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European Biopharmaceutical Review

Mesenchymal Stem Cells 2.0

It was Heraclitus of Ephesus (535-475 BC) who first proclaimed that the world exists in a constant flow of change. This is even more apparent when it comes to science and technology. While the developers and proprietors of consumer-targeted products and gadgets know this well, in drug development the bottleneck of regulatory approval ensures that the flow is linear and only towards improvement.

A drug will not be approved unless it is better – or, in some cases, safer – than existing ones. As a result, while we witness very rapid changes in the nature of drugs being discovered and those under development, there is much less variability in the marketplace.

New Therapeutic Techniques

The majority of marketed therapeutics are small molecule-based, followed by biologics such as monoclonal antibodies (mAbs). Therapeutic modalities such as cell and gene therapies still represent a relatively insignificant fraction of the global drug market. Mesenchymal stem cells (MSCs) were first described by Arnold Caplan in the early 1990s and have proven to be a popular therapeutic modality, and the subject of at least 346 clinical trials (1,2). However, so far, there have only been two conditional marketing authorisations for Prochymal©.

The advancement of an entirely new and very different therapeutic technology is a complex undertaking. A key complication is the necessary optimisation of all parameters of technology and all associated products, to achieve sufficient efficacy for clinical benefit and, ultimately, marketing authorisation. Such a goal is not achievable overnight, but is, and must be, the result of systematic experimentation; a lengthy continuum of change and evolution.

The development of mAb therapeutics was a lengthy progression from murine, to chimeric, humanised and fully human, and continues in full flow toward better glycosylation, bi- and multi-specific antibodies, alternative antibody scaffolds, and armed antibodies that carry therapeutic payloads, such as anti-cancer agents. Cell-based therapies must follow the same path of progress and improvement.

Prochymal (remestemcel-L) arguably represents the first generation of MSCbased therapeutics. It is based on MSCs purified from the bone marrow of donors and expanded to generate thousands of doses. It was developed by Osiris Therapeutics, but is now owned by Mesoblast Ltd, and has so far obtained conditional market authorisation in Canada and New Zealand for graft versus host disease, and is being tested for multiple other indications. Many investigators have sought to build upon the groundwork laid by Prochymal and exploit the therapeutic potential of MSCs, while adding their contribution to the field through various improvements and modifications of the basic cell-product.

Technology Evolution

The purification and expansion methodologies are now known to be critical, and have the potential to fundamentally change the nature of cells and the resultant therapeutic product.

Purification Methods

These have been the subject of investigation, and there have been multiple examples where the selection of specific cell sub-populations contained in bone marrow has yielded different cell types – potentially, also with changed characteristics. Therefore, by purifying MSCs expressing the marker Stro1, it has been possible to purify cells which may be progenitors of bone marrow MSCs, and which have been named mesenchymal precursor cells (MPCs) (3). This cell type is also in late-stage clinical development by Mesoblast Ltd for multiple indications.

Culture Conditions

The culturing conditions can have a profound effect on the nature of cells. In 2001, Catherine Verfaillie pioneered a new way to expand MSCs that confers new characteristics to the resulting cell population, which have been called multipotent adult progenitor cells (4). These cells are the basis of Multistem©, another promising product in late-stage clinical development by Athersys, Inc. Here, cells appear to have the ability to continuously self-renew, and it is possible to generate many doses from the same bone marrow donor.

There now exist many methods that exploit alterations of culturing conditions to introduce changes in the nature and functionality of cells for therapeutic purposes. It might be possible to tailor the properties of cells for particular indications.

Hypoxic Preconditioning
Developing MSCs in hypoxic conditions has been shown to increase their ability to treat ischemic disease, as they do not suffer from hypoxic shock in the ischemic site. Specifically, in the case of bone marrow-derived MSCs, some evidence suggests that hypoxic culture conditions may mimic the bone marrow microenvironment more closely, making them potentially better at targeting inflamed sites and expressing higher levels of anti-inflammatory cytokines.

Cytokine Pre-Treatment

Additionally, treating cells with proinflammatory cytokines has shown promise in generating cells with enhanced immunomodulatory capacity, as they are pre-primed (5-8). MSCs are known to respond to pro-inflammatory environments by secreting an assortment of immunomodulatory cytokines (9). Therefore, they are able to control the immune cells involved, by blocking their activation and causing them to become immunomodulatory themselves. This function is boosted by the expression of adhesion molecules on the surface, and enables them to attach and penetrate inflamed vasculature and reach the site of inflammation (10). However, many of those priming cytokines will also push the cells toward certain differentiation pathways, so it is important to maintain a tight control between preconditioning and differentiation (11).

