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Originally published In Press as doi:10.1074/jbc.M706190200 on October 17, 2007

J. Biol. Chem., Vol. 283, Issue 2, 1156-1166, January 11, 2008
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Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528*Formula

Koki Makabe{ddagger}12, Takeshi Nakanishi{ddagger}1, Kouhei Tsumoto{ddagger}3, Yoshikazu Tanaka{ddagger}3, Hidemasa Kondo§, Mitsuo Umetsu{ddagger}, Yukiko Sone{ddagger}, Ryutaro Asano{ddagger}, and Izumi Kumagai{ddagger}4

From the {ddagger}Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11-606, Aoba-ku, Sendai 980-8579, Japan and the §Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan

Received for publication, July 27, 2007 , and in revised form, September 21, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Structural and functional analyses of antigen-antibody protein-protein interactions have revealed that complementarity-determining regions (CDRs)5 in the variable domains of antibodies play a critical role in the specificity and affinity of the antibodies for their targets by means of shape and charge complementarities (1-4). Antibodies have a common fold (the immunoglobulin fold), and hypervariable CDRs are located on one edge of the framework region (1-4). Grafting of the CDRs of antibodies onto the frameworks of other antibodies has been attempted (5-9). Especially interesting from diagnostic and therapeutic viewpoints is the humanization, or reshaping, of murine antibodies, whereby a set of CDRs from murine antibodies are transplanted to appropriate scaffolds of human antibodies to reduce immune responses against murine antibodies in human hosts (10-20).

However, grafting of the six CDRs of murine antibodies onto appropriate frameworks of human antibodies often results in reduced affinity or specificity for the target antigen (10, 14, 16). The pioneering work of Foote and Winter (21) has suggested that antibody residues in the β-sheet framework underlying the CDRs play a critical role in adjustment of the loop structures of the CDRs. Although these residues, referred to as Vernier zone residues, do not directly interact with the antigen, careful selection of these residues may prove essential for the success of loop transplants in antibodies, and variation of these sites may also have a role in shaping the diversity of structures found in the primary repertoire and a role in affinity maturation (21).

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with an intracellular tyrosine kinase domain (22-24). High level overexpression of the EGFR has been found in many tumors and is considered to be generally correlated with critical factors in the development of tumors (25-28). To date, several approaches involving regulation of the EGFR have been attempted for cancer immunotherapy, some of which are now in clinical trials (29-33).

In previous work, we focused on antibody-based adoptive immunotherapy (34-38). Recently, we successfully constructed an Ex3 diabody, a fusion protein of the variable region (Fv) of an anti-CD3 antibody and an anti-EGFR antibody (39, 40). We also found that humanization of 528, a murine antibody specific for the human EGFR (41), led to a substantial reduction of the antibody's affinity for its target.

In this study, we focused on Vernier zone residues of 528 to determine what is predominantly responsible for the reduction of affinity that occurs upon humanization. Several mutants of the variable domains of humanized 528 (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. The interactions between mutants and the soluble extracellular domain of EGFR (sEGFR) were investigated using isothermal titration calorimetry (ITC). On the basis of results obtained, we discuss the role that Vernier zone residues may play in the high affinity of antibodies for their targets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of CDR-grafted (Humanized) 528 Antibody—Anti-human EGFR murine antibody 528 was humanized by means of the CDR-grafting method. First, sequences of human immunoglobulin variable regions were identified from the DDBJ data base (available on the World Wide Web) by using the BLAST sequence program. We chose the sequences of human variable light (VL) and variable heavy (VH) chains with the highest homology with the 528 framework regions. Amino acid sequences of BR55-2 (accession ID A25561 [GenBank] ; 88% identity in framework) and Xu-12 (accession ID AF062257 [GenBank] ; 68% identity in framework) were selected for the VL framework and the VH framework, respectively. Fig. 1 shows the amino acid sequences of the variable domain fragment of anti-human EGFR murine antibody 528 (m528Fv) and of humanized 528Fv (h528Fv). The genes encoding h528Fv were chemically synthesized by a method based on overlap extension PCR as described previously (38, 42). The NcoI + SacII-digested PCR products were cloned into pUC19-based plasmid pRA (43). The designed DNA sequences encoding h528VH and h528VL are shown in supplemental Fig. S1.

Site-directed Mutagenesis—Site-directed mutagenesis was accomplished as described previously (44). The DNA primers used for mutagenesis are listed in supplemental Table S1. The mutants constructed in this study are summarized in Table 2.


