Michael G. Mulkerrin

Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody with a Human IgG1 Fc

Rituxan (Rituximab) is a chimeric mAb with human IgG1 constant domains used in the therapy of non-Hodgkin’s B cell lymphomas. This Ab targets B cells by binding to the cell-surface receptor, CD20. In our investigation of the mechanism of B cell depletion mediated by Rituximab, we first constructed mutants of Rituximab defective in complement activation but with all other effector functions intact. Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1 when challenged with either human, rabbit, or guinea pig complement. Alanine substitution at positions E318 and K320 in Rituximab had little or no effect on C1q binding and complement activation, whereas alanine substitution at positions D270, K322, P329, and P331 significantly reduced the ability of Rituximab to bind C1q and activate complement. We have also observed that concentrations of complement approaching physiological levels are able to rescue >60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs.

Engineered Antibodies with Increased Activity to Recruit Complement

This manuscript describes two sites in a human IgG1 that, when mutated individually or in combination, result in a dramatic increase in C1q binding and complement-dependent cytotoxicity activity. These two residues, K326 and E333, are located at the extreme ends of the C1q binding epicenter in the CH2 domain of a human IgG. A mutation to tryptophan at K326 debilitates Ab-dependent cell-mediated cytotoxicity activity. In addition, substitutions of the residues E333 with serine and of K326 with tryptophan in a human IgG2 confer biological activity in the complement-dependent cytotoxicity assay in which the wild-type IgG2 is inactive. This study reveals that the residues K326 and E333 play a significant role in the control of the biological activity of an IgG molecule and can rescue the activity of an inactive IgG isotype.

Influence of histidine on the stability and physical properties of a fully human antibody in aqueous and solid forms.

AbstractPurpose. The aim of the study was to investigate the effect of histidine on the stability and physical properties of a fully human anti-IL8 monoclonal antibody (ABX-IL8) in aqueous and solid forms. Methods. Using a fractional factorial design, we tested many excipients, including histidine, sucrose, and other commonly used excipients, on the stability and physical properties of the antibody in both liquid and lyophilized forms. Antibody stability and physical properties were evaluated using size-exclusion high-performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-polyacryla- mide gel electrophoresis (SDS-PAGE), and a viscometer. Residual moisture content was determined by coulometric Karl Fischer titrator. Differential scanning calorimetry (DSC) was used to detect the glass transition temperatures (Tg) of the solid cakes and melting temperatures (Tm) of the antibody in liquid formulations. Fourier-transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure. Results. Increasing the histidine concentration in the bulk solution inhibited the increases of high-molecular-weight (HMW) species and aggregates upon lyophilization and storage. In addition, histidine bulk enhanced solution stability of the antibody under freezing and thermal stress conditions, as evidenced by the lower levels of aggregates. Furthermore, histidine reduced viscosity of the antibody solution, which is desirable for the manufacture of the dosage form. However, high concentrations of histidine in liquid formulations led to coloration of the solution and high levels of aggregates on storage at elevated temperature (40°C) after the formulations were exposed to stainless steel containers during bulk freezing-thawing. Conclusions. Histidine enhanced the stability of ABX-IL8 in both aqueous and lyophilized forms. Histidine also improved the physical properties such as reducing the solution viscosity. Liquid formulations containing high concentrations of histidine should not be stored in stainless steel tanks at elevated temperatures.

Unfolding and Inactivation

One of the first criteria used for identifying and classifying proteins was the tendency of these “albuminoid” substances to respond to heating by coagulating in the manner of egg white albumin (see discussion in Fruton, 1972). By the early 1900s, thermally induced coagulation was being studied with purified proteins. By the 1930s, heat- and denaturant-induced coagulation was understood to involve two steps: a heat-dependent denaturation* step associated with an increase in solution viscosity and chemical accessibility of side-chain groups, and a heat-independent, but pH and salt-dependent, aggregation/precipitation step involving newly exposed groups (Wu, 1931; Mirsky and Pauling, 1936; Anson, 1938). A number of these coagulated proteins could be restored to the solubility and other properties of their native states by use of denaturing solvents followed by a return to native conditions (Neurath et al., 1944; Anson, 1945). The early observation of very high temperature coefficients for protein coagulation set this process apart from standard chemical reactions and was compared to the temperature dependence of state changes such as melting (Anson, 1938). Whereas the rates of chemical reactions might increase by a factor of two to three with a 10°C increase in temperature, the rate of coagulation was found to increase by factors of hundreds with a 10°C increase (Chick and Martin, 1912). The developing concepts of the nature of protein structure and stability in the first decades of this century depended greatly on such data.