Wouter Van't Hof

Potency assay development for cellular therapy products: an ISCT∗ review of the requirements and experiences in the industry

The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development.

Development of adult pluripotent stem cell therapies for ischemic injury and disease

Over the past 5 years, adult pluripotent stem cell lines have been isolated from multiple organs and tissues in laboratories worldwide. Adult pluripotent stem cells are capable of regenerating tissues of all three primitive germ layers and express pluripotency markers, such as Oct4 or telomerase, which are associated with the primitive stem cell properties of embryonic stem cells. As our collective understanding of the biology of these unique cells has improved, so has our ability to isolate, expand and subsequently evaluate them as therapeutics in preclinical models of acute injury and disease. Pluripotent adult stem cells, as opposed to tissue-restricted adult stem cells, such as mesenchymal stromal cells; have extensive replicative capacity enabling large-scale clinical expansion. This is essential to achieving consistent clinical response data and enabling the cost-effective production necessary for commercial adoption. In addition, investigators have reported effective use of allogeneic adult pluripotent stem cells in acute ischemic injury models in the heart and brain, supporting the ‘off the shelf’ product concept for this cellular therapy. In this article, the authors review preclinical animal data demonstrating the benefit of pluripotent adult progenitor cells in the treatment of ischemic injuries of the heart, vascular system and CNS.

Clinical scale expanded adult pluripotent stem cells prevent graft-versus-host disease

Adherent bone marrow adult stem cells have been used in the treatment of GVHD. In this study, we investigate the capacity of a newly characterized population of stem cells, the Multipotent Adult Progenitor Cells (MAPC), to modulate acute GVHD. These cells were derived from bone marrow cells and grown extensively without evidence for replicative senescence or loss of differentiating capacity. MAPC significantly decreased mortality of acute GVHD. Moreover, they were non immunogenic and they were not sensitive to NK-lysis. When these cells were added to a mixed lymphocyte reaction (MLR), a dose-dependent suppression of T cell proliferation was observed that was non-MHC restricted, was reversible upon removal of MAPC from culture and was mediated by soluble factors. These data show that in vitro expanded adult stem cells can efficiently control an allo-reactive response associated with acute GVHD, that they are immuno-privileged and present strong immunosuppressive properties.

Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohns disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production

BACKGROUND AIMS Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. METHODS AND RESULTS Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. CONCLUSIONS A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

Concise Review: Guidance in Developing Commercializable Autologous/Patient-Specific Cell Therapy Manufacturing

Cell therapy is poised to play an enormous role in regenerative medicine. However, little guidance is being made available to academic and industrial entities in the start‐up phase. In this technical review, members of the International Society for Cell Therapy provide guidance in developing commercializable autologous and patient‐specific manufacturing strategies from the perspective of process development. Special emphasis is placed on providing guidance to small academic or biotech researchers as to what simple questions can be addressed or answered at the bench in order to make their cell therapy products more feasible for commercial‐scale production. We discuss the processes that are required for scale‐out at the manufacturing level, and how many questions can be addressed at the bench level. The goal of this review is to provide guidance in the form of topics that can be addressed early in the process of development to better the chances of the product being successful for future commercialization.

Regenerative stromal cell therapy in allogeneic hematopoietic stem cell transplantation: current impact and future directions.

Regenerative stromal cell therapy (RSCT) has the potential to become a novel therapy for preventing and treating acute graft-versus-host disease (GVHD) in the allogeneic hematopoietic stem cell transplant (HSCT) recipient. However, enthusiasm for using RSCT in allogeneic HSCT has been tempered by limited clinical data and poorly defined in vivo mechanisms of action. As a result, the full clinical potential of RSCT in supporting hematopoietic reconstitution and as treatment for GVHD remains to be determined. This manuscript reviews the immunomodulatory activity of regenerative stromal cells in preclinical models of allogeneic HSCT, and emphasizes an emerging literature suggesting that microenvironment influences RSC activation and function. Understanding this key finding may ultimately define the proper niche for RSCT in allogeneic HSCT. In particular, mechanistic studies are needed to delineate the in vivo effects of RSCT in response to inflammation and injury associated with allogeneic HSCT, and to define the relevant sites of RSC interaction with immune cells in the transplant recipient. Furthermore, development of in vivo imaging technology to correlate biodistribution patterns, desired RSC effect, and clinical outcome will be crucial to establishing dose-response effects and minimal biologic dose thresholds needed to advance translational treatment strategies for complications like GVHD.