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  		TABLE 2
Mutants constructed in this study

 
Production and Expression of h528Fv and sEGFR—The genes encoding the VH and VL chains of h528Fv and its mutants were inserted into the T7-based expression vector, pRA, for bacterial co-expression of the Fvs (39, 40). Escherichia coli strain BL21 (DE3) was transformed with the vector constructed and then incubated at 37 °C in 2x YT broth with 100 mg of ampicillin per liter of culture. When the optical density reached 0.8 at a wavelength of 600 nm, isopropyl-1-thio-β-D-galactopyranoside was added to the culture at a final concentration of 1 mM to induce expression of the target protein, and the cells were further grown overnight. The culture supernatant was salted out with ammonium sulfate at 80% saturation, and the precipitates were collected by centrifugation at 7000 x g for 30 min at 4 °C. The protein precipitates were dissolved in 50 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and dialyzed against the same buffer for 2 days. The precipitates formed during dialysis were removed by centrifugation at 10,000 x g for 15 min at 4 °C. The supernatant was loaded onto a TALON immobilized metal affinity chromatography column (Invitrogen) previously equilibrated with the same buffer. The column was washed with 50 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and 1 mM imidazole, and then the adsorbed protein was eluted with 50 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and 150 mM imidazole. Further purification was performed by anion exchange chromatography (Resource S; GE Healthcare, Tokyo, Japan) followed by size exclusion chromatography on Superdex200pg (26/60; GE Healthcare, Tokyo, Japan).

A C-terminal hexahistidine tag-fused sEGFR was expressed by Chinese hamster ovary cells, which were kindly provided by Dr. Tsutomu Arakawa (Alliance Protein Laboratories). Cells were grown in a medium composed of RPMI1640 (Sigma) containing 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin. For large scale production, cells were grown in Dulbecco's modified Eagle's medium containing 1% nonessential amino acid solution (Invitrogen) for 3 days. Nickel-charged His-bind resin (1% volume; Novagen Inc., Madison, WI) was added to the supernatant of the culture medium and allowed to batch bind at 4 °C for more than 12 h and then loaded to the column. After washing with 50 mM Tris-HCl (pH 8.0) containing 200 mM NaCl and 10 mM imidazole, the elution buffer (50 mM Tris-HCl (pH 8.0) containing 200 mM NaCl and 10 mM imidazole) was added for elution of the protein of interest. Final purification was performed by size exclusion chromatography on Superdex200pg (26/60; GE Healthcare).

Matrix-assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry—Mass spectra were measured on a REFLEX III MALDI-TOF mass spectrometer (Bruker Analytische, GmbH, Germany) equipped with a nitrogen laser (337 nm). Sinapic acid was applied as a matrix and dissolved to saturation in water/acetonitrile (2:1, v/v) containing 0.067% trifluoroacetic acid. Sample solutions from each stage were mixed with the sinapic acid-saturated solution in a 1:1 (v/v) ratio, and then 1 µl of the mixed solution was loaded onto the sample target. After co-crystallization on the target, the crystals were washed two times with 2 µl of water containing 0.1% trifluoroacetic acid to remove salts. Analysis was performed in positive and linear modes with an accelerating voltage of 27 kV, and 200 scans were averaged.

Preparation of m528Fab Fragment—The Fab fragment of m528 was generated by papain digestion of m528 IgG (subtype IgG2a) with an ImmunoPure Fab Preparation Kit (Pierce). The Fab fragment was separated from undigested IgG and the constant domain fragment (Fc) by means of a Protein A column. The flow-through fraction was collected and used for crystallization.

Crystallization of h528Fv and m528Fab—All crystallization experiments were performed using the vapor diffusion method at 20 °C. Crystal screening was performed by the sitting drop vapor diffusion method using Crystal Screens I and II, Salt Rx (Hampton Research, Aliso Viejo, CA), and Wizards 1 and 2 (Emerald Biostructures, Bainbridge Island, WA). Well-diffracted crystals were obtained in hanging drops equilibrated against a reservoir solution consisting of 3 M sodium chloride and 0.1 M Tris-HCl (pH 7.5) for h528Fv and 1.4 M ammonium sulfate, 0.01 M cadmium chloride, and 0.1 M Tris-HCl (pH 7.1) for m528Fab. The crystallization drops contained equal volumes (2 µl) of reservoir and purified protein solution (20 mg/ml for h528Fv and 36 mg/ml for m528Fab in 10 mM Tris-HCl, pH 8).

X-ray Data Collection and Structure Determination—Diffraction data for h528Fv were obtained at the NW-12 beamline at KEK PF-AR (Japan). Crystals of h528Fv were flash-cooled to 100 K by using paratone-N oil as a cryoprotectant. The protein crystallized in the space group P65 with the following unit cell dimensions: a = b = 63.28 and c = 225.34. The resolution was 2.1 Å. Data were processed with HKL2000 and SCALA of the CCP4 program suite (45). The structure of h528Fv was solved by molecular replacement with the MOLREP program of CCP4 (46). The search models used were the VH domain of humanized anti-CD40 ligand antibody (Protein Data Bank code 1I9R) and the VL domain of anti-HIV protease antibody (Protein Data Bank code 1CL7), respectively. After rigid body refinement with the REFMAC program (47), refinement was carried out with the simulated annealing and energy minimization protocols in the CNS1.1 program (48). The model was rebuilt using the Xfit module in XtalView (49) against a composite omit map calculated in CNS. Shape complementarity (SC) coefficients, excluding water molecules, were calculated using SC (50) as implemented in the CCP4 suite with a 1.7-Å probe. Graphics were generated using Pymol (available on the World Wide Web).

