Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

High resolution transmission electron microscopy of age-hardenable Au-Cu-Zn alloys for dental applications.

Microstructures of age-hardenable AuCu-Zn pseudobinary alloys for dental applications were studied by means of high resolution transmission electron microscopic (HRTEM) observation and X-ray diffraction study. HRTEM study revealed that the appearance frequency of antiphase boundaries (APBs) per unit volume of the AuCu II superstructure effectively increased by Zn addition to AuCu, which may be the reason for that high hardness was maintained for a long time in AuCu-Zn alloys. The disordered APBs zone in the AuCu II superstructure had wavy characteristics and fluctuated within regular range. With increasing Zn content in AuCu-Zn alloys, the fluctuation range of APBs' width became narrower, thus random APBs' spacing and irregular APBs' shape of AuCu II superstructure changed to comparatively regular APBs' spacing and shape. Due to the APBs' wavy characteristics, spacing between successive APBs, M, was not constant but scattered, and the magnitude of the scattering of M value decreased with increasing Zn content. By Zn addition to AuCu, phase transformation from a disordered alpha phase to AuCu II phase was greatly accelerated, which made it possible for the AuCu-Zn alloy to have excellent age-hardenability at relatively low temperature like intraoral temperature.

Factors affecting the optical properties of Pd-free Au-Pt-based dental alloys.

The optical properties of experimental Au-Pt-based alloys containing a small amount of In, Sn, and Zn were investigated by spectrophotometric colorimetry to extract factors affecting color of Au-Pt-based high-karat dental alloys. It was found that the optical properties of Au-Pt-based alloys are strongly affected by the number of valence electrons per atom in an alloy, namely, the electron:atom ratio, e/a. That is, by increasing the e/a-value, activities of reflection in the long-wavelength range and absorption in the short-wavelength range in the visible spectrum apparently increased. As a result, the maximum slope of the spectral reflectance curve at the absorption edge, which is located near 515 nm (approximately 2.4 eV), apparently increased with e/a-value. Due to this effect, the b*-coordinate (yellow-blue) in the CIELAB color space considerably increased and the a*-coordinate (red-green) slightly increased with e/a-value. The addition of a third element with a higher number of valence electrons to the binary Au-Pt alloy is, therefore, effective in giving a gold tinge to the parent Au-Pt alloy. This information may be useful in controlling the color of Au-Pt-based dental alloys.

Ordering behaviors and age-hardening in experimental AuCu-Zn pseudobinary alloys for dental applications.

Age-hardening mechanisms and related ordering behaviors of the experimental (AuCu)(1-x)Zn(x) alloys with x < or = 0.2 were investigated for dental applications. The addition of Zn to equiatomic AuCu greatly increased the age-hardening rate and delayed overaging. It was suggested that the quenched-in excess vacancies were greatly related to the age-hardening rate in the AuCu-Zn pseudobinary alloys. In these alloys, the hardness became maximum during the very initial stage of ordering, and with the development of ordered phase, the hardness began to decrease. Transmission electron microscopy revealed that the age-hardening of AuCu-Zn pseudobinary alloys is caused by lattice distortion that occurred during the very early stage of atomic ordering. The addition of Zn to AuCu effectively increased the density of antiphase boundaries per unit volume of the AuCu II superstructure. This is suggested to be the main cause for the retardation of the overaging in the alloys containing Zn of 5 at% or more. This pronounced effect of Zn addition to AuCu alloy on its age-hardening characteristics may be advantageous for obtaining stable mechanical properties of dental casting gold alloys.

Effects of Zn addition to AuCu on age-hardening behaviors at intraoral temperature.

The effect of Zn addition to AuCu on the age-hardening rate at the intraoral temperature was investigated to find out the proper condition for high age-hardening rate. The increase in hardness of Zn-added alloys during aging at 37 degrees C was due to the atomic ordering. With an increase in Zn concentration, hardness of a sample under the as-quenched condition decreased, but the age-hardening rate obviously increased. When Zn content was fixed, a higher solution treatment temperature was more effective for the age-hardening at 37 degrees C. It was suggested that the formation energy of a vacancy considerably decreased with an increase in Zn content. It is reasonable to consider that the amount of quenched-in excess vacancies are markedly increased with an increase in Zn content when the solution treatment temperature was fixed. By transmission electron microscopic observations, it was revealed that the formation of the AuCu II superstructure contributed to the age-hardening at 37 degrees C in the high zinc content alloy.