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Autophagy is an evolutionarily conserved catabolic process where the proteins and organelles of a cell are degraded using the lysosome. Defective autophagy has been implicated in mammary tumorigenesis as autophagy deficient beclin1+/- mice develop mammary hyperplasias at a higher frequency than their wild-type counterparts and beclin1 is monoallelically deleted in 40% of human breast tumors. Phospho(S73)-K8, a modification required for keratin filament reorganization under stress, in autophagy-deficient cell lines, and mouse mammary tissues. We conclude that interfering with the phosphorylation of K8 at this residue however, does not appear to alter the reorganization of the keratin network under stress and therefore, are unable to investigate the role of this phosphorylation site in the context of autophagy deficiency. On the other hand, using gene expression analysis, we have identified increased Keratin 6 expression in autophagy-deficient cell lines, tumors generated from these cell lines and mouse mammary gland tissues. We are currently attempting to understand the association between of Keratin 6 expression and autophagy-deficiency in mammary tumorigenesis.

Antibodies are glycoproteins specifically binding to a variety of antigens and today extensively used in prevention, diagnosis and treatment of diseases. Carcinomas originate from epithelial tissues and are the most common forms of human malignancies. In the necrotic areas of carcinomas significant amounts of keratins (K) are found extracellularly. They remain there due to their low solubility and can be used as targets in immuno-targeting and -therapy. Single-chain fragment variable (scFv) may display several advantages over intact IgGs in various applications but their use in immunotherapy of tumors could be limited. Their small size and monovalency may result in low accumulation of the scFv in the tumors. The targeting efficiency of antibodies needs to be evaluated in vitro prior to in vivo studies in order to exclude poor candidates. In vitro models should preferably resemble the in vivo situation as much as possible. In the present study a scFv variant (TS1-218) of the anti-keratin 8 (K 8) monoclonal antibody TS1 (mAb TS1) was constructed and characterized using site-directed mutagenesis. In addition, the valency of the TS1-218 and one of its mutants, HE1-Q, were increased by construction of a covalently linked divalent single-chain fragment variable (sc(Fv)2). To improve the yield of the antibody fragments, the expression host was changed from E. coli to P. pastoris and culture conditions were optimized using Design of Experiments (DoE). Furthermore, a HeLa HEp-2 multicellular tumor spheroid (MCTS) in vitro model was established. The functionality of the radiolabeled TS1-218 alone and in immunocomplex with its anti-idiotype scFv, αTS1 scFv, were evaluated in the MCTS model and compared to a tumor xenograft nude mouse model. The targeting efficiency of the scFv and sc(Fv)2s were also investigated using MCTS. TS1-218 in immunocomplex with αTS1 scFv displayed a significantly higher uptake than the TS1-218 alone in both MCTS and tumor xenografts. The sc(Fv)2s, DiT