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European Biopharmaceutical Review

Life and Limb

Patient-specific cell therapies minimise the risk of rejection and increase the likelihood of integration with the surrounding tissues. In addition, they can help to avoid the controversies associated with other sources of stem cells.

Medicinal therapies began with the use of natural products that could help cure or alleviate disease. Early pharmaceutical companies standardised preparations of these products, and formulations of purified active small molecules followed. More recently, biologics – including proteins and nucleic acids – have been developed.We are currently in the midst of yet another advancement in the progression of therapeutic possibilities: the medical use of a complex biochemical product – living cells.

The astute reader may well note that the use of cells as therapy is not new. Patients regularly receive cells each time a blood transfusion or bone marrow transplant is performed. In these cases, however, the cells or tissues from the donor are intended as a substitute for identical cells in the recipient. The remarkable thing about many novel cell therapies in development is that the cells do not come from the same organ in which they have their therapeutic effect. This overview will focus on one such novel cell therapy: the use of bone marrow-derived cells – and more specifically, autologous cells – to treat severe peripheral arterial disease.

Autologous versus Allogeneic

As with bone marrow transplants, bone marrow cell therapies may be derived from the individual who will receive the cells (autologous) or from the donor individual (allogeneic). With autologous cell therapy, the patient being treated undergoes a bone marrow aspirate to collect the material needed for a ‘patient-specific’manufactured product. Allogeneic cells can be banked from volunteer donors and retrieved as needed ‘off the shelf’. Autologous cells, derived from the recipient’s own bone marrow, are thus histocompatible with the patient being treated, and will not be rejected by the immune system. In contrast, allogeneic cells have surface antigens that can be recognised as foreign by the recipient’s immune system.The potential for immunological toxicities has thus presented a higher regulatory safety hurdle for allogeneic products.

Autologous therapies appear to be more likely to gain regulatory approval, and so enter medical practice, before allogeneic therapies. An autologous cell product for the immunotherapy treatment of metastatic hormone refractory prostate cancer, sipuleucel-T (Provenge®), was approved in 2010 by the US Food and Drug Administration.

Stem, Progenitor, Precursor and Mature Cells

If cells originate from one tissue and produce their therapeutic benefit in a different tissue, then one of two things may be expected to occur. Firstly, the cells may change from their original type into a new type that is found within the target tissue. This transformation is called ‘differentiation’ and can occur via an ex vivo manipulation process, or in situ within the target tissue environment itself. Secondly, the cells may provide physiological activities within the target tissue that contribute to reversal of disease processes without fully differentiating into a new cell type. Cells in the first category are often called stem cells. Cells in the second category include cells that are committed to a given tissue lineage but have not yet fully matured – ‘progenitor’ or ‘precursor’ cells – as well as mature cells. It should be noted that these terms are somewhat fluid in the scientific literature and may overlap.

Peripheral Arterial Disease

Peripheral arterial disease (PAD) is typically caused by atherosclerosis that results in obstruction of blood flow that causes tissue ischaemia, depriving cells of adequate oxygen and nutrients. It is estimated that 27 million individuals in Europe and North America have PAD, which can produce pain while walking (claudication), pain at rest, and the development of non-healing wounds and tissue death (gangrene)(1). Treatment for PAD involves addressing the underlying atherosclerosis; in more severe cases, patients are candidates for surgical revascularisation, which includes bypass grafting, angioplasty and stent placement. In the severest form of PAD, known as critical limb ischaemia (CLI), patients have high risk for amputation of affected limbs. The life expectancy of patients with CLI is worse than for most cancers and some CLI patients have no revascularisation options. Patients with CLI literally risk losing life and limb to their disease: approximately 30 per cent have a major amputation (above the ankle) within one year, and 25 per cent do not survive (1).

Circulating Cells that Target Tissue Injury

It has been long understood that the bone marrow is the origin of blood cells. Specifically, haematopoietic stem cells in the bone marrow generate all of the cells in the blood including red blood cells, white blood cells and platelets.More recently it has been recognised that the bone marrow also produces other types of circulating cells that home to sites of injury in order to promote new blood vessel formation (angiogenesis) (2). These cells were shown to incorporate into active angiogenic sites in animal models of ischaemia. The first major clinical trial of bone marrow cells in PAD was published in 2002 (3). In this study, patients with bilateral leg ischaemia were injected with bone marrow-mononuclear cells (BMMC) into one leg, and peripheral blood-mononuclear cells (PBMC) in the other. The BMMC-treated legs had better functional outcomes in comparison with the PBMC-treated legs. The report concluded “autologous implantation of bone marrow-mononuclear cells could be safe and effective for achievement of therapeutic angiogenesis”. There have been numerous subsequent studies in animal models and in humans demonstrating activity of bone marrow-derived cells in ischaemic vascular disease (4).

Mechanisms of Activity

The complete explanation of how bone marrow cells exert their therapeutic effect has not yet been determined. There is evidence to indicate that several physiological functions may be involved. The best-characterised mechanism in animal models has been an increase in the formation of new blood vessels and associated limb perfusion. Decreased muscle atrophy, preservation of limb function and of the limbs themselves has also been noted, as has enhanced wound healing.

