The Affinity Maturation of Anti-4-hydroxy-3-nitrophenylacetyl Mouse Monoclonal Antibody

et al. 2008

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 antihuman 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.Structural and functional analyses of antigen-antibody protein-protein interactions have revealed that complementaritydetermining 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 th...

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confidence: 89%

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

Makabe

Nakanishi

et al. 2008

“…Many studies show that hydrogen bonds play a significant role in a wide range of biomolecular interactions in terms of generating affinity and specific recognition (26 -31). It is also reported that hydrogen bonds can make a favorable enthalpic contribution to protein-ligand interactions (32)(33)(34). Thus, the subject of hydrogen bonding is of major interest in biological research.…”

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confidence: 99%

Contribution of Asparagine Residues to the Stabilization of a Proteinaceous Antigen-Antibody Complex, HyHEL-10-Hen Egg White Lysozyme

Yokota

Shiroishi

et al. 2010

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.The specific recognition of ligands by proteins is a fundamental biological phenomenon (1), and the interaction between antigen and antibody in the immune system is a typical example. Antibodies acquire their affinity and specificity for various target antigens by changing the amino acid residue composition of their six hyper-variable regions, known as complementarity-determining regions (CDRs) 3 (2). Despite the small number of amino acid residues composing the CDRs, antibodies can precisely bind a large number of target antigens (3). Some of these residues, serine and asparagine, for example, are located at specific positions in the CDRs in the germ line protein (supplemental Fig. S1). Many structural studies have shown that these residues are essential for the affinity and specificity of the antigen-antibody interaction (4 -9, 11).Recent high resolution analyses of three-dimensional structures of protein-ligand complexes, including the antigen-antibody complexes, and analyses of the kinetic and thermodynamic parameters underlying these interactions (12-19) have indicated that protein-ligand interactions require a good geometric fit according to the lock-and-key (20) and induced fit models (21) as well as a high degree of complementarity of hydrophobic and polar parts of each binding site (17,22,2...

“…However, thermodynamic analysis of antigen-antibody interaction for analyzing precisely the contribution of noncovalent forces to the interaction is most suitable (17)(18)(19)(20)(21)(22), especially when combined with site-directed mutagenesis of structurally well defined ones (23)(24)(25). We have focused on the interaction of hen egg white lysozyme (HEL 1 ) with the anti-HEL monoclonal antibody Hy-HEL10 interaction as a model of proteinaceous antigen-antibody interaction (26,27).…”

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confidence: 99%

Role of Salt Bridge Formation in Antigen-Antibody Interaction

Ogasahara

Ueda

et al. 1996

For elucidation of the role of salt bridge formation in the antigen-antibody complex, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, the structure of which has been well characterized and forms one salt bridge (Lys97 of HEL and Asp32 of HyHEL10 heavy chain variable region (VH)), was investigated. Asp32 of VH was substituted with Ala, Asn, or Glu by site-directed mutagenesis, and the interaction between HEL and the mutant fragments of the variable region of light chain was investigated by inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. Inhibition assay indicated that these mutations lowered the inhibition only slightly. Thermodynamic study indicated that the negative enthalpic change in the interaction between each of the mutant variable regions of light chain and HEL was significantly increased, although the association constant was slightly decreased, suggesting that these mutations increased the entropy change upon antigen-antibody binding. These results indicate that the role of salt bridge formation in the HyHEL10-HEL interaction is to lower the entropic loss due to binding. In the mutant proteins, the numbers of residues that were perturbed structurally on binding increased, suggesting that the salt bridge suppresses excess structural movement of the antibody upon binding.A major goal of molecular biology is to describe biological phenomena in terms of interaction between molecules. Recently, thanks to progress in structural biology, several quaternary structures of biomolecular complexes have been clarified, and now a number of biological events can be described precisely at the atomic level. Among them, DNA-repressor (1), enzyme-inhibitor (2, 3) and antigen-antibody interactions (4, 5) have been most extensively investigated at the atomic level.Antigen-antibody interactions have been studied in detail not only from an immunological but also a biochemical viewpoint as a model of protein-ligand interaction. The most striking feature of antigen-antibody interactions is the creation of strict specificity and high affinity of antibodies for their antigens. It is known that unique conformations and special locations on the surfaces of proteinaceous antigens are recognized by antibodies (6). Among the amino acids located on protein surfaces, it is believed that antibodies recognize charged residues by forming (1) hydrogen bonds and (2) salt bridges and that salt bridge plays a significant role in the interaction (7-9).For detailed elucidation of antigen-antibody interactions, a number of approaches have been attempted, including structural analysis (10 -13) and calculation and modeling (14 -16). However, thermodynamic analysis of antigen-antibody interaction for analyzing precisely the contribution of noncovalent forces to the interaction is most suitable (17)(18)(19)(20)(21)(22), especially when combined with site-directed mutagenesis of structurally well defined ones (23-25). We have focused on the interaction of hen egg white lysozym...