The tumor necrosis factor related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF superfamily that initiates apoptosis of tumor cells through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of tumor cells but not normal cells makes it an attractive agent for cancer therapy. However, many cancer types have developed resistance mechanisms, such as dysfunctions of proapoptotic proteins.

Hereby, we report a novel molecule - AD-O56.9, which is composed of the soluble fragment of TRAIL (acting as both carrier and also effector) fused with a cationic, alpha-helical (KLAKLAK)2 antimicrobial peptide (acting as effector). The (KLAKLAK)2 peptide fused to protein transduction domain can induce cancer cell death by triggering mitochondrial membrane permeabilization and swelling, resulting in the release of cytochrome c and induction of apoptosis. It creates also the capacity to cause aggregation of mitochondria that is also a mechanism of cytotoxic action. (KLAKLAK)2 peptide is equipped with protein transduction domain domain, to increase its internalization. To allow separation of TRAIL fragment from the effector peptide domain specifically in the tumor environment, we linked these two domains with a sequence motif recognized by MMP and uPa proteases, present in tumor cells membranes or their proximity.


AD-O56.9 protein was produced in E. coli and purified by IEC. The molecule was characterized biochemically and biophysically. MTT assay was used to estimate killing of carcinoma cells. Flow cytometric analysis was used to evaluate influence of the AD-O56.9 on plasma and mitochondrial membrane integrity, caspase 3 activation, PARP cleavage, as well as on the cell cycle of cancer cells. The tumoricidal activity of AD-O56.9 was evaluated in NOD/SCID mice bearing different types of tumor xenografts.


AD-O56.9 exhibited cytotoxic effect on various cancer cell lines, both TRAIL-sensitive and TRAIL-resistant, but showed no toxic effect on normal cells. This protein was also highly cytotoxic against primary cancer cells. The component that overcomes resistance to TRAIL is RRRRRRRR(KLAKLAK)2 peptide, but only as a component of AD-O56.9 fusion protein. Analyzing cell cycle and plasma membrane integrity in relatively sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549) we showed that AD-O56.9 induced apoptosis in those cells. This protein led to activation of caspase 3, cleavage of PARP as well as caused strong depolarization of mitochondrial membrane. AD-O56.9 administration caused significant regression of TRAIL-sensitive MIA PaCa-2, OE19, Colo205 and TRAIL-resistant HepG2.


AD-O56.9 is able to induce cell death in many cancer cell lines, even TRAIL resistant and causes tumor regression in mice bearing human tumors. Obtained results make this molecule worth of further preclinical development.

Citation Format: Bartlomiej Maciej Zerek, Michal Szymanik, Piotr Kamil Rozga, Anna Pieczykolan, Marlena Galazka, Katarzyna Bukato, Albert Jaworski, Katarzyna Poleszak, Sebastian Dominik Pawlak, Malgorzata Teska-Kaminska, Wojciech Strozek, Jerzy Szczepan Pieczykolan. AD-O56.9: A fusion of TRAIL/Apo2L with a membrane disrupting peptide as a novel anticancer therapeutic. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2773. doi:10.1158/1538-7445.AM2014-2773