Abstract 3796: Biochemical and biophysical characterization of AD-O51.4 a novel anticancer biological therapeutic agent with dual mechanism of action
Authors: Pawlak Sebastian D. , Pieczykolan Jerzy S. , Zerek Bartlomiej , Poleszak Katarzyna , Teska-Kaminska Malgorzata , Galaska Marlena , Szymanik Michal , Jaworski Albert , Pieczykolan Anna , Bukato Katarzyna , Strozek Wojciech , Rozga Piotr K. 
Published: May 2015

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