Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments

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Abstract
The murine monoclonal antibody 4D5 (anti-p185HER2) inhibits the proliferation of human tumor cells overexpressing p185HER2 in vitro and has been "humanized" [Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W.-L. T., Rowland, A. M., Kotts, C., Carver, M. E., & Shepard, H. M. (1992) Proc. Natl. Acad. Sci. U.S.A (in press)] for use in human cancer therapy. We have determined the antigen binding thermodynamics and the antiproliferative activities of chimeric 4D5 Fab (ch4D5 Fab) fragment and a series of eight humanized Fab (hu4D5 Fab) fragments differing by amino acid substitutions in the framework regions of the variable domains. Fab fragments were expressed by secretion from Escherichia coli and purified from fermentation supernatants by using affinity chromatography on immobilized streptococcal protein G or staphylococcal protein A for ch4D5 and hu4D5, respectively. Circular dichroism spectroscopy indicates correct folding of the E. coli produced Fab, and scanning calorimetry shows a greater stability for hu4D5 (T(m) = 82-degrees-C) as compared with ch4D5 Fab (T(m) = 72-degrees-C). K(D) values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the DELTA-H and DELTA-C(p) for binding were determined by using isothermal titration calorimetry. ch4D5 Fab and one of the humanized variants (hu4D5-8 Fab) bind p185HER2-ECD with comparable affinity (DELTA-G-degrees = - 13.6 kcal mol-1). The enthalpy changes associated with binding, however, are considerably different (ch4D5 Fab DELTA-H = -17.2 +/- 1.5 kcal mol-1; hu4D5-8 Fab DELTA-H = -12.9 +/- 0.4 kcal mol-1), which suggests a significant difference in the mechanism of antigen binding. This difference may be important for antiproliferative activity since ch4D5 Fab retains activity whereas hu4D5-8 Fab is inactive. These results suggest that K(D) measurements alone are insufficient in an attempt to reproduce the activity of a murine antibody in a humanized form. Analysis of the thermodynamic data using an empirical method [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2236-2240] indicates that differences in the hydrophobic or vibrational contributions to binding cannot account for the observation of equivalent DELTA-G but differing DELTA-H. The hydrophobic contribution to antigen binding is equivalent for ch4D5 and hu4D5-8 Fab and is consistent with burial of about 960 angstrom2 of nonpolar surface area upon complex formation.