Animal ischaemia models are valuable research tools, but there are important differences between them and the disease they are modelling. Rodent models of limb ischaemia are generated by surgical ligation of major arteries and typically take between four to 12 weeks. In contrast, PAD is a chronic ischaemic disease that develops over years, if not decades, and is overwhelmingly the result of atherosclerosis, a metabolic-inflammatory disease of the arterial wall that has well understood risk factors, including dyslipidaemia, hypertension, diabetes and smoking. PAD has been recently understood as a form of repetitive ischaemia-reperfusion injury, wherein increased metabolic demand due to exercise causes temporary ischaemia that is followed by reperfusiontype injury with reactive oxygen species-mediated damage when blood flow again meets demand after exercise ends. Inflammatory processes associated with atherosclerosis are also thought to contribute.

It is therefore possible, perhaps even likely, that the activity of bone marrow-derived cells in human disease may be multifactorial. Therapeutic benefit in PAD may result from – and require – more than ‘simple’ angiogenesis. Modification of underlying disease processes, including the initiating atherosclerosis and the chronic end-organ inflammatory and fibrotic changes caused by prolonged cycles of ischaemia-reperfusion may be equally important. They may also be necessary to promote angiogenesis in the context of chronic advanced vascular disease. In support of this, haemodynamic measures of increased perfusion have not always correlated with harder outcomes of efficacy, such as survival, amputation and wound progression in clinical trials (5).

Types of Bone Marrow-Derived Therapies

The bone marrow contains haematopoietic cells of monocyte/macrophage, lymphocyte, megakaryocyte and granulocyte lineages, and mesenchymal stem (or stromal) cells (MSCs) that give rise to pericytes, smooth muscle cells, endothelial progenitor cells (EPCs) and fibroblast-like cells. The activity of BMMC could theoretically be the result of individual activities of these cell subsets, or of an interacting combination of subsets, or even both – individual subsets may have activity that could be enhanced when combined with other subsets. These possibilities have led to the development of three types of autologous bone marrow-derived cell therapy in PAD: freshly isolated bone marrow cells, fractionated bone marrow cells, and culture-expanded cell products.

Freshly isolated bone marrow preparations are made from minimally processed aspirates. The majority of red cells are removed, the cells are washed and the volume is reduced to generate BMMC for administration at the point of care in a single patient visit. The relative proportion of cell subsets remains the same as in the bone marrow aspirate. Since activity is generally understood to be present in cell subsets that constitute a small minority of the aspirate, a large total number of cells must be administered. Consequently, freshly isolated BMMC preparations typically require a large volume of bone marrow, up to 600 mL, which often requires general anaesthesia for collection. Since there is a mixture of cells, there exists the potential for synergistic functions of different cell populations. However, standardisation of the composition of such preparations beyond total viable cell number is not feasible; the preparation may therefore vary considerably between individuals. There may also be considerable differences in how physicians choose to administer them.

Fractionated bone marrow cell preparations are based on the hypothesis that the therapeutic effects of BMMC in PAD are concentrated in a single definable sub-population of cells. Consequently, a product made only of that cell population would be expected to have beneficial and possibly more potent effects. To produce this kind of product, bone marrow cells are concentrated using methods that separate cells according to surface molecule expression (the ‘phenotype’). Examples of fractionated therapies include EPCs, MSCs, haematopoietic stem cells expressing the surface protein CD34, and monocyte/macrophages. Since these are minority cell populations, a large volume of bone marrow aspirate is again often required. Fractionation allows standardisation according to surface phenotype, making a reproducible product feasible, and decreasing the total number of administered cells. In addition, it requires relatively little processing time between aspirate collection and administration of the cells.With fractionation, activity is limited to the selected cell population; this may limit effectiveness in a multi-faceted disease like PAD/atherosclerosis. In addition, synergistic interaction with the excluded cell populations is not possible.

Culture-expanded cell products are produced by placing collected bone marrow aspirate cells into a bioreactor system under controlled conditions, and harvesting them after a specified time.An example of a culture-expanded autologous bone marrow-derived cell therapy being developed to treat PAD is ixmyelocel-T, a product in advanced clinical development. Ixmyelocel-T is manufactured from a small volume (roughly 60mL) bone marrow aspirate by culturing in a fully-closed and highly-automated cell manufacturing system for approximately 12 days under cGMP (current Good Manufacturing Practices) conditions (6).This process expands the number of mesenchymal cells,monocytes and macrophages while retaining many of the mononuclear cells from the original bone marrow (see Figure 1).This combination of cells is capable of biological activities that promote remodelling of tissue, resolution of inflammation and angiogenesis.After culture, the cells are harvested from the system, washed, packaged, shipped and administered to the individual from whom the bone marrow was collected.

Ixmyelocel-T has been investigated in a Phase 2 study in 72 patients with CLI with no available option for revascularisation (7). In the trial – a prospective, randomised, double-blinded, placebo controlled, multi-centre study – ixmyelocel-T or placebo was injected into the muscles of each patient’s ischaemic leg. The primary analysis endpoint was to determine if the treatment was safe.Additional endpoints evaluated the effectiveness of treatment by assessing the time to first occurrence of treatment failure, defined as any of the following events: above ankle amputation, death from any cause, newly developed gangrene, or doubling of wound size. Following this was the assessment of amputation-free survival, defined as avoidance of above ankle amputation or death from any cause.

