Effector molecules of the apoptosis-inducing signaling cascade as active components in cytotoxic antibody fusion proteins for cancer therapy

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Major objective of this project is the generation of highly active, immunotoxin-like antibody fusion proteins, that utilize a cell-death inducing effector protein of human origin to eliminate tumor cells. The potential utility of immunotoxins for cancer therapy was demonstrated in clinical studies with recombinant molecules, that employ a bacterial toxin as a cytotoxic effector. However, these studies also identified the high immunogenicity of the bacterial protein domain as a critical limitation. We previously described prototypic, humanized antibody fusion proteins, that approach the high specificity and cell killing activity of current immunotoxins. Such molecules contain the human pro-apoptotic serine protease Granzyme B (GrB) as a cytotoxic effector, fused to cell recognition domains that bind to the clinically relevant tumor-associated antigens ErbB2 and EGF receptor. Based on such prototypic molecules, we will introduce well directed modifications intended to eliminate remaining nonspecific cell binding activity of the effector domain, and extend specific cell recognition to other important surface antigens on tumor cells such as CD20 and EpCAM. Furthermore, efficient release from endosomes after uptake, and intracellular routing to critical target substrates will be improved by including additional functional domains. To enhance antitumoral activity, individual modifications will be combined, and such optimized molecules will be tested in human tumor xenograft models in vivo. We expect that principles established during this project will not only result in tumor-specific GrB fusion proteins suitable for further development towards clinical applications, but will also provide a basis to improve immunotoxin-like reagents derived from other human pro-apoptotic molecules.