title: Targeted delivery of a designed sTRAIL mutant results in superior apoptotic activity towards EGFR-positive tumor cells.
abstract: Previously, we have shown that epidermal growth factor receptor (EGFR)-selective delivery of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), by genetic fusion to antibody fragment scFv425, enhances the tumor-selective pro-apoptotic activity of sTRAIL. Insight into the respective contribution of the agonistic receptors TRAIL-R1 and TRAIL-R2 to TRAIL-induced apoptosis may provide a rational approach to further optimize TRAIL-based therapy. Recently, this issue has been investigated using sTRAIL mutants designed to selectively bind to either receptor. However, the relative contribution of the respective TRAIL receptors, in particular TRAIL-R1, in TRAIL signaling is still unresolved. Here, we fused scFv425 to designed sTRAIL mutant sTRAILmR1-5, reported to selectively activate TRAIL-R1, and investigated the therapeutic apoptotic activity of this novel fusion protein. EGFR-specific binding of scFv425:sTRAILmR1-5 potently induced apoptosis, which was superior to the apoptotic activity of scFv425:sTRAIL-wt and a nontargeted MOCK-scFv:sTRAILmR1-5. During cotreatment with cisplatin or the histone deacetylase inhibitor valproic acid, scFv425:sTRAILmR1-5 retained its superior pro-apoptotic activity compared to scFv425:sTRAIL-wt. However, in catching-type Enzyme-Linked ImmunoSorbent Assays with TRAIL-R1:Fc and TRAIL-R2:Fc, scFv425:sTRAILmR1-5 was found to not only bind to TRAIL-R1 but also to TRAIL-R2. Binding to TRAIL-R2 also had functional consequences because the apoptotic activity of scFv425:sTRAILmR1-5 was strongly inhibited by a TRAIL-R2 blocking monoclonal antibody. Moreover, scFv425:sTRAILmR1-5 retained apoptotic activity upon selective knockdown of TRAIL-R1 using small inhibitory RNA. Collectively, these data indicate that both agonistic TRAIL receptors are functionally involved in TRAIL signaling by scFv425:sTRAILmR1-5 in solid tumor cells. Moreover, the superior target cell-restricted apoptotic activity of scFv425:sTRAILmR1-5 indicates its therapeutic potential for EGFR-positive solid tumors.
authors.affiliation: Laboratory for Tumor Immunology, Section Medical Biology, Department of Pathology and Laboratory Medicine, University Medical Center Groningen, Hanzeplein 1, Groningen, The Netherlands.
authors.forename: Edwin
authors.initials: E
authors.lastname: Bremer
authors.forename: Marco
authors.initials: M
authors.lastname: de Bruyn
authors.forename: Douwe F
authors.initials: DF
authors.lastname: Samplonius
authors.forename: Theo
authors.initials: T
authors.lastname: Bijma
authors.forename: Bram
authors.initials: B
authors.lastname: ten Cate
authors.forename: Lou F M H
authors.initials: LF
authors.lastname: de Leij
authors.forename: Wijnand
authors.initials: W
authors.lastname: Helfrich
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis; D001665:Binding Sites; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000819:agonists / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D008024:Ligands; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D066246:ErbB Receptors