In an interim analysis that was conducted after all subjects completed six months of follow-up, there was no difference in adverse or serious adverse events between the ixmyelocel-T and control groups. There was a statistically significant lengthening in time to treatment failure in patients that received ixmyelocel-T, producing a risk reduction of 56 per cent compared to controls. This was reflected in a much longer median time to reach any of the treatment failure events in the ixmyelocel-T group than in the control group. There was a risk reduction of 24 per cent for the occurrence of amputation or death that was not statistically significant; however the study was not statistically powered to demonstrate efficacy. Based on these results, Phase 3 studies of ixmyelocel-T in CLI patients are expected to begin later this year.



Impact on Clinical Practice


The discovery that the bone marrow generates circulating cells that participate in tissue remodelling,modulation of inflammation and angiogenesis has led to investigation of bone marrow-derived therapies for peripheral vascular disease. The development of bone marrow cell therapy products is also being undertaken in other ischaemic cardiovascular diseases, including cardiomyopathy and myocardial infarction. The rationale for collecting bone marrow cells, boosting their numbers and activity, and re-administering them to the location in which they are needed has moved from the theoretical to the practical. The entrance of these therapies into clinical practice may well be just around the corner, providing treatment alternatives, and in some cases, options where there were none before.

References
  1. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA and Fowkes FG, TASC II Working Group, Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II), Eur J Vasc Endovasc Surg 33(1): S1-75, 2007
  2. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G and Isner JM, Isolation of putative progenitor endothelial cells for angiogenesis, Science 275(5,302): pp964-967, 1997
  3. Tateishi-Yuyama E et al, Imaizumi T and therapeutic angiogenesis using cell transplantation (TACT) study investigators, Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial, Lancet 360(9,331): pp427-435, 2002
  4. Lawall H, Bramlage P and Amann B, Stem cell and progenitor cell therapy in peripheral artery disease: a critical appraisal, Thromb Haemost 103(4): pp696-709, 2010
  5. European Agency for the Evaluation of Medical Products, Committee for Proprietary Medical Products, Note For Guidance on Clinical Investigation of Medicinal Products for the Treatment of Peripheral Arterial Occlusive Disease, CPMP/EWP/714/98 Rev 1, April 2002
  6. Goltry K, Hampson B, Venturi N and Bartel R, Large-scale Production of Adult Stem Cells for Clinical Use: Emerging Technology Platforms for Stem Cells, John Wiley & Sons, Inc: pp153-168, 2009
  7. Powell RJ et al, Interim analysis results from the RESTORE-CLI, a randomized, double-blind multi-center Phase II trial comparing expanded autologous bone marrow-derived tissue repair cells and placebo in patients with critical limb ischemia, J Vasc Surg, accepted for publication


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Ronnda L Bartel is Chief Scientific Officer at Aastrom Biosciences. She joined the company in 2006 and is responsible for R&D and manufacturing and engineering operations. Ronnda has more than 20 years of research and product development experience and most recently was Executive Director, Biological Research at MicroIslet and Vice president, Scientific Development at StemCells Inc. She has also worked as Senior Director of Science and Technology at SRS Capital and was Senior Principle Scientist of Cell Biology at Advanced Tissue Sciences. Ronnda holds a PhD in Biochemistry from the University of Kansas, completed postdoctoral work at the University of Michigan and received a BA in Chemistry and Biology from Tabor College. Email: rbartel@aastrom.com

Sharon Watling joined Aastrom in February 2010 and is responsible for clinical development, clinical operations and regulatory affairs. Sharon has over 12 years of experience in clinical development, with an emphasis on translational and early stage development, as well as development of clinical strategies. Her industry career started in late stage development within Warner-Lambert/Parke Davis and evolved while at Pfizer to include an early clinical leadership role in cardiovascular-metabolic diseases. Following Pfizer, she was Site Leader and Senior Director, Clinical Development at Metabasis, Inc. She received a Doctor of Pharmacy Degree from the University of Michigan, College of Pharmacy. Email: swatling@aastrom.com

Eric Kaldjian began working with Aastrom as a consultant in May 2010 and participates in a range of clinical development and translational medicine functions. He also serves as the Tissue Bank Medical Director. Eric’s pharmaceutical experience includes clinical development in oncology and transplant with Hoffmann-La Roche and pathobiology, toxicological pathology and exploratory oncology clinical programmes at Parke-Davis and Pfizer. In biotechnology, he has directed clinical genomics and biomarkers programs at Gene Logic and was the Chief Scientific Officer at Transgenomic. Eric earned his MD and trained in pathology at the University of Michigan. He completed a research fellowship in at the National Cancer Institute and is certified in anatomic pathology by the American Board of Pathology. Email: ekaldjian@aastrom.com
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Ronnda L Bartel
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Sharon Watling
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Eric Kaldjian
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