Effector molecules of the apoptosis-inducing signaling cascade as active components in cytotoxic antibody fusion proteins for cancer therapy
Final Report Abstract
Cancer cells are frequently insensitive to apoptotic stimuli. Targeted delivery of a potent apoptosis inducer into such cells that can activate multiple targets within the apoptotic cascade may effectively overcome apoptosis resistance and selectively induce cancer cell death. One such strategy with therapeutic potential is based on recombinant granzyme B (GrB) fused to tumor-targeting ligands. The serine protease GrB is naturally produced by cytotoxic T lymphocytes and NK cells, where it resides together with other granzymes in cytotoxic granules. Upon target cell recognition and induction of degranulation, GrB is released in a directed fashion. GrB then binds to the surface of target cells, at least in part mediated by electrostatic interactions via positively charged surface loops on the GrB molecule. Following cellular uptake aided by the pore-forming protein perforin, GrB rapidly induces target cell apoptosis via caspase-dependent and caspase-independent mechanisms. We investigated intrinsic and ligand-mediated cell binding, and antitumoral activity of recombinant proteins employing wildtype or surface charge-modified human GrB fused to the natural epidermal growth factor receptor (EGFR) ligand transforming growth factor α (TGFα). We mutated two cationic heparin-binding motifs responsible for non-selective electrostatic interactions of GrB with cell surface structures, and fused the resulting GrBcs derivative to TGFα. Recombinantly produced GrBcs-TGFα (GrBcs-T) and a corresponding TGFα fusion protein employing wildtype GrB (GrB-T) displayed similar enzymatic activity and targeted cytotoxicity against EGFR-overexpressing breast carcinoma cells. However, unspecific binding of the modified GrBcs-T variant to EGFR-negative cells was dramatically reduced, preventing the sequestration by non-target cells in mixed cell cultures and increasing tumorcell specificity. In a similar approach we investigated targeted delivery of recombinant human apoptosis inducing factor (AIF) as an alternative for GrB to selectively induce tumor cell death. We generated a bacterially expressed fusion protein (termed 5-E-AIFΔ100), which contains a single-chain antibody fragment for specific binding to tumor cells expressing the tumor-associated surface antigen ErbB2 (HER2), linked to a C-terminal AIF fragment via the protein translocation domain of Pseudomonas exotoxin A. This 5-E-AIFΔ100 molecule displayed specific binding to ErbB2-expressing cells and facilitated efficient cell killing in the presence of an endosomolytic activity. Our data demonstrate that surface charge-modified GrBcs is superior to wildtype GrB as part of tumor-specific fusion proteins. Similar to GrB, AIF can be employed as an alternative effector for the generation of immunotoxin-like molecules with effector domains of human origin. Applicability of such reagents is currently limited by low yields of recombinant proteins and the requirement for an exogenous endosome escape activity for effective uptake by target cells. These issues are being addressed in ongoing work.
Publications
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Cisplatin-resistant neuroblastoma cells express enhanced levels of EGF receptor and are highly sensitive to treatment with EGF receptor-specific recombinant toxins. Clin. Cancer Res. 14: 6531-6537, 2008
Michaelis M, Bliss J, Arnold SC, Hinsch N, Rothweiler F, Deubzer HE, Witt O, Langer K, Doerr HW, Wels WS, Cinatl jr J
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Induction of programmed cell death in ErbB2/HER2- expressing cancer cells by targeted delivery of apoptosis inducing factor. Mol. Cancer Ther. 8: 1526-1535, 2009
Mahmud H, Dälken B, Wels WS
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Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein. PLoS ONE 5: e14404, 2010
Dälken B, Jabulowsky RA, Oberoi P, Benhar I, Wels WS
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Potent antitumoral activity of TRAIL through generation of tumortargeted single-chain fusion proteins. Cell Death Dis. 1: e68, 2010
Schneider B, Münkel S, Krippner-Heidenreich A, Grunwald I, Wels WS, Wajant H, Pfizenmaier K, Gerspach J
