Characterization of recombinant antibodies for cancer therapy by infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to study the structure of the recombinant antibodies 1E10, anti-CD20 and hR3, which are used as anti-cancer therapeutic drugs. We tested their sensitivity against different conditions and treatments such as pH, temperature, freeze-thaw cycles and drying, which are relevant for the practical usefulness of the drugs. All antibodies were stable against moderate temperature increases (up to 50 °C) and pH changes (range 5-9). 1E10 was sensitive to extreme pH values (pH 3 and 12), whereas hR3 was most sensitive to temperature (at and above 60 °C). We did not observe any significant changes upon freeze-thaw and drying treatments. The secondary structure content of all three antibodies was estimated to be similar to that of IgG with ∼64% ß-sheet, 0% α-helix and ∼36% other structure.

Immunohistochemical evaluation of H-R3 a novel humanized monoclonal antibody that neutralizes the EGF-receptor.

The epidermal growth factor receptor (EGF-R) is an important growth regulator of epithelial cancer cells, overexpressed by several human tumors and scantly detectable in most normal tissues. The introduction of monoclonal antibodies (Mabs) and more recently engineered humanized Mabs have greatly expanded the therapeutic potential of this modality of cancer treatment. The present study was designed to compare the specificity of the murine and humanized anti-EGF-R Mabs. Biotinylated Mabs were tested in samples of fetal and adult normal and neoplastic tissues by ABC peroxidase method. All fetal tissues studied were positive for both Mabs, showing 2 different staining patterns, one homogeneous and finely granular in cytoplasm and another grosser with intense labeling in both membrane and cytoplasm. A similar recognition pattern was exhibited in adult normal tissues, where an intense reactivity was also evidenced in skin, tongue, gastrointestinal tract, renal tubules, and breast gland epithelium. In tissues from genitourinary and central nervous system, a faint staining was demonstrate, whereas those from cardiovascular and lymphoid tissues proved to be negative. These Mabs exhibited a heterogeneous and strong membrane and cytoplasm staining in neoplastic cells from lung, breast, and head and neck cancer. On the basis of these results, we conclude that the humanized (h-R3) and murine (egf/r3) anti-EGF-R Mabs show a very similar immunohistochemical pattern of recognition of fetal, adult, and neoplastic tissues. Also h-R3 Mab is a novel candidate for the development of an immunotherapeutic approach suitable for the treatment of tumors with EGF-R overexpression.