Genetic Modification


Unquestionably, genetic modification is the most controllable, specific and targeted method to generate cellbased therapeutics with improved characteristics, tailored to each indication. Many of the above methods are able to modify the characteristics of the cells, but their effects are often short-lived and, in many cases, not extensive enough to yield sufficient benefit. Therapeutic genes can be transferred into the cells using one of many different gene vectors, resulting in permanent or transient expression of the gene, as required. As a result, genetically modified MSCs (gmMSCs) have been increasing in popularity, and represent a rapidly growing and credible new wave of MSC therapeutics. Now, gmMSCs can be called a true next-generation in the field.

Transduction and Expression

There are multiple technologies and methodologies able to transfer therapeutic genes to cells such as MSCs. Viral vectors based on modified noninfective viruses are popular, as they are very efficient and can yield stable genetic modification (transduction) of the target cell. This is a considerable advantage because it means that cells can be genetically modified prior to expansion, and all their progeny will carry the therapeutic transgene.

Some viral vectors, non-viral chemical vectors and technologies such as electroporation do not cause stable transduction. They do offer the potential of generating transient gene expression, although their use in large-scale manufacturing is challenging, due to the need to transduce large amounts of cells at the end of the production process. This can be prohibitively expensive. However, novel modalities – such as zinc-finger nucleases (ZFNs), transcription activatorlike effector nucleases, and the clustered regulatory interspaced short palindromic repeat GeneArt© system – offer the possibility of stable integration with non-viral transduction technologies. Such systems hold great promise for next-generation cell therapies that do not rely on the use of viruses, but are at early stages and, with the exception of ZFNs, have not yet entered the clinic.

In addition to the above methods, alternatives also exist that only lead to a transient gene expression. For example, Jeffrey Karp’s laboratory has developed an mRNA transduction system, whereby only the mRNA for the desired protein is added to cells. When this addition runs out, there will be no more production of the therapeutic protein. This method was used to express interleukin-10 as a therapeutic immunomodulatory protein alongside P-selectin glycoprotein ligand-1 and Sialyl-Lewis X for enhanced adhesion to inflamed vasculature and targeting of inflammation (12). This is an exciting development, although its scale-up suffers from the same cost restrictions as non-viral gene transduction methods mentioned above.

Therapeutic Uses

The use of genetic modification on MSCs effectively merges the fields of cell and gene therapy, and generates considerable synergies of efficacy. By exploiting the well-defined tropism of MSCs to sites of inflammation, injury or tumours, expression of the transgene is directed to the disease site where it is needed, overcoming a long-standing limitation of in vivo gene therapy: that of delivery. On the other hand, the field of cell therapy benefits greatly by these new cells with added or improved functionality.

There is virtually no limit to the new functions and uses that can be added to MSCs via genetic modification. For example, addition of the vascular endothelium growth factor, hepatocyte growth factor or the endothelial nitric oxide synthase genes have all shown potential to enhance their therapeutic benefits in ischemic disease (13-16). Likewise, the specific tissue targeting of cells can be enhanced – or simply accelerated – when they express specific receptors, such as the CXCR4 chemokine receptor (17).

There are also many examples of gmMSCs being used in oncology. An early report on gmMSCs for cancer therapy has been described where MSCs were transduced to express interferon IFN-b to treat melanoma xenografts in mice (18). Intravenous injection of the transfected MSCs led to reduced tumour growth and prolonged survival of tumour-bearing mice. To date, human gmMSCs from various human, mouse and rat tissues have been evaluated for tumour therapy. The expression of diverse therapeutic genes has been engineered into MSCs to allow a targeted release of these agents in multiple animal cancer models (19-20).

While genetic modification can be permanent, the expression of the therapeutic gene does not have to be. The most advanced application of genetic modifi cation relies on the use of conditional promoters to ensure the expression of the therapeutic gene is limited to a particular site – such as an inflammatory environment – and not the entire body. This highly targeted gene expression is a second element of specificity that, coupled with the natural homing characteristics of the cells, results in a very precise therapeutic.

apceth GmbH is initiating clinical trials with gmMSC therapeutics. These cells are loaded with a suicide gene that is only activated in sites of damage and inflammation. Tumours are highly inflamed tissue with large areas of tissue damage and therefore recruit MSCs in high numbers. gmMSCs are attracted into the tumours, where the suicide gene herpes simplex virus-thymidine kinase (HSV-TK) becomes expressed. This occurs under the gene promoter for RANTES, a gene that is activated under conditions of inflammation. HSV-TK activates the pro-drug ganciclovir to its toxic form, so when Agenmestencel-treated patients are given ganciclovir, it will become activated only within the tumour and kill all the cells in the vicinity (21).