Diffraction data for m528Fab were obtained at the BL-6A beamline at KEK PF (Japan). Crystals of m528Fab were flash-cooled to 100 K by using 25% glycerol as a cryoprotectant. The m528Fab crystallized in the space group P62 with the following unit cell dimensions: a = b = 126.60 and c = 68.28. The resolution was 2.3 Å. The structure was determined as described for h528Fv. The search models used were the VH and VL domains of h528Fv and the CH1 and CL domains of anti-TGF{alpha} antibody (Protein Data Bank code 1E4X), respectively. Data and refinement statistics are summarized in Table 1.


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  		TABLE 1
Crystallographic data for m528Fab and h528Fv

Highest resolution shells are shown in parentheses.

 
Isothermal Titration Calorimetry—Thermodynamic parameters for the interactions between the sEGFR domain and m528Fv, h528Fv, and several mutants were determined by microtitration calorimetry using a VP-ITC from MicroCal Inc. (Northampton, MA) (51). The experimental conditions were as follows. sEGFR at a concentration of 2 µM in phosphate-buffered saline (pH 7.2) containing 0.005% Tween 20 in a calorimeter cell was titrated with a 20-µM solution of the Fv in the same buffer. The ligand solution was injected 25 times in 10-µl portions over a period of 15 s. Data acquisition and subsequent nonlinear regression analysis were done in terms of a simple binding model, using the MicroCal ORIGIN software package.

The enthalpy changes ({Delta}H) and binding constants (Ka) for the antigen-antibody interactions were directly obtained from the experimental titration curves. The Gibbs energy changes, {Delta}G =-RT ln Ka, and the entropy changes, {Delta}S = ({Delta}G + {Delta}H)/T, for the associations were calculated from the {Delta}H and Ka values. The heat capacity changes ({Delta}Cp) were estimated from the temperature dependence of the enthalpy changes.

Estimation of Protein Concentration—The concentration of sEGFR was estimated by using Formula = 9.53 (cm-1 M-1). The concentration of m528Fv was estimated by using Formula = 17.2 (cm-1 M-1), and the concentration of h528Fv was estimated by using Formula = 17.2 (cm-1 M-1) for the wild type and mutants except for those including a Y27D mutation. Formula = 16.8 (cm-1 M-1) was used for the mutants, including Y27D (52).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Construction, Preparation, and Functional Characterization of the Fv Region of Humanized Anti-human EGFR Murine Antibody 528
Anti-human EGFR murine antibody 528 was humanized by the CDR grafting method. We selected human antibody frameworks with the highest homology to the framework of m528Fv, and CDR regions defined by Kabat et al. (53) were grafted onto the frameworks selected. Murine and human heavy chain V genes are classified into three known clans (54). Both VH genes of parental murine antibody 528 (J558 family) and selected human antibody framework Xu-12 (VH1 family) belong to clan I (55). The DNA sequences designed were chemically synthesized as described under "Experimental Procedures" (Fig. 1 and supplemental Fig. S1).

The Fv fragments were expressed using an E. coli secretory expression system and were purified from the culture supernatant by immobilized metal affinity chromatography and subsequent size exclusion chromatography. Final yields of m528Fv and h528Fv were ~1.0 mg/liter of culture (Fig. 2). For functional characterization of the Fvs, c-Myc peptide tag was fused to the C terminus of both chains. Flow cytometric analyses using some EGFR-positive cells and anti-c-Myc peptide tag antibody 9E10 indicated that both Fv fragments had binding profiles identical to that of IgG, and specific inhibition of the binding of the Fvs to the cell surface antigen by the IgG was confirmed (data not shown) (37-39, 41). These results suggest that m528Fv and h528Fv recognized the same epitope structure of EGFR as the parental 528 IgG.

Thermodynamic Analysis of the Interaction between sEGFR and 528 Antibody Fragments
To investigate the interactions between sEGFR and the antibody fragments constructed, we performed thermodynamic analyses by means of ITC. A Chinese hamster ovary cell expression system was used for production of sEGFR. The molecular weight of the purified sEGFR was confirmed using MALDI-TOF mass spectroscopy. Although the polypeptide chain of sEGFR is estimated to be ~68 kDa, the expressed and purified sEGFR had a broad distribution of molecular masses around 90 kDa (data not shown), which suggests that sEGFR was heavily glycosylated. This result agrees with those in previous reports (56, 57).

