- Austrianova Singapore Pte Ltd., 2 International Business Park, The Strategy @ IBP #09-04, Singapore 609930, Singapore; salmons@sgaustria.com
- Institute of Virology, Department of Pathobiology, University of Veterinary Medicine, 1210 Vienna, Austria
* Correspondence: walter@gunzburg.biz
Abstract: Background: The use of encapsulated cells for the in vivo delivery of biotherapeutics is a promising new technology to potentiate the effectiveness of cell-based therapies for veterinary and human application. One use of the technology is to locally activate chemotherapeutics to their short-lived highly active forms. We have previously shown that a stable clone of HEK293 cells overexpressing a cytochrome P450 enzyme that has been encapsulated in immunoprotective cellulose sulphate beads can be implanted near solid tumours in order to activate oxazaphosphorines such as ifosfamide and cyclophosphamide to the tumour-killing metabolite phosphoramide mustard. The efficacy of this approach has been shown in animal models as well as in human and canine clinical trials. In these previous studies, the oxazaphosphorine was only given twice. An analysis of the Kaplan–Meier plots of the results of the clinical trials suggest that repeated dosing might result in a significant clinical benefit. Aims: In this study, we aimed to (i) demonstrate the stable long-term expression of cytochrome P450 from a characterized, transfected cell clone, as well as (ii) demonstrate that one implanted dose of these encapsulated cytochrome P450-expressing cells is capable of activating multiple doses of ifosfamide in animal models. Methodology: We initially used cell and molecular methods to show cell line stability over multiple passages, as well as chemical and biological function in vitro. This was followed by a demonstration that encapsulated HEK293 cells are capable of activating multiple doses of ifosfamide in a mouse model of pancreatic cancer without being killed by the chemotherapeutic. Conclusion: A single injection of encapsulated HEK293 cells followed by multiple rounds of ifosfamide administration results in repeated anti-tumour activity and halts tumour growth but, in the absence of a functioning immune system, does not cause tumour regression
1. Introduction
The use of suicide gene prodrug combinations, also known as gene-directed enzyme prodrug therapy (GDEPT), is a common strategy for the treatment of solid tumours [1,2]. We have been focusing on a modified form of GDEPT where encapsulated cells expressing the enzyme encoded by the cytochrome P450 2B1 gene are placed in the vicinity of tumours in order to locally activate prodrugs of the oxazaphosphorine family such as ifosfamide and cyclophosphamide [3]. Local activation is required for maximum anti-tumour effects since the CYP 2B1-activated forms of the oxazaphosphorines (4-hydroxy-amides) are short-lived and need to pass through the cell membrane of tumour cells before they spontaneously decay to the anti-tumour active phosphoramide mustard and the tissue toxic form, acrolein [4]. Phosphoramide mustard causes DNA alkylation and irreversible cross-linkages between and within strands at the guanine N-7 position [5]. DNA cross-linking is not toxic per se, but when cells attempt to divide, they die [6]. Although DNA alkylation and cross-linking occur in many cells, its cytotoxic effects are thus limited to rapidly dividing cells such as tumour cells. The intra- and interstrand DNA linkages are, however, also cytotoxic since besides blocking DNA replication, they also block essential processes such as DNA transcription [7,8]. The mechanism of cell death has been thought to be via apoptosis, although death primarily via necrosis, rather than apoptosis, has been shown in cell culture [9].
The encapsulation of contact-inhibited cells at high cell density not only confines them to the site at which they are implanted and protects them from clearance by the host immune system, but it also prevents their replication, so that they themselves are not killed by the activated prodrug [4,10,11].
Local encapsulated cell-mediated activation has been shown to give better anti-tumour effects (tumour shrinkage, enhanced median survival) even when low doses of oxazaphosphorines, such as ifosfamide, are used, thereby mitigating the usual side effects that are observed with standard doses in two human clinical trials for pancreatic cancer [12]. These data are supported by another clinical trial using encapsulated cell-mediated local activation of cyclophosphamide in dogs with spontaneously occurring mammary cancer [13].
The previously reported clinical trials of this encapsulated cell therapy for the treatment of pancreatic cancer were performed using one administration of encapsulated CYP 2B1-expressing cells, and subsequently, two rounds of systemic ifosfamide treatment [12]. However, since the cells in the capsule are non-dividing but metabolically active, they should be able to continue to convert multiple doses of ifosfamide. In this study, we wanted
to determine whether (i) encapsulated cells in general can survive and remain viable for long periods after implantation into mice, and (ii) whether multiple rounds of ifosfamide can be delivered and activated by the encapsulated cells without the encapsulated cells eventually being killed.
2. Materials and Methods
2.1. Parental HEK293 Cell Lines
2.2. Transfection with Expression Vector and Selection of Clones
2.3. IVIS Imaging
2.4. Resorufin CYP2B1 Enzymatic Expression Assay
2.5. Western Blotting of CYP2B1 Protein
2.6. XTT Metabolic Activity Assay
2.7. Cell-in-a-Box Encapsulation
2.8. Cell Killing via “by-Stander” Effect
2.9. Tumour Cell Line
2.10. Animal Experiments
2.11. Statistical Analysis
Read full article here >> Life: Long-Term Survival of Cellulose Sulphate
About Austrianova
Austrianova, (www.austrianova.com) part of the SG Austria Group, is a biotech company with a global footprint and headquarters in Singapore. Austrianova utilizes a novel and proprietary technology for the encapsulation of living mammalian (Cell-in-a-Box®) and bacterial (Bac-in-a-Box®) cells. Cell-in-a-Box® protects the encapsulated human or animal cells from rejection by the immune system and allows cells to be easily transported, stored and implanted at specific sites in patients. The technology, which has been proven safe and efficacious in clinical trials carried out in Europe, allows companies to develop a wide range of standard cells as one-for-all living pharmaceuticals. Bac-in-a-Box® is a similar protective device adapted for encapsulation of bacteria and yeast, including probiotics, and has both human food as well as animal feed applications due to its ability to extend storage under lyophilized conditions and to protect encapsulated bacteria against destruction by stomach acid. Austrianova now also offers GMP4Cells that includes competitively priced Master Cell Bank and Working Cell Bank production as well as “Fill and Finish” services for cell therapy products (such as stem cell therapies, biologics produced from cells e.g. vaccines, antibodies, recombinant proteins etc).