Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.
Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK), to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin) for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3) indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells.
Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, United States of America.
Sujatha P
SP
Koduvayur
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, United States of America.
Ying
Y
Su
Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, United States of America.
Brian K
BK
Kay
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, United States of America.|The Jesse Brown VA Medical Center, Chicago, IL, United States of America.
Arnon
A
Lavie
D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D049109:Cell Proliferation; D003131:Combined Modality Therapy; D003842:Deoxycytidine Kinase / Q000235:genetics*; D016503:Drug Delivery Systems; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D010766:Phosphorylation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology
D003842:Deoxycytidine Kinase; D018719:Erb-b2 Receptor Tyrosine Kinases; D014740:Vidarabine; C508053:ERBB2 protein, human; C024352:fludarabine