We carried out an ITC at three temperatures under the conditions described under "Experimental Procedures" (Fig. 3). Thermodynamic parameters (25 °C and pH 7.2) calculated from the titration curves are summarized in Table 3, and the temperature dependence of the enthalpy changes due to binding is shown in Fig. 4. The affinity of h528Fv for sEGFR was less than 1/40 that of the m528Fv fragment (Table 3). At 25 °C, the negative enthalpy change (-{Delta}H) of the h528Fv-sEGFR interaction was 18.9 kJ mol-1 less than that for m528Fv binding, and the negative entropy change was ~9.6 kJ mol-1 higher. The heat capacity changes ({Delta}Cp) of the interactions of m528Fv and h528Fv, estimated from the temperature dependence of the enthalpy changes, were -2.02 and -1.44 kJ mol-1 K-1, respectively.


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  		TABLE 3
Thermodynamic parameters of antibody fragment-sEGFR interactions at 25 °C and pH 7.2

Ka, binding constant; {Delta}G, {Delta}H, {Delta}S, and {Delta}Cp, changes in Gibbs energy, binding enthalpy, entropy, and heat capacity, respectively. {Delta}{Delta}H and T{Delta}{Delta}S are the differences of binding enthalpy and entropy between mutant and h528Fv. Experimental protocols are described under "Experimental Procedures."

 


Figure 1
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  		FIGURE 1.
Amino acid sequences of m528Fv and h528Fv. *, identical amino acids; red, CDRs; blue, Vernier zone residues.

 


Figure 2
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  		FIGURE 2.
Expression and purification of 528Fv. The case of wild-type h528Fv is shown. The gel concentration was 15%. The molecular size markers are indicated on the left. Lane 1, culture supernatant of E. coli BL21(DE3)-expressed Fv fragment; lane 2, purified fractions.

 
Crystal Structures of m528Fab and h528Fv
The structures of m528Fab and h528Fv were determined in antigen-free form at resolutions of 2.3 and 2.1 Å, respectively, by the molecular replacement method. The crystallographic asymmetric units contain one Fab in the m528Fab crystal and two Fvs in the h528Fv crystal. Data and refinement statistics are summarized in Table 1. The R and Rfree values of the final model of m528Fab were 0.191 and 0.228, and those of h528Fv were 0.273 and 0.301, respectively.

The overall structures of m528Fab and h528Fv are shown in Fig. 5. A relatively large groove is present on the CDRs. h528Fv was superimposed on the Fv part of m528Fab. The root mean square (r.m.s.) deviations of the C{alpha} atoms between the Fv portions of m528Fab and h528Fv and r.m.s. deviations of the C{alpha} atoms when the CDRs were superimposed are summarized in Tables 4 and 5, respectively. The results demonstrate that the overall structure of h528Fv is similar to that of m528Fab, including the CDR structures (Table 5, "All CDRs fit"). No major changes in the CDR loop structures were observed when the CDRs were superimposed (Fig. 6). The structures of these CDRs can be classified into the canonical structural classes (58). CDR-L1 (16 residues) has a 5-amino acid insertion after position 27 and thus belongs to canonical structure 4. CDR-L2 (7 residues) is in the hairpin loop and classified into canonical structure 1. CDR-L3 (9 residues) belongs to canonical structure 1. CDR-H1 (5 residues) and CDR-H2 (17 residues) belong to canonical structures 1 and 2, respectively. CDR-H2 has one amino acid insertion after position 52 and contains a 4-residue loop.


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  		TABLE 4
r.m.s. deviations in the C{alpha} atom of each chain (Å)

The r.m.s. deviations were obtained by superimposing the C{alpha} atom coordinates of each polypeptide chain (VL or VH) or of all chains on the corresponding chain of the wild-type complex. r.m.s. deviations were calculated with LSQKAB and COMPAR in the CCP4 suite. Bold indicates the criteria for the values of r.m.s. deviations.

 


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  		TABLE 5
r.m.s. deviations in the C{alpha} atom of each CDR loop or chain (Å)

The r.m.s. deviations were obtained by superimposing the C{alpha} atom coordinates of each CDR loop (VL or VH) or all CDR loops on the corresponding loops of the wild-type complex. r.m.s. deviations were calculated with LSQKAB and COMPAR in the CCP4 suite. Bold indicates the criteria for the values of r.m.s. deviations.

 
No marked changes in the relative orientations of VL and VH were noted for m528Fv and h528Fv (Table 4, "Fv fit"). However, the r.m.s. deviations of the VL and VH chains of h528Fv were 1.03 and 1.56 Å, respectively, when the VH and VL chains of h528Fv were superimposed on those of m528Fv (Table 4, "VH fit" and "VL fit"). When the CDR loops of the VH and VL chains of h528Fv were superimposed on the corresponding loops of m528Fv, the r.m.s. deviations of the CDR loops were 0.84 and 1.58 for the VL and VH chains, respectively (Table 5, "CDR-H fit" and "CDR-L fit").

The contact areas of the VH-VL interfaces for m528Fv and h528Fv were 1569 and 1497 Å2, respectively. The decreases in the contact areas originated mainly from the interactions around residues 39, 42, and 45 in the VH chain (Fig. 7).