Fresh Potential

The development of gmMSC technology represents a significant improvement and a crucial milestone in the path of MSCs toward clinical and commercial success. The potential of this next generation of therapeutics is obvious. While it potentially carries additional regulatory requirements and may increase the cost of manufacturing, it is highly likely that the benefits will outweigh the limitations. Ultimately, the cell therapy field will be judged on the benefit it delivers to patients, and this should remain the prime aim of all stakeholders.

References

1. Caplan A, Mesenchymal stem cells, J Orthop Res 9: pp641-650, 1991
2. Visit: www.clinicaltrials.gov
3. Gronthos S et al, Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow, J Cell Sci 116(Pt9): pp1,827-1,835, 2003
4. Reyes M and Verfaillie CM, Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells, Ann N Y Acad Sci 938: pp231-233, 2001
5. Bukulmez H et al, A125: Immunomodulatory factors produced by mesenchymal stem cells after in vitro priming with danger signals, Arthritis Rheumatol 66(Suppl.11): S163, 2014
6. Carrero R et al, IL1B induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-B, Stem Cell Rev 8(3): pp905-916, 2012
7. Herrmann JL et al, Preconditioning mesenchymal stem cells with transforming growth factor-alpha improves mesenchymal stem cellmediated cardioprotection, Shock 33(1): pp24-30, 2010
8. Kalwitz G et al, Gene expression profi le of adult human bone marrow-derived mesenchymal stem cells stimulated by the chemokine CXCL7, Int J Biochem Cell Bio 41(3): pp649-658, 2009
9. Murphy MB et al, Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine, Exp Mol Med 15: 45: e54, 2013
10. Ren G et al, Inflammatory cytokineinduced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression, J Immunol 1: 184(5): pp2,321-2,328, 2010
11. Kotake S and Nanke Y, Effect of TNF on osteoblastogenesis from mesenchymal stem cells, Biochim Biophys Acta 1,840(3): pp1,209-1,213, 2014
12. Levy O et al, mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sitesof inflammation, Blood 3: 122(14): e23-32, 2013
13. Fierro FA et al, Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy, Stem Cells 29(11): pp1,727-1,737, 2011
14. Yang J et al, Effects of myocardial transplantation of marrow mesenchymal stem cells transfected with vascular endothelial growth factor for the improvement of heart function and angiogenesis after myocardial infarction, Cardiology 107(1): pp17-29, 2007
15. Su GH et al, Hepatocyte growth factor gene-modifi ed bone marrow-derived mesenchymal stem cells transplantation promotes angiogenesis in a rat model of hindlimb ischemia, J Huazhong Univ Sci Technolog Med Sci 33(4): pp511-519, 2013
16. Zhao YD et al, Rescue of monocrotalineinduced pulmonary arterial hypertension using bone marrow-derived endotheliallike progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease, Circ Res 4: 96(4): pp442-450, 2005
17. Cheng Z et al, Targeted migration of mesenchymal stem cells modifi ed with CXCR4 gene to infarcted myocardium improves cardiac performance, Mol Ther 16(3): pp571-579, 2008
18. Studeny M et al, Bone marrow derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors, Cancer Res 1: 62(13): pp3,603-3,608, 2002
19. Bao Q et al, Mesenchymal stem cell-based tumor-targeted gene therapy in gastrointestinal cancer, Stem Cells Dev 1: 21(13): pp2,355-2,363, 2012
20. Collet G et al, Trojan horse at cellular level for tumor gene therapies, Gene 10: 525(2): pp208-216, 2013

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Stefanos Theoharis joined apceth GmbH as Chief Business Officer in 2013 from Antisense Pharma (now Isarna), where he was Head of Business Development. Previously, he held the position of Business Development Director with Roche, where he focused on deals and alliances on emerging technologies. Stefanos holds a PhD in Cell and Gene Therapy from Imperial College London and has broad experience in the life sciences sector, Big Pharma, biotech, investment banking and academia.
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