Heart Grafts Tolerized Through Third-Party Multipotent Adult Progenitor Cells Can Be Retransplanted to Secondary Hosts With No Immunosuppression

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal‐type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor‐ and organ recipient‐independent, third‐party cell product. We report that stable allograft survival can be achieved following third‐party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long‐term accepted heart grafts recovered from MAPC‐treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC‐mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC‐mediated allograft acceptance differs mechanistically from drug‐induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC‐based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third‐party MAPC therapy after liver transplantation.

Single and Multiple Dose MultiStem (Multipotent Adult Progenitor Cell) Therapy Prophylaxis of Acute Graft-versus-Host Disease in Myeloablative Allogeneic Hematopoietic Cell Transplantation: A Phase 1 Trial

We conducted a multicenter, phase 1 dose escalation study evaluating the safety of the allogeneic multipotent adult progenitor cell (MAPC, MultiStem, Athersys, Inc., Cleveland, OH) stromal product administered as an adjunct therapy to 36 patients after myeloablative allogeneic hematopoietic cell transplantation (HCT). Patients received increasing doses of MAPC (1, 5, or 10 million cells per kilogram recipient weight) as a single i.v. dose on day +2 after HCT (n = 18), or once weekly for up to 5 doses (1 or 5 million cells per kilogram; n = 18). Infusional and regimen-related toxicities were assessed for 30 days after the last MAPC dose. Of 36 allogeneic HCT donors (17 related and 19 unrelated), 35 were 6/6 HLA matched. MAPC infusions were well tolerated without associated infusional toxicity, graft failure, or increased incidence of infection. Median times to neutrophil (n = 36) and platelet (n = 31) engraftment were 15 (range, 11 to 25) and 16 (range, 11 to 41) days, respectively. The overall cumulative incidences of grades II to IV and III and IV acute graft-versus-host disease (GVHD) at day 100 were 37% and 14%, respectively (n = 36). In the group that received the highest single MAPC dose (10 million cells/kg), day 100 incidence of grade II to IV GVHD was 11.1% (1 of 9) with no observed cases of grade III and IV GVHD. We found no evidence for MHC class II allogeneic antibody induction, although some patients showed an increase in serum anticlass I titers compared with baseline. MAPC contribution to blood chimerism was negligible. These phase I data support the safety of stromal stem cell therapy and suggest that MAPC should be tested prospectively as a novel therapeutic option for GVHD prophylaxis after HCT.

Human multipotent adult progenitor cells transcriptionally regulate fucosyltransferase VII

BACKGROUND AIMS Targeted recruitment of leukocytes to sites of inflammation is a crucial event in normal host defense against pathogens, and attachment to and rolling on activated endothelial cells is a prerequisite first step for eventual leukocyte extravasation into sites of inflammation. These key events are mediated by interactions between glycosylated ligands expressed on leukocytes and selectins expressed on activated endothelium. Cell surface expression of selectin ligands on leukocytes is regulated by the rate-limiting enzyme fucosyltransferase VII (Fut7), and in its absence extravasation of leukocytes is severely inhibited. Multipotent adult progenitor cells (MAPCs) are an adherent cell population isolated from adult bone marrow. Intravenous administration of MAPCs provided functional improvement in multiple pre-clinical models of injury or disease, but the mechanisms by which these outcomes were achieved remain poorly understood. METHODS In vitro cell analysis studies including fluorescence-activated cell sorting, messenger RNA analysis, T-cell proliferation assays and endothelial cell binding assays were performed. RESULTS The in vitro cell analysis studies characterized the ability of MAPCs to secrete factors that transcriptionally attenuate expression of Fut7 in T cells, blocking the terminal fucosylation event in the biosynthesis of selectin ligands and reducing T-cell binding to endothelial cells. CONCLUSIONS This study presents the first example of a distinct regulatory mechanism involving transcriptional down-regulation of Fut7 by MAPCs that could modulate the trafficking behavior of T cells in vivo.