Thermodynamic Analysis of Complex Formation of h528Fv Framework Mutants with EGFR
To address the role of the Vernier zone residues in the high affinity of antibodies for their targets, we constructed mutants of h528Fv in which some of the Vernier zone residues were mutated to the corresponding residues of the parental murine antibody. The Vernier zone residues of the VL of h528Fv were completely identical to those of the parental murine one (Figs. 1 and 8); therefore, we focused on the Vernier zone residues of the heavy chain at the following target sites: HTyr27, HMet48, HMet69, HArg71, HThr73, and HAla93 (Fig. 8). Additionally, HArg66, which underlay the CDR-H2 and could have an effect on the conformation of the loop, was selected as the target site. The residues at these sites of h528Fv were substituted with those of m528Fv, and the mutants were prepared as wild type using a bacterial expression system. The mutants constructed are listed in Table 2. Thermodynamic analyses of the interactions between sEGFR and the mutant Fvs were performed by ITC, and the results are summarized in Table 3.


Figure 3
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  		FIGURE 3.
Isothermal titration calorimetry of the interactions between sEGFR and m528Fv (A) and h528Fv (B). Top, typical calorimetric titration of sEGFR (2.5 µM) with 25 µM antibody fragments at pH 7.2 and 25 °C; bottom, integration plot of the data calculated from the raw data. The solid line corresponds to the best fit curve obtained by least squares deconvolution.

 


Figure 4
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  		FIGURE 4.
Temperature dependence of the enthalpy change of the interactions between Fvs and sEGFR. Typical results are shown. Circle, h528Fv; cross, m528Fv; triangle, HM48I (Mu2); diamond, HR66KR71V (Mu4); square, HM48IR66KR71V (Mu6).

 
Mu1 (HY27D)—Mutation of HTyr27 to Asp led to a substantial increase in the negative enthalpy change (-{Delta}H) of the association with sEGFR; the {Delta}H value was almost the same as that of h528Fv. However, because of the enhanced entropy loss relative to h528Fv, the affinity of Mu1 for the target was not improved by the mutation. The heat capacity change ({Delta}Cp) was -2.15 kJ mol-1 K-1, which was 0.1 kJ mol-1 K-1 more negative than that of m528Fv.

Mu2 (HM48I)—Mutation of HMet48 to Ile led to a substantial increase in the negative enthalpy change of the association with sEGFR, and the {Delta}H value was almost the same as that of the parental m528Fv. However, because of the enhanced entropy loss, the affinity of Mu1 for the target was not improved by the mutation. The heat capacity change was -3.66 kJ mol-1 K-1, which was 1.6 kJ mol-1 K-1 more negative than that of m528Fv.

Mu3 (HA93T)—Mutation of HAla93 to Thr led to a substantial increase in the negative enthalpy change of the association with sEGFR. However, because of the enhanced entropy loss, the affinity of Mu2 for the target was not improved by the mutation. The heat capacity change was -2.99 kJ mol-1 K-1, which was 1.0 kJ mol-1 K-1 more negative than that of m528Fv.

Mu4 (HR66K/R71V)—We substituted HArg66 and HArg71 with the corresponding residues in m528Fv. These sites are located in the framework H3 region (FR-H3) (i.e. the reverse site of the antigen-binding sites of the CDR-H2). The favorable enthalpy change for Mu1 and Mu2 was almost completely compensated for by the unfavorable entropy change. {Delta}Cp of the interaction between Mu4 and sEGFR was almost the same as that of h528Fv.

Mu5 (HM48I/A93T)—We combined Mu2 with Mu3. Substitution of HAla93 with Thr led to reduction of the favorable enthalpy change due to the mutation in H48M. The heat capacity change ({Delta}Cp) was -1.50 kJ mol-1 K-1, which was 0.5 kJ mol-1 K-1 more negative than that of m528Fv.

Mu6 (HM48I/R66K/R71V)—We combined Mu2 (HM48I) with Mu4 (HR66KR71V). The negative enthalpy change was increased by 8 kJ mol-1 compared with that of Mu4. The enthalpy change was compensated for by the increase in entropy loss, and thus the affinity for the target was not changed by an additional mutation at site 48 of VH. The heat capacity change ({Delta}Cp) was -3.34 kJ mol-1 K-1, which was 1.3 kJ mol-1 K-1 more negative than that of m528Fv.

Mu7 (HY27D/M48I/R66K/R71V)—We combined Mu6 with Mu1 (HY27D). The enthalpic advantage due to the three mutations (HM48I/R66K/R71V) was substantially reduced by substitution of HAla93 with Thr, and the negative value of the heat capacity change ({Delta}Cp) was substantially reduced compared with that of m528Fv.


