IDENTIFICATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY 14C5 - A potential agent for radioimmunodetection and -therapy

摘要

In search of new antibody therapeutics for inhibition of metastatic cancer, the foci of this research were to identify and further characterize antigen 14C5 as potential target for monoclonal antibody 14C5 (mAb 14C5)-based imunotherapies and to explore the targeting properties of radioiodinated mAb 14C5 for radioimmunodetection and –therapy. In the first part of the thesis we show that antigen 14C5 is the αvβ5 integrin receptor. Immunocytochemical analysis and flow cytometry analysis show that the expression of antigen 14C5 is present in varying degrees on neoplastic human cell lines from different origins. Antigen 14C5 expression on lung and colon carcinoma tissues mainly occurred in the stroma and on stromal fibroblasts surrounding the tumour cells and to a lesser extent at the tumour cells. Further studies on the expression of antigen 14C5 have to show whether antigen 14C5 is an indicator of the fibroblast cancer-associated phenotype. The second part of the thesis explored the potential of radioiodinated mAb 14C5 as agent for radioimmunodetection and –therapy. Radioiodinated mAb 14C5 binds its antigen with high affinity, shows good and specific tumour uptake in vivo and good planar gamma camera imaging quality. However, the blood clearance is slow. Therefore, Fab and F(ab’)2 fragments of intact mAb 14C5 were generated. Radioiodinated F(ab’)2 still binds antigen 14C5 with high affinity, while radioiodinated Fab looses affinity. Fragments showed increased tumour-to-blood and tumour-to-muscle ratios, but the overall tumour uptake of both fragments was significantly reduced. Internalization can lead to a rapid loss of the radiolabel from the tumour cell. We investigated the fate of the 125I-labelled mAb 14C5-antigen complex. Studies were also conducted with 125I-labeled F(ab’)2 and Fab fragments. Genetic engineering provides powerful tools for manipulating the structure and pharmacokinetic properties of antibodies in order to obtain optimal targeting properties. In this context, the variable heavy and variable light chain genes of mAb 14C5 were sequenced and the complement determining regions characterizing mAb 14C5 were identified. This opens the way to a broad range of applications including altering pharmacokinetics, reducing immunogenicity, the use of a variety of isotopes, conjugation of drugs or toxins, the design of bispecific antibodies and pretargeting strategies.