugated magnetic beads came from Promega (Tokyo, Japan). HEL was obtained from Seikagaku-kogyo Inc. (Tokyo, Japan) and TEL from Sigma. Horseradish peroxidase-conjugated anti-M13 mouse antibody came from Amersham Pharmacia Biotech, Inc., and 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid (ABTS) was purchased from Wako Fine Chemicals Inc. (Osaka, Japan). The variable region of the light chain (VL) fragment of HyHEL-10 was prepared as described previously (31.Ueda H. Tsumoto K. Kubota K. Suzuki E. Nagamune T. Nishimura H. Schueler P.A. Winter G. Kumagai I. Mahoney W.C. Nat. Biotechnol. 1996; 14: 1714-1718Crossref PubMed Scopus (141) Google Scholar), and the mutated HyHEL-10 Fv fragments (HY58A and HY58F) were prepared as described (25.Rajpal A. Taylor M.G. Kirsch J.F. Protein Sci. 1998; 7: 1868-1874Crossref PubMed Scopus (33) Google Scholar). DNA oligonucleotides for randomization of HyHEL-10 VH were ordered from the Nippon Gene Laboratory (Sendai, Japan). All other reagents were of biochemical research grade. The CDR-H2 of HyHEL-10 was randomized by PCR. The plasmid, pTZPsFv2, constructed for the phage display of HyHEL-10 single chain Fv fragment (33.Maenaka K. Furuta M. Tsumoto K. Watanabe K. Ueda Y. Kumagai I. Biochem. Biophys. Res. Commun. 1996; 218: 682-687Crossref PubMed Scopus (31) Google Scholar) was used as a template. Prior to use for PCR, the back primer CDR-H2RANBACK (5′-CGCCTCGAGTACATGGGCTACGTCAGCNNKNNKGGCNNKACCNNKTACAACCCC3′) and the forward primer HyHELFOR (5′-ACCCGCGGAGACGGTGACGAGGGTGCC3′) were biotinylated using a 5′-biotinylation kit (Amersham Pharmacia Biotech) and mixed to a final concentration of 25 pmol/ml. N and K represent the mixtures of AGCT and GT, respectively. The PCR reaction was carried out in 100 μl of 250 nm dNTP, with 25 pmol of each primer, 10 ng pTZPsFv2 as a template, and 0.5 units of Taq DNA polymerase (Perkin-Elmer) in the manufacturer's recommended buffer. The PCR reaction was cycled 35 times (at 94 °C for 1 min, 55 °C for 1.5 min, and 72 °C for 2 min) using a thermal cycler MP3000 (Takara). The PCR-amplified product was subjected to agarose-gel electrophoresis, recovered from the gel with Geneclean II, and digested with XhoI and SacII. The randomized VH fragment was purified using streptavidin beads and ligated with the vector, pTZPsVH2, at the XhoI-SacII site using T4 DNA ligase. The resulting substance was designated pTZPsVH2-ran. Phage was recovered by polyethylene glycol precipitation (31.Ueda H. Tsumoto K. Kubota K. Suzuki E. Nagamune T. Nishimura H. Schueler P.A. Winter G. Kumagai I. Mahoney W.C. Nat. Biotechnol. 1996; 14: 1714-1718Crossref PubMed Scopus (141) Google Scholar, 33.Maenaka K. Furuta M. Tsumoto K. Watanabe K. Ueda Y. Kumagai I. Biochem. Biophys. Res. Commun. 1996; 218: 682-687Crossref PubMed Scopus (31) Google Scholar) and resuspended in 500 μl of phosphate-buffered saline (PBS) per 20 ml of culture. In order to select the phage antibody libraries, streptavidin magnetic beads were used. In the first step of the procedure, 100 μl of phage VH were mixed with soluble biotinylated TEL and purified HyHEL-10 VL chain at room temperature for 1 h. Next, 50 μl of streptavidin-conjugated paramagnetic beads was added and rapidly mixed. After an incubation period of 10 min, a magnet (Promega) was used to select out the beads and attached phage. The beads were washed stringently 5 times with 1000 μl of PBST (where PBST is PBS containing 0.05% Tween 20) and 5 more times with 1000 μl of PBS. The bound phages were eluted with 500 μl of 100 mm glycine (pH 2.0) containing 200 mmNaCl, rapidly neutralized with 100 mm Tris-HCl (pH 8.5) containing 500 mm NaCl, and used to reinfect into early log phase Escherichia coli JM109. After 1 h, 1000 μl of the culture was plated and incubated at 37 °C overnight. Clones on the plates were transferred into the LB culture containing 100 mg/liter ampicillin, infected with helper phage, M13KO7, and incubated overnight at 37 °C. The phage was prepared by centrifugation and polyethylene glycol precipitation and was subjected to the next selection stage. Following the four stages, the clones on plates were cultured, and phage VH was prepared according to the method described previously (31.Ueda H. Tsumoto K. Kubota K. Suzuki E. Nagamune T. Nishimura H. Schueler P.A. Winter G. Kumagai I. Mahoney W.C. Nat. Biotechnol. 