Figure 5
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  		FIGURE 5.
Overall structures of the Fv portions of m528Fab and h528Fv. The structure of h528Fv, for which the C{alpha} coordinates of the VL chain are superimposed on the C{alpha} coordinates of m528Fv, is superimposed on the structure of m528Fv (gray). Blue, heavy chain of h528Fv; green, light chain of h528Fv; gray, Fv portion of m528Fab.

 


Figure 6
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  		FIGURE 6.
CDR loop structures of the light (A) and heavy chains (B) of m528Fab and h528Fv. The C{alpha} coordinates of the CDR loops are superimposed. Green and blue, h528Fv; gray, m528Fab.

 


Figure 7
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  		FIGURE 7.
Local structures of the VH-VL interfaces. Blue, heavy chain of h528Fv; green, light chain of h528Fv; gray, m528Fab. Residue numbers are according to Kabat et al. (53). The parentheses indicate m528Fab residues.

 
Mu8 (HR66K/R71V/A93T)—We combined Mu4 (HR66K/R71V) with Mu3 (HA93T). The combined mutation led to substantial reduction of the enthalpic advantage.

Mu9 (HR66K/M69K/R71V/T73R)—HMet69 and HThr73 of Mu4 (HR66K/R71V) were substituted with the corresponding murine residues. The mutations increased the negative enthalpy change by -15 kJ mol-1; however, the affinity for the target was not improved. The heat capacity change ({Delta}Cp) was -3.15 kJ mol-1 K-1.

Mu10 (HM48I/R66K/M69K/R71V/T73R)—HMet48 of Mu9 was substituted with Ile. The favorable enthalpy changes and entropy loss were reduced by the additional mutation, and no significant change in the Gibbs energy was observed. The heat capacity change ({Delta}Cp) was almost identical to that for h528Fv.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Thermodynamic Consequences of Humanization of 528—The parental murine antibody, 528 IgG, inhibited the binding of m528Fv and h528Fv to EGFR expressed on cancer cells in a flow cytometric assay (data not shown), suggesting that the epitopes of the Fvs were identical to that of the parental IgG. The thermodynamic analyses of the binding of m528Fv or h528Fv to sEGFR indicated that humanization led to reduction of the affinity, due to an unfavorable enthalpy change. Enthalpy changes generally relate to the formation of noncovalent bonds, such as hydrogen bonds, ionic bonds, and van der Waals interactions at binding interfaces (59). Thus, the decrease in the negative enthalpy change due to humanization of 528 was probably caused by differences in the formation of noncovalent bonds, including bonds with interfacial water molecules, upon complex formation.


Figure 8
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  		FIGURE 8.
Schematic representation of Vernier zone residues. A, overall structures of the Fv portions of m528Fab and h528Fv; B, local structures around the target residues. Both the main chain and the side chains from the target residues in this work and only the main chain from the other residues were shown. Blue, heavy chain of h528Fv; green, light chain of h528Fv; red, Vernier zone residues of h528Fv; gray, m528Fab. Residue numbers are according to Kabat et al. (53). The parentheses indicate m528Fab residues.

 
Crystal Structures of m528Fab and h528Fv—To determine whether humanization produced conformational changes in the antibody fragment, we determined the crystal structures of m528Fab and h528Fv. We were unable to obtain a crystal of m528Fv appropriate for structural analyses, so we prepared a Fab fragment of m528 from the parental murine 528 IgG via papain digestion and subsequent purification. Crystal structures of m528Fab and h528Fv indicate that the conformations of all of the CDR loops and Vernier zone residues were almost completely conserved after humanization of m528. The interfacial residues between VH and VL were relatively conserved in going from m528Fv to h528Fv, except for the regions around sites 39, 42, and 45 of VH (data not shown). SC values between the VH and VL chains of m528Fv and h528Fv were 0.754 and 0.735, respectively, which suggests that the shape complementarities in the VH-VL interface were retained after humanization. These results suggest that no major conformational changes were introduced by humanization, either in the side chains or the main chains of the CDR structures. However, the r.m.s. deviations of the VL and VH chains of h528Fv were 1.03 and 1.56 Å, respectively, when VH and VL of h528Fv were superposed on those of m528Fv (Table 4, "VH fit" and "VL fit"). When the CDR loops of the VH and VL chains of h528Fv were superimposed on the corresponding loops of m528Fv, the r.m.s. deviations of the CDR loops were 0.84 and 1.58 for VL and VH, respectively (Table 5, "CDR-H fit" and "CDR-L fit"). Previous reports on structural analyses of anti-lysozyme antibody fragments have indicated that r.m.s. deviations of Fv and Fab fragments are within 1 Å (60, 61); therefore, we observed slight but critical humanization-induced changes in the relative orientations of VL and VH.

