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

• Published in: Biochemistry (Easton) 1992-06, Vol.31 (24), p.5434-5441
• Main Authors: Kelley, Robert F, O'Connell, Mark P, Carter, Paul, Presta, Leonard, Eigenbrot, Charles, Covarrubias, Michael, Snedecor, Brad, Bourell, James H, Vetterlein, David
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, immunoglobulins; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - metabolism; Biological and medical sciences; Calorimetry, Differential Scanning; Circular Dichroism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - metabolism; Mice; Molecular immunology; Monoclonal antibodies; Motion; Protein Conformation; Protein Denaturation; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - immunology; Proto-Oncogene Proteins - metabolism; Radioimmunoassay; Receptor, ErbB-2; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Solubility; Structure-Activity Relationship; Thermodynamics; X-Ray Diffraction
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00139a003

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 (Tm = 82 degrees C) as compared with ch4D5 Fab (Tm = 72 degrees C). KD values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the delta H and delta Cp 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).