1996; 14: 1714-1718Crossref PubMed Scopus (141) Google Scholar). Dideoxy reactions for DNA sequencing were performed using an auto-read sequencing Kit (Amersham Pharmacia Biotech) according to the manufacturer's recommendations. The analysis of DNA sequences was performed using an ALF express auto-read sequencer (Amersham Pharmacia Biotech). Open-sandwich ELISA was performed according to the method described by Ueda et al. (31.Ueda H. Tsumoto K. Kubota K. Suzuki E. Nagamune T. Nishimura H. Schueler P.A. Winter G. Kumagai I. Mahoney W.C. Nat. Biotechnol. 1996; 14: 1714-1718Crossref PubMed Scopus (141) Google Scholar). One hundred microliters of 10 μg/ml HyHEL-10 VL fragment in PBS was applied to each well of the microtiter plates and incubated for 1 h at room temperature. After removing the solution, 200-μl SuperBlock (Pierce) was added to each well and incubated for 1 h at room temperature. After discarding the buffer, 100 μl of VH-phage, which had been mixed with TEL or HEL and prediluted with 1 volume of binding buffer 30 min before, was added to each well and incubated for 1 h at room temperature. After washing twice with PBST, 100 μl of 5000 times diluted horseradish peroxidase-anti-M13 (Amersham Pharmacia Biotech) was added, and the mixture was incubated for 1 h. After three more washings with PBST, 200 μl of 50 mm sodium/succinate buffer containing 10 mg/ml ABTS and 0.01% H2O2 was added and the mixture incubated for 1 h. The absorbance was measured at 415 nm using a microplate reader (Bio-Rad, type 550), with 630 nm as the control. A transformant E. coli strain BL21 (DE3) (34.Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (5309) Google Scholar) harboring a plasmid clone was grown at 28 °C in 2× YT (35.Sambrook J. Fritsch F.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) supplemented with 200 μg/ml ampicillin, until the early stationary phase. To induce the expression of soluble Fv fragment, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.1 mm, and the culture was grown overnight at 28 °C. Two types of sample, bacterial supernatant and periplasmic fractions, were separated from 200 ml of the culture as follows (24.Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1996; 271: 32612-32616Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). After the removal of the culture supernatant by centrifugation at 6,000 × g for 15 min at 4 °C, the cell pellet was resuspended in 10 ml of 20 mm Tris-HCl (pH 7.5), 0.5 m sucrose, and 0.1 mm EDTA and incubated for 5 min at room temperature. Then, 40 ml of water was added to give an osmotic shock, and the cells were left on ice for 30 min. The cells were collected by centrifugation at 7,000 ×g for 60 min at 4 °C, and the supernatant was saved as the periplasmic sample. The supernatant and periplasmic samples were salted out with ammonium sulfate at 80% saturation, and the precipitates were collected by centrifugation at 7,000 × g for 30 min. The protein precipitates were dissolved in phosphate-buffered saline (PBS) and dialyzed against the same buffer for 2 days. The precipitates formed during dialysis were removed by centrifugation at 10,000 ×g for 15 min. The supernatant was loaded onto a TEL-Sepharose column, in which about 10 mg of TEL/ml of gel was bound to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech), previously equilibrated with the same buffer. The column was washed with the same buffer, 100 mm Tris-HCl (pH 8.5) containing 500 mm NaCl; then the adsorbed protein was eluted with 100 mm glycine buffer (pH 2.0). The eluate was rapidly neutralized with 1 m Tris-HCl (pH 7.5). The Fv fragment obtained from affinity chromatography was further purified on a Superdex 75 Prep Grade (inner diameter 1.6 × 40 cm) equilibrated with 50 mm Tris-HCl (pH 7.5) containing 200 mm NaCl and finally dialyzed overnight against 50 mm phosphate buffer (pH 7.2) containing 200 mmNaCl. All the proteins in the supernatant were precipitated with 6% trichloroacetic acid and 0.083% sodium deoxycholate and subjected to protein analysis by SDS-PAGE in the buffer system described by Laemmli (36.