Thermodynamic Consequences of Mutations in Vernier Zone Residues of h528Fv—For improvement of the affinity or specificity of humanized antibodies, Vernier zone residues of humanized antibodies can be substituted with the residues of murine antibodies. To determine the role of Vernier zone residues of humanized antibodies, we chose the following target sites: HTyr27, HMet48, HArg66, HMet69, HArg71, HThr73, and HAla93 (Fig. 8). First we analyzed four point mutants (Y27D (Mu1), M48I (Mu2), A93T (Mut3), and R66K/R71V (Mut4)), because these sites have already been studied in other antibody systems (62-64). The thermodynamic parameters of the interactions between the mutants and sEGFR indicate that these mutations substantially increased the negative enthalpy changes. However, the mutations improved affinity for the target only slightly, which indicates that unfavorable entropy changes were enhanced. We then combined some mutations; however, we observed no marked improvement of the affinity for sEGFR, although the enthalpic advantage varied depending on the combination of mutations. These results suggest that the effects of point mutations on the affinity were not incremental; thus, combination of mutations may correlate with structural rearrangement of the antigen-binding region of the antibody.

Note that substitutions of Met48 and Ala93 with the corresponding murine residues led to an increase in the negative values of the heat capacity change. Negative heat capacity changes in protein-protein interaction are observed frequently and are believed to result from coverage of the protein hydrophobic surface (65-68). However, pioneering works by Morton and Ladbury (69), Spolar and Record (70), and Frier et al. (65) have suggested the correlation of other factors, such as restriction of water molecules at the interface and recognition-coupled structural changes due to induced fitting (71, 72), with the large negative values of the heat capacity change. The heat capacity changes due to antigen binding of the h528Fv mutants (Mu2, Mu3, Mu6, and Mu9) did not fall within the typical range for antigen-antibody interaction, which suggests significant changes of the structure of water molecules or the binding surface.

Entropy Changes Resulting from Conformational Changes upon Binding of Antibody Fragments with sEGFR—According to the considerations of Murphy et al. (73), the total {Delta}S of binding is given by Equation 1,

Formula(Eq.1)
where {Delta}Ssolv represents the change in entropy derived from solvent release upon binding, {Delta}Sconf is the change in the entropy resulting from conformational changes due to formation of the antigen-antibody complex, and {Delta}Scrat is the cratic entropy change. {Delta}Ssolv is given by Equation 2,

Formula(Eq.2)
where Ts is the temperature at which the desolvation entropy change is considered to be 0 (Ts = 386 K), and {Delta}Scrat can be considered to have a constant value (-33 kJ mol-1 K-1) (73). It has been suggested that the overall conformational entropy loss is, to a large extent, compensated for by the desolvation entropy. Therefore, the present data can be interpreted as follows.

In the interaction between sEGFR and m528Fv, {Delta}Cp is estimated to be -2.02 kJ mol-1 K-1 (Table 3), and from Equation 2, {Delta}Ssolv is calculated to be 0.523 kJ mol-1 K-1. From the experimental results, the total entropy change is -0.0977 kJ mol-1 K-1, and {Delta}Sconf is estimated to be -0.587 kJ mol-1 K-1 from Equation 1. In the interaction between sEGFR and h528Fv, {Delta}Ssolv is estimated to be 0.373 kJ mol-1 K-1, which is 0.150 kJ mol-1 K-1 smaller than the value for m528Fv. This result indicates that the entropic advantage gained by desolvation is reduced by CDR grafting (i.e. humanization). The total entropy change is estimated to be -0.0654 kJ mol-1 K-1, and {Delta}Sconf is calculated to be -0.405 kJ mol-1 K-1, which was 0.182 kJ mol-1 K-1 smaller than the value for m528Fv, which indicates that the negative entropy change generated by the conformational changes was substantially reduced. These observations indicate that humanization of m528Fv led to reduction of the entropy loss due to the conformational changes.

We then calculated {Delta}Ssolv and {Delta}Sconf values of the interactions between sEGFR and the constructed mutants (Table 6). All mutants except for Mu7 and Mu10 had favorable {Delta}Ssolv and unfavorable {Delta}Sconf values. These results suggest that conformational changes were increased by the mutations and that the entropy loss due to the enhanced conformational changes was not completely compensated for by the increased desolvation entropy change. Note that point mutation of Vernier zone residues led to a substantial increase in the conformational entropy change and that combination of these mutations led to reduction of the conformational entropy loss. For instance, M48I and A93T mutations increased the conformational entropy loss (see Mu2 and Mu3, respectively); however, combination of these mutations (Mu5) led to a decrease in the conformational entropic disadvantage (Table 6).


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  		TABLE 6
Entropy changes of the interaction between sEGFR and 528 antibody fragments at 25 °C

{Delta}{Delta}Ssolv and {Delta}{Delta}Sconf are the differences of solvation and conformational entropies between mutant and h528Fv. ND, not determined.

 


Figure 9
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  		FIGURE 9.
Enthalpy-entropy compensation plot of the interactions between h528Fv mutants and sEGFR. Solid diamond, m528Fv; double circle, h528Fv; open circles, h528Fv mutants.

