Association Energetics of Cross-Reactive and Specific Antibodies

Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204-4004, USA.
Biochemistry (Impact Factor: 3.02). 02/2009; 48(6):1390-8. DOI: 10.1021/bi801901d
Source: PubMed


HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.

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    • "Many have endeavored to understand the molecular mechanisms of antibody polyreactivity or multi-specificity. Antibodies have been shown to bind distinct antigens that are conserved chemically and/or structurally with high affinity [13], [14], [15], [16], [17], [18], [19], [20]. However, a single antibody able to interact with more than one antigen epitope devoid of homology may have greater impact on the antigen recognition capacity of the immune repertoire. "
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    ABSTRACT: The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2) antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF) to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.
    Full-text · Article · Apr 2011 · PLoS ONE
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    • "st in the complexes of HH8 with both HEL and JQL , smallest in those formed by HH26 , and intermediate in those of HH10 . This is not possible unless HH8 exhibits an ability to undergo the greatest conforma - tional rearrangement during association , albeit at a cost of large configurational entropic penalty , reflecting its inherent flexibility ( Mohan et al . , 2000 ) . The opposite of this situation is true of HH26 . Both these experimental results support the hypothesis that the three antibodies could exhibit different degrees of flexibilities . The retention ( as in HH26 ) or elimination ( as in HH8 ) of sequences capable of mediating salt bridges during the process of affinity maturation may we"
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    ABSTRACT: Three antibodies, HyHEL-8 (HH8), HyHEL-10 (HH10), and HyHEL-26 (HH26) are specific for the same epitope on hen egg white lysozyme (HEL), and share >90% sequence homology. Their affinities vary by several orders of magnitude, and among the three antibodies, HH8 is the most cross-reactive with kinetics of binding that are relatively invariable compared to HH26, which is highly specific and has quite variable kinetics. To investigate structural correlates of these functional variations, the Fv regions of HH8 and HH26 were homology-modeled using the x-ray structure of the well-characterized HH10-HEL complex as template. The binding site of HH26 is most charged, least hydrophobic, and has the greatest number of intramolecular salt bridges, whereas that of HH8 is the least charged, most hydrophobic and has the fewest intramolecular salt bridges. The modeled HH26-HEL structure predicts the recently determined x-ray structure of HH26, (Li et al., 2003, Nat. Struct. Biol. 10:482-488) with a root-mean-square deviation of 1.03 A. It is likely that the binding site of HH26 is rendered rigid by a network of intramolecular salt bridges whereas that of HH8 is flexible due to their absence. HH26 also has the most intermolecular contacts with the antigen whereas HH8 has the least. HH10 has these properties intermediate to HH8 and HH26. The structurally rigid binding site with numerous specific contacts bestows specificity on HH26 whereas the flexible binding site with correspondingly fewer contacts enables HH8 to be cross-reactive. Results suggest that affinity maturation may select for high affinity antibodies with either "lock-and-key" preconfigured binding sites, or "preconfigured flexibility" by modulating combining site flexibility.
    Full-text · Article · Nov 2003 · Biophysical Journal
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    ABSTRACT: Thermodynamic and structural studies addressed the increased affinity due to L-chain somatic mutations in the HyHEL-10 family of affinity matured IgG antibodies, using ITC, SPR with van't Hoff analysis, and X-ray crystallography. When compared to the parental antibody H26L26, the H26L10 and H26L8 chimeras binding to lysozyme showed an increase in favorable DeltaG(o) of -1.2+/-0.1 kcal mol(-1) and -1.3+/-0.1 kcal mol(-1), respectively. Increase in affinity of the H26L10 chimera was due to a net increase in favorable enthalpy change with little difference in change in entropy compared to H26L26. The H26L8 chimera exhibited the greatest increase in favorable enthalpy but also showed an increase in unfavorable entropy change, with the result being that the affinities of both chimeras were essentially equivalent. Site-directed L-chain mutants identified the shared somatic mutation S30G as the dominant contributor to increasing affinity to lysozyme. This mutation was not influenced by H-chain somatic mutations. Residue 30L is at the periphery of the binding interface and S30G effects an increase in hydrophobicity and decrease in H-bonding ability and size, but does not make any new energetically important antigen contacts. A new 1.2-A structure of the H10L10-HEL complex showed changes in the pattern of both inter- and intra-molecular water bridging with no other significant structural alterations near the binding interface compared to the H26L26-HEL complex. These results highlight the necessity for investigating both the structure and the thermodynamics associated with introduced mutations, in order to better assess and understand their impact on binding. Furthermore, it provides an important example of how backbone flexibility and water-bridging may favorably influence the thermodynamics of an antibody-antigen interaction.
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