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (218014) Google Scholar). Thermodynamic parameters for the interactions between HEL and the wild-type or mutant Fv fragments were determined by microtitration calorimetry using an OMEGA titration calorimeter (37.Wiseman T. Williston S. Brandts J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2495) Google Scholar) from MicroCal Inc. (Northampton, MA). The experimental conditions were as follows. The Fv fragment at a concentration of 5 μm in 50 mm phosphate buffer (pH 7.2) containing 200 mm NaCl in a calorimeter cell was titrated with a 125 μm solution of HEL in the same buffer at four different temperatures (25, 30, 35, and 40 °C). The ligand solution was injected 16 times in portions of 7 μl during a period of 15 s. Thermogram data were analyzed using the computer program (Origin) supplied by Microcal Inc. The enthalpy change (ΔH) and binding constant (K a) on antigen-antibody interaction are directly obtainable from the experimental titration curve. Gibbs energy change (ΔG = −RT ln K a) and the entropy change (ΔS = (−ΔG+ΔH)/T) on the association could be calculated from ΔH and K a. Heat capacity change (ΔCp) was estimated from the temperature dependence of enthalpy change. The concentration of HEL or TEL was estimated using A2801% = 26.5 (38.Imoto T. Johnson L.N. North A.C.T. Phillips D.C. Rupley J.A. Enzymes. 1972; 7: 665-868Crossref Scopus (795) Google Scholar) and that of each mutant HyHEL-10 Fv fragment was estimated using quantitative amino acid analysis. Fig.1 shows the amino acid residues of lysozymes recognized by HyHEL-10 (epitope). Four amino acid replacements exist as follows: His15 (Leu in TEL), Arg73 (Lys), Val99 (Ala), and Asp101 (Gly). His15 and Arg73 are located at the edge of the complex and are solvent-exposed from x-ray crystallography (20.Kondo H. Shiroishi M. Matsushima M. Tsumoto K. Kumagai I. J. Biol. Chem. 1999; 274: 27623-27631Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 21.Padlan E.A. Silverton E.W. Sheriff S. Cohen G.H. Smith-Gill S.J. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5938-5942Crossref PubMed Scopus (518) Google Scholar). Conversely, Val99 and Asp101 are located in the center of the antigen-antibody interface. The research reported here focused on CDR-H2 of HyHEL-10, as the region around Asp101 of HEL was primarily recognized by CDR-H2 (20.Kondo H. Shiroishi M. Matsushima M. Tsumoto K. Kumagai I. J. Biol. Chem. 1999; 274: 27623-27631Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To this end, a DNA primer was designed for construction of a CDR-H2 library (see “Experimental Procedures), and some of the residues in CDR-H2 of HyHEL-10 wild-type (Tyr53, Ser54, Ser56, and Tyr58) were completely randomized. Mutation was introduced by the general PCR method. The amplified fragments were ligated into phagemid vector, pTZPsVH2, and the synthetic CDR-H2 library can have about 105 (20 × 20 × 20 × 20) members. In this study, 1.5 × 105 identical transformants were obtained. Before being selected, more than 50 clones on plates were sequenced, confirming that clones are not biased (data not shown). The CDR-H2 libraries were subjected to a selection on the basis of the mechanism of Fv fragment stabilization under coexistent antigens using streptavidin magnetic beads. Phage VH was mixed with soluble biotinylated turkey egg white lysozyme (TEL) and purified HyHEL-10 VL chain, forming a ternary complex upon incubation for an hour at 37 °C. The resulting ternary complex was captured by streptavidin-conjugated paramagnetic beads, followed by selection using a magnet. The beads were then washed meticulously, and the adsorbed phage particles were recovered by acid elution and used to reinfect into early log phase E. coli cells. The prepared phage was subjected to the next selection stage. ELISA analysis (31.Ueda H. Tsumoto K. Kubota K. Suzuki E. Nagamune T. Nishimura H. Schueler P.A. Winter G. Kumagai I. Mahoney W.C. Nat. Biotechnol. 1996; 14: 1714-1718Crossref PubMed Scopus (141) Google Scholar) using selected phages and soluble VL fragment clearly indicated that clones with increased affinity toward TEL were enriched, and the number of clones increased during selection (TableI). After four rounds of panning, clones on the culture plates were randomly picked up, and the DNA sequences of the clones were determined. Five clones, i.e.Tyr53 → Ser/Ser54 → Phe/Ser56/Tyr58 → Phe (designated as SFSF); Tyr53 → Ala/Ser54 → Glu/Ser56→ Ala/Tyr58 → Phe (AEAF); Tyr53 → Ala/Ser54 → Gly/Ser56 → Tyr/Tyr58 → Phe (AGYF); Tyr53 → Glu/Ser54 → Thr/Ser56 → Lys/Tyr58 → Phe (ETKF); and Tyr53 → Ala/Ser54 → Leu/Ser56 → Thr/Tyr58 → Phe (ALTF) were found to be enriched (TableII). ELISA analyses have indicated that the clones selected showed a higher ELISA signal for TEL than wild-type Fv (Table II). ELISA analysis without VL fragment clearly indicates that only VH wild-type fragment or selected mutants have an insignificant affinity toward lysozymes (data not shown), suggesting that ternary complex formation is necessary for specific recognition of target antigen.Table IEnrichment of clones during selectionPanning againstPBSTurkey egg-white lysozymecfu/ml cultureaNumbers