DEVELOPING BLF1 AS A NOVEL IMMUNOTOXIN FOR CANCER TREATMENT

One approach to enhancing the natural properties of antibodies is by conjugating them to toxins, especially where tumours have developed resistance to previous therapeutic regimes. Despite considerable research on immunotoxins, to date only diphtheria toxin has been approved for clinical use. One problem is that even with antibody targeting, there is a risk of damage to normal cells through inappropriate uptake of toxins. A novel small toxin, Burkholderia Lethal Factor 1 or BLF1, which we recently identified, is selective for rapidly dividing cells and is usually only active if it is deliberately introduced into the cytoplasm. These features give BLF1 significant advantages over previous toxins that have been investigated. BLF1 acts specifically to inhibit the initiation of translation mediated by intiation factor eIF4A. Rapidly dividing cells such as cancer cells are particularly dependent on eIF4A mediated translation. In this study, we further investigated the activity of BLF1 across different cellular models and the role of macropinocytosis in uptake of BLF1 by some cell types that are directly sensitive to the toxin. We demonstrated that high rates of macropinocytosis correlated strongly with direct sensitivity to BLF1 and that BLF1 is primarily active against rapidly dividing cells, consistent with the known role of elF4A. We also developed model systems for assessing the potential of BLF1 for development as an immunotoxin using antibodies directed against the tetraspanin proteins CD9 and CD63. CD63 in particular was selected as a model antigen because it is rapidly internalised on antibody binding, which is an important feature of immunotoxins. These non-covalently linked BLF1 immunoconjugates were useful for assessing antigen targeting and internalisation; however, it was not really possible to demonstrate significant specific effects on cell growth. This may have been due to inefficient uptake of the toxin, problems associated with intracellular trafficking (e.g. poor endosomal escape) or instability of the conjugates. This thesis also describes the successful production of chemically cross-linked BLF1/anti-CD63 and BLF1/anti-CD9 immunotoxins (IMT) that demonstrated targeting and significant effects on the growth of human cancer cell lines. Further investigations revealed that targeted BLF1 IMT could be co-administered with saponin, which may work synergistically to enhance their potency as a combinatorial therapy. Determination of intracellular trafficking of BLF1 IMT suggests that different internalization routing may be involved in the uptake of IMT for a specific cell type or targeting antigen.