XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic

RESEARCH ARTICLE XTEN as Biological Alternative to PEGylation Allows Complete Expression of a ProteaseActivatable Killin-Based Cytostatic Akvile Haeckel1, Franziska Appler2, Angela Ariza de Schellenberger1, Eyk Schellenberger1* 1 Department of Radiology, Charité - Universitätsmedizin Berlin, Berlin, Germany, 2 nanoPET Pharma, Berlin, Germany a11111 * Abstract OPEN ACCESS Citation: Haeckel A, Appler F, Ariza de Schellenberger A, Schellenberger E (2016) XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic. PLoS ONE 11(6): e0157193. doi:10.1371/ journal.pone.0157193 Editor: Marco Rito-Palomares, Tecnologico de Monterrey, MEXICO Received: April 11, 2016 Accepted: May 25, 2016 Published: June 13, 2016 Copyright: © 2016 Haeckel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Increased effectiveness and reduced side effects are general goals in drug research, especially important in cancer therapy. The aim of this study was to design a long-circulating, activatable cytostatic drug that is completely producible in E. coli. Crucial for this goal was the novel unstructured polypeptide XTEN, which acts like polyethylene glycol (PEG) but has many important advantages. Most importantly, it can be produced in E. coli, is less immunogenic, and is biodegradable. We tested constructs containing a fragment of Killin as cytostatic/cytotoxic element, a cell-penetrating peptide, an MMP-2 cleavage site for specific activation, and XTEN for long blood circulation and deactivation of Killin. One of three sequence variants was efficiently expressed in E. coli. As typical for XTEN, it allowed efficient purification of the E. coli lysate by a heat step (10 min 75°C) and subsequent anion exchange chromatography using XTEN as purification tag. After 24 h XTEN-Killin reduced the number of viable cells of HT-1080 tumor cell line to 3.8 ±2.0% (p<0.001) compared to untreated controls. In contrast, liver derived non-tumor cells (BRL3A) did not show significant changes in viability. Our results demonstrate the feasibility of completely producing a complex protease-activatable, potentially long-circulating cytostatic/cytotoxic prodrug in E. coli—a concept that could lead to efficient production of highly multifunctional drugs in the future. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the German Research Foundation, Deutsche Forschungsgemeinschaft (DFG) in the form of salaries for FA and AH (KFO 213: SCHE 1416/2-2). nanoPET Pharma is the current employer of FA. Her contribution for this manuscript was during her PhD program when she was employed in our group at the Charité - University Medicine Berlin. nanoPET Pharma did not support this project, was not involved, and has no rights. The funders had no role in study Introduction Developing new cytostatic drugs with enhanced anticancer effectiveness but reduced side effects in non-cancerous tissues is still the central goal of cancer research. One strategy to achieve this goal is to modulate the biodistribution of active drugs by reducing their accumulation in critical organ tissues sensitive to side effects while enhancing their uptake into malignant tumors. This can be accomplished by enhancing the hydrophilic properties through coupling of polymers like polyethylene glycol (PEG) to the active agent in order to reduce the unspecific uptake by normal tissues, which additionally can increase the circulation time in PLOS ONE | DOI:10.1371/journal.pone.0157193 June 13, 2016 1 / 13 XTEN-Killin design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Apart from the fact that Volker Schellenberger, the inventor of the XTEN technology and the CEO of Amunix, is ES’s cousin, the authors have no financial benefit or share of profits of Amunix. na-noPET Pharma is the current employer of FA. FA contributed to this manuscript during her PhD program when she was employed in our group at the Charité - University Medicine Berlin. nano-PET Pharma did not support this project, was not involved, and has no rights to any of the results. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The German Research Foundation DFG provided financial for the salaries of FA and AH. DFG did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. blood and the interval between drug applications. Furthermore, uptake of PEGylated substances into tumors is promoted by the enhanced permeability and retention (EPR) of tumors resulting from leaky tumor blood vessels and altered lymphatic vessels. This so-called EPR effect facilitates the transport and retention of macromolecules (larger than 40 kDa) in tumor tissues [1,2] and has been reported to reduce side effects [3–5]. A clinical example is a PEGylated liposome-encapsulated form of doxorubicin (Doxil), which has lower cardiac toxicity [6]. However, even though immunogenicity of PEGylated drugs is considered to be very low, repeated treatment can result in induction of antibodies against PEG [7]. Moreover, antibodies against PEG were found in 22–25% of healthy blood donors up from 0.2% two decades ago, which could be the consequence of increasing use of PEG in cosmetics, therapeutics, and processed food [8]. Another way to reduce side effects is to develop inactive prodrugs that are specifically activated in the tumor area. The inclusion of cleavage sites of proteases that are highly expressed in tumors, e.g. matrix metalloproteinases MMP-2 and MMP-9, provides an option for building activatable drugs [9]. For example, doxorubicin was coupled to a MMP 2/9-cleavable peptide resulting in a therapeutically inactive prodrug. When injected, this prodrug could be activated by the increased MMP activity in the tumor, whereas the toxicity to other tissues, particularly the heart, was reduced [10]. To achieve substantial tumor uptake of drugs that do not penetrate plasma membranes themselves, so-called cell-penetrating peptides (CPP) [11], also known as protein transduction domains (PTDs), have been developed. Such peptides, derived from proteins (penetratin, Tat peptide), as well as synthetic polyarginines have been shown to efficiently deliver biologically active molecules into the cell [12]. The aim of this study was to develop a fusion protein that combines the aforementioned features in a way that allows complete synthesis in E. coli. The key to accomplishing this goal was to replace PEG by the unstructured (...truncated)