of clones on culture plates during selection were shown.ELISAbExperimental conditions of ELISA have been described under “Experimental Procedures.” The values shown are the absorbance at 415 nm after 1 h color development at room temperature and averages of at least three independent analyses.cfu/ml cultureaNumbers of clones on culture plates during selection were shown.ELISAbExperimental conditions of ELISA have been described under “Experimental Procedures.” The values shown are the absorbance at 415 nm after 1 h color development at room temperature and averages of at least three independent analyses.Input5 × 10110.05 ± 0.015 × 10110.05 ± 0.02After 1st3 × 1040.05 ± 0.023 × 1060.10 ± 0.032nd5 × 1070.20 ± 0.053rd5 × 10110.25 ± 0.044th5 × 10110.28 ± 0.05a Numbers of clones on culture plates during selection were shown.b Experimental conditions of ELISA have been described under “Experimental Procedures.” The values shown are the absorbance at 415 nm after 1 h color development at room temperature and averages of at least three independent analyses. Open table in a new tab Table IIDeduced amino acid sequences of selected clones and ELISACloneaDesignation of the clones have been mentioned in the text. For instance, SFSF clone has Ser, Phe, Ser, and Phe at sites 53, 54, 56, and 58 of VH, respectively.FrequencebTwenty clones are randomly picked up, and the sequences have been determined. Sixteen clones have the correct open reading frame, whereas four clones have frame-shift mutations./20Mutated site in VHELISAcExperimental conditions of ELISA have been mentioned under “Experimental Procedures.” Only values obtained in the caase of which 10 μg/ml of lysozymes solution are used, and the averages of at least three independent analyses have been shown. Errors are ± S.D.53545658TELHELWTTyrSerSerTyr0.11 ± 0.020.15 ± 0.02SFSF5SerPheSerPhe0.18 ± 0.030.14 ± 0.03AEAF3AlaGluAlaPhe0.28 ± 0.050.14 ± 0.04AGYF3AlaGlyTyrPhe0.14 ± 0.030.14 ± 0.03ETKF3GluThrLysPhe0.18 ± 0.020.06 ± 0.02ALTF2AlaLeuThrPhe0.21 ± 0.050.15 ± 0.03a Designation of the clones have been mentioned in the text. For instance, SFSF clone has Ser, Phe, Ser, and Phe at sites 53, 54, 56, and 58 of VH, respectively.b Twenty clones are randomly picked up, and the sequences have been determined. Sixteen clones have the correct open reading frame, whereas four clones have frame-shift mutations.c Experimental conditions of ELISA have been mentioned under “Experimental Procedures.” Only values obtained in the caase of which 10 μg/ml of lysozymes solution are used, and the averages of at least three independent analyses have been shown. Errors are ± S.D. Open table in a new tab To characterize the selected clones, secretory expression of soluble Fv fragments of five clones has been attempted, and three clones (SFSF, AEAF, and ETKF) were highly expressed in E. coli, which was similar to the case of wild-type Fv. The clones were purified by affinity chromatography using TEL-Sepharose, and this was followed by gel filtration using Superdex 75 Prep Grade (Fig.2). Purity greater than 95% was obtained using these steps, and purified Fv fragment was subjected to a precise analysis. It has been shown that the HyHEL-10 Fv fragment inhibits the enzymatic activity of its antigen, HEL, in the presence of a slight molar excess of the Fv fragment, resulting from the binding of the Fv to the active site of HEL (39.Ueda Y. Tsumoto K. Watanabe K. Kumagai I. Gene (Amst.). 1993; 129: 129-134Crossref PubMed Scopus (41) Google Scholar). First, the inhibitory activity of HyHEL-10 Fv for TEL was examined. As shown in Fig.3 A, HyHEL-10 Fv can inhibit the enzymatic activity of TEL, although the inhibition is rather lower than in the case of HEL. Next, inhibition of the enzymatic activity of TEL and HEL by mutants selected was investigated (Fig. 3). Upon comparison with wild-type Fv, these clones selected showed enhanced inhibition toward TEL under the conventional conditions of the inhibition assay. On the other hand, the three mutants had a rather large reduction of inhibition toward HEL (Fig. 3). In order to investigate the interactions between mutants selected and lysozymes from a thermodynamic viewpoint, isothermal titration calorimetry of the association between mutant Fv fragments and lysozymes was performed under the same conditions as described before (23.Tsumoto K Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24.Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1996; 271: 32612-32616Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Typical profile of