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Background 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. Methods 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. Results 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. Conclusions 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

Although standard chemotherapy is still one of the most effective treatment for many types of cancer - often causes side effects. Chemotherapeutic agents primarily damage cancer cells with a rapidly dividing and growing profile, thereby also destroy healthy cells with such characteristics, which leads to side effects. Scientists are continually working to identify new, safer drugs, methods of administering chemotherapy, and combinations of existing treatments that have fewer side effects. In recent years, drug delivery systems (DDS) based on a concept of conjugation anticancer agents to carrier protein have been developed, achieving a better clinical response and tolerability. Protein-drug conjugates represent a whole new concept for cancer treatment which, although highly effective, cause much fewer sides effects than traditional chemotherapy. The present work is aimed to create a new conjugate molecule which essentially demonstrates greater effect than the summarized effects of its components due to specific targeting to cancer cells. We generated AD-O64.4 complex molecule, consisting of fusion protein INF- γ - TRAIL/Apo2L conjugated with the anti-mitotic agent SN-38 via the thioether linker. Carrier protein is a fusion molecule which is composed of soluble fragment of TRAIL (acting as a targeting carrier and effector) fused with artificial dimer of IFN-γ (acting as effector). SN-38, a topoisomerase I inhibitor with low nanomolar potency, the active metabolite of irinotecan, cannot be given directly to patients because of its toxicity and poor solubility. Linking SN-38 to the carrier fusion protein results in selective drug delivery to tumors which consequently leads to increased amount of the drug reaching the tumors and minimized damage of healthy cells. Analysis of the conjugate was performed by LC-MS and resulted in determination of drug-protein ratio. Agregation studies of the molecule complex were conducted by DLS and fluorescent protein aggregation assay. Cytotoxicity was evaluated with MTT assay and efficacy was performed on female SCID mice xenograft model bearing Human Uterine Sarcoma (MES-SA/Dx5). Our results show an effective process of conjugation of anticancer compound SN-38 to the carrier protein which leads to formation of an active complex with enhanced efficacy. AD-O64.4 exhibited cytotoxic effect on various cancer cell lines, (IC50 about 10 ng/ml), but showed no toxic effect on normal cells. This conjugate was also highly cytotoxic against primary cancer cells. AD-O64.4 was highly active (up to 75% tumor remission) against subcutaneous multiple drug resistant, MES-SA/Dx5, tumor xenografts in severe combined immunodeficient mice with better activity compared to the carrier protein alone, as well as to combination of the carrier protein with free-drug. Overall, these results represent a novel and a valuable system for drug delivery to the tumor and its microenvironment. Citation Format: Wojciech Strozek, Anna Pieczykolan, Bartlomiej Zerek, Michal Szymanik, Albert Jaworski, Marlena Galazka, Katarzyna Bukato, Piotr Rozga, Sebastian Pawlak, Katarzyna Poleszak, Malgorzata Teska-Kaminska, Jerzy Pieczykolan. AD-O64.4 - a novel bioconjugate for tumor-targeted drug delivery. [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 4471. doi:10.1158/1538-7445.AM2014-4471

Background Cancer growth and development is tightly related to both new vessels formation for tissue remodeling and inhibition of anti-apoptotic signals. Vascular endothelial growth factor (VEGF) is important for vascular development in physiological and pathological processes. Blockade of VEGF pathway has been shown to inhibit both pathological angiogenesis and tumor growth. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been under intense scientific evaluation because of its ability to induce apoptosis in cancer cells while omitting normal cells. However, in most cases it wasn't potent enough to become the effective single therapy agent. The AD-O51.4 is a novel anticancer fusion protein. It consists of a recombinant variant of TRAIL/Apo2L fragment, which is linked to the repeated antiangiogenic effector peptide sequence derived from the 6th exon of VEGF ligand. The peptide sequences are separated by a motif recognized by tumor-specific proteases (MMP's, uPa). The structure and biophysical properties of AD-O51.4 should be mostly derived from TRAIL/Apo2L - the carrier component of the fusion molecule. The AD-O51.4 should be targeted to both types: VEGF and TRAIL receptors and lead to sequestration of the VEGF receptors on malignant and endothelial cells making them susceptible to apoptosis induced through TRAIL/Apo2L dead receptors. Methods The AD-O51.4 has been produced in E. coli cells and purified using IEX chromatography. Its structure and biophysical properties were verified using circular dichroism (CD), size-exclusion chromatography (SEC), proteolytic digestion of activation sequence and finally direct N-terminus sequencing. The VEGF and TRAIL/Apo2L receptors specificity was confirmed using surface plasmon resonance (SPR). Antitumor activity was analyzed on a panel of established cancer cell lines and xenograft model of human cancer. Results The analysis of AD-O51.4 revealed secondary structure rich with beta-sheets and a trimeric form of the fusion molecule what is typical for TRAIL/Apo2L ligand. The molecule also displays strong specific binding for both classes of receptors: VEGF and TRAIL/Apo2L what confirms potential antiangiogenic and proapoptotic properties. Sequencing and specific digestion endorsed molecule identity. Finally specific cytotoxic and antitumor activities were confirmed for AD-O51.4. Conclusion We demonstrated that AD-O51.4 fusion protein has well established structure corresponding to its main component TRAIL/Apo2L. The structure of VEGF-derived peptides determines its specific interaction with therapeutic targets and as a consequence its antitumor properties. The obtained results confirm that a combination of two effectors in one protein molecule may be an effective way of anticancer compounds development. Citation Format: Sebastian D. Pawlak, Jerzy S. Pieczykolan, Bartlomiej Zerek, Katarzyna Poleszak, Malgorzata Teska-Kaminska, Marlena Galaska, Michal Szymanik, Albert Jaworski, Anna Pieczykolan, Katarzyna Bukato, Wojciech Strozek, Piotr K. Rozga. Biochemical and biophysical characterization of AD-O51.4 a novel anticancer biological therapeutic agent with dual mechanism of action. [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 3796. doi:10.1158/1538-7445.AM2014-3796