Construction and Characterization of Novel, Completely Human Serine Protease Therapeutics Targeting Her2/neu
2013
Immunotoxins containing bacterial or plant toxins have shown promise in cancer-targeted therapy, but their long-term clinical use may be hampered by vascular leak syndrome and immunogenicity of the toxin. We incorporated human granzyme B (GrB) as an effector and generated completely human chimeric fusion proteins containing the humanized anti-Her2/neu single-chain antibody 4D5 (designated GrB/4D5). Introduction of a pH-sensitive fusogenic peptide (designated GrB/4D5/26) resulted in comparatively greater specific cytotoxicity although both constructs showed similar affinity to Her2/neu-positive tumor cells. Compared with GrB/4D5, GrB/4D5/26 demonstrated enhanced and long-lasting cellular uptake and improved delivery of GrB to the cytosol of target cells. Treatment with nanomolar concentrations of GrB/4D5/26 resulted in specific cell killing accompanied by induction of apoptosis mechanisms and efficient downregulation of the PI3K/Akt and Ras/ERK pathways. The endogenous presence of the GrB proteinase inhibitor 9 (PI-9) did not impact the response of cells to the fusion construct. Surprisingly, tumor cells resistant to Lapatinib or Herceptin, and cells expressing MDR-1 resistant to chemotherapeutic agents demonstrated no cross-resistance to the GrB-based fusion proteins. Administration (i.v., tail vein) of GrB/4D5/26 to mice bearing BT474 M1 breast tumors resulted in significant tumor suppression. In addition, tumor tissue excised from GrB/4D5/26-treated mice demonstrated excellent delivery of GrB to tumors and a dramatic induction of apoptosis compared with saline treatment. This study clearly demonstrated that the completely human, functionalized GrB construct can effectively target Her2/neu expressing cells and displays impressive in vitro and in vivo activity. This construct should be evaluated further for clinical use.
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