 
Effect of Enthalpy-Entropy Compensation on Improvement of Affinity via Mutation—T{Delta}S values for the binding of h528Fv and mutants to sEGFR are plotted against the corresponding {Delta}H values in Fig. 9. A linear relationship was observed with a slope of 1.04 (correlation coefficient, R2 = 0.993). The dashed line represents an energy compensation line for m528Fv with a slope of 1.00. This linear relationship, referred to as entropy-enthalpy compensation, has been described in many systems 74-78. A favorable enthalpy change of a mutant compared with h528Fv is compensated for by an unfavorable entropy change, and an unfavorable enthalpy change is compensated for by a favorable entropy change, which makes the difference in the free energy change ({Delta}{Delta}G) of antigen binding smaller than expected.

In the system we describe here, the values for the free energy of antigen binding for h528Fv and its mutants ranged from -43.7 to -39.8 kJ mol-1, and these values were far from the value for m528Fv (-50.9 kJ mol-1). The energy plot of h528Fv and its mutants showed an apparent compensation relationship (Fig. 9, dashed line), and the plot for m528Fv is far from the line (Fig. 9, solid line). These results suggest the existence of an energetic barrier (79) that originates from enthalpy-entropy compensation and cannot be overcome by mutations in Vernier zone residues. The results of thermodynamic analyses suggest that mutations in the framework regions of the antibody may affect hydrated structures of the antigen-free antibody. Mutations in the Vernier zone residues may contribute to structural adjustment of the CDR regions upon binding; however, enthalpy-entropy compensation may interfere with the improvement of the antibody affinity for its target. Enthalpy-entropy compensation has been observed in previous pioneering studies of the thermodynamics of affinity maturation of antibodies; an energetic barrier originating from the relationship between enthalpy and entropy changes is overcome by an increase in negative enthalpy changes, a decrease in negative entropy changes during the affinity maturation process, or both (80). Note that affinity maturation of antibodies leads to a decrease in the structural changes of the antibody upon binding (81). The present results show that some mutations in the Vernier residues increased the negative heat capacity changes, and this increase may be correlated with changes in the hydrated structures of the antibody.

Conclusions—Our present results indicate that substitution of Vernier zone residues of the humanized anti-EGFR antibody 528 with the corresponding murine residues did not improve the affinity of antigen binding, and thermodynamic analyses suggest that the effects of structural changes due to mutations on the association of the antibody with the target were cancelled out by entropy-enthalpy compensation. These results suggest that Vernier zone residues made enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Note that the mutated sites in this study were in framework residues and not in the interfacial areas of antigen binding; the CDR residues of m528Fv were completely conserved in h528Fv. Crystal structures of antigen-free antibody fragments clearly indicate that no major conformational changes were introduced into the CDR loop structures by humanization. These observations suggest that the structural rearrangement that occurs when the antibody recognizes the antigen makes a critical contribution to the high affinity of the antibody for its target.

Finally, note that the relative orientation of VH and VL in m528Fv was changed by humanization. A critical contribution of variable domain interactions to high specificity and affinity of antibodies for targets has been proposed (60, 61, 82-84). Structural analyses of the sEGFR-528Fv complexes would allow for precise description of the interaction from structural and thermodynamic viewpoints.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1WT5 and 2Z4Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (to K. T. and I. K.). Additional support was provided through Grants-in-Aid for Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan and through the Proposal-based R&D Promotion Program and the Industrial Technology Research Grant Program in 2003 from the New Energy and Industrial Technology Development Organization of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Fig. S1. Back

1 Both authors contributed equally to this work. Back

2 Present address: Dept. of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637. Back

3 Present address: Dept. of Medical Genome Sciences, Graduate School of Engineering, University of Tokyo, Kashiwanoha, Kashiwa 277-8562, Japan. Back

4 To whom correspondence should be addressed. Tel.: 81-22-795-7274; Fax: 81-22-795-6164; E-mail: kmiz{at}kuma.che.tohoku.ac.jp.

5 The abbreviations used are: CDR, complementarity-determining region; CDR-H, CDR in heavy chain; CDR-L, CDR in light chain; EGFR, epidermal growth factor receptor; sEGFR, soluble extracellular domain of human EGFR; m528Fv, variable domain fragment of murine 528 antibody; h528Fv, variable domain of humanized 528 antibody; m528Fab, Fab fragment of murine antibody 528; ITC, isothermal titration calorimetry; VH, heavy chain of variable domain; VL, light chain of variable domain; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Fv, variable domain fragment of antibody; r.m.s., root mean square; SC, shape complementarity. Back


   ACKNOWLEDGMENTS
 
We thank N. Sakabe, S. Wakatsuki, M. Suzuki, and N. Igarashi of the Photon Factory for kind help with the data collection.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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R. Asano, Y. Sone, K. Ikoma, H. Hayashi, T. Nakanishi, M. Umetsu, Y. Katayose, M. Unno, T. Kudo, and I. Kumagai
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