ISCT Telegraft Column: Mesenchymal Stromal Cell (MSC) Product Characterization and Potency Assay Development

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1 ISCT Telegraft Column: Mesenchymal Stromal Cell (MSC) Product Characterization and Potency Assay Development University of Wisconsin-Madison, Production Assistance for Cellular Therapies (PACT) Over the last decade mesenchymal stromal cells (MSCs), derived from bone marrow or other tissues have generated a lot of excitement in the field of regenerative medicine and cellular therapy. These cells have the potential to differentiate into a variety of cell types, ability to migrate to sites of injury or inflammation after intravenous infusion, stimulate proliferation and survival of resident progenitor cells, and promote recovery of damaged tissues through a variety of mechanisms including secretion of cytokines and growth factors. An intriguing property of ex vivo expanded MSCs is their ability to modulate the immune responses in vitro and in vivo through interactions with a broad range of immune cells including T-and B lymphocytes, natural killer cells and dendritic cells, monocytes and macrophages. For example, a variety of in vitro studies, have shown that MSCs have the ability to suppress activation and proliferation of T- lymphocytes, and thus making them ideally suited for use in immune-mediated diseases such as graft-versus-host disease (GVHD) after bone marrow transplantation 1. Also, in contrast to most pharmacological agents that target single pathophysiological pathways, MSCs have both immune modulation and tissue regeneration properties. This dual mode of action provides a unique advantage in conditions where tissue damage and immunological disturbances play a major role in tissue pathology. MSCs are currently produced at several PACT sites for a wide variety of clinical indications including GVHD, bronchiolitis obliterans after lung transplantation, acute lung injury, myocardial infarction, and stroke. Methodologies Used for MSC Production One of the main caveats in the interpretation of clinical outcomes of MSC cellular therapy studies is the lack of reliable assays that define MSC products and their biological potencies. It is widely known that different culture systems and conditions can dramatically affect the characteristics of individual MSC products, yet there is no standard culture methodology that is utilized for any particular clinical application. It is expected that a host of different culture methodologies will continue to be utilized until a particular defined set of culture conditions produces a consistent MSC product. To date, there is a paucity of data about the potential effects of different culture conditions on the performance and presumed immunomodulatory and regenerative properties of MSCs. The current differences in culture conditions likely influence biological activity (potency) of MSCs thus making comparisons between clinical outcomes of MSC therapy unrealistic. A potency assay could lend value in elucidating differences in manufacturing processes on the quality of MSCs and thus establish standardized procedures using best practices in manufacturing clinical-grade MSC cell banks. General Guidelines for Potency Assay Development for Cellular Products 1 P a g e

2 Assay Development Strategy- Potency assay development for biological products should be initiated during the later stages of product development but prior to clinical development. Implementation of putative potency assay(s) at the time of a clinical product development will prove beneficial in establishing the relationship between assay parameters, cell product lot-to-lot variability, and clinical outcome. Key parameters in potency assay development include the systematic evaluation and validation of accuracy, reproducibility, and specificity while preserving assay sensitivity. The collection of historic assay data through the repetitive use of a potency assay will contribute to a better understanding of cellular product attributes such as activity, quality, and consistency. These data may also be used to support future specifications, changes in manufacturing processes or raw materials, and evaluation of product stability. The evaluation of a multiple potency assay strategy is strongly encouraged during preclinical and early clinical product development. Development of Multiple Assays- Cellular products are inherently complex therapeutics with multiple active ingredients and biological functions, therefore, limiting the potency development to monitoring a single factor would be unrealistic. Factors thought to be responsible for biological function or suspected primary mode of action are logical candidates to monitor as potential indicators of potency. For example, if the function of a cellular product is to provide the patient with immunosuppressive benefit, then monitoring the cellular product for factors associated with immunosuppression warrant further evaluation as a potency assay candidate. During the early stages of assay development it is important to evaluate different assay formats, readouts, and kinetics to enhance the success of identifying a reliable potency assay. Importantly, in vitro potency data analysis needs to be performed in parallel with obtaining clinical outcomes to discern a correlation between the two. Benefits of Early Assay Development- The relationship between potency and clinical effectiveness will remain a challenge due to factors common to cellular therapy products including unknown in vivo fate of a cellular product, multiple active ingredients, inherent variability of starting materials, and complex mechanism of action. Cellular assays are typically not quantitative or not sufficiently robust or precise and therefore more recent approaches such as multiplexing should be evaluated to identify a collective characterization profile for any particular cellular product. Ultimately, the use of a combination of assays may be required to assess true product potency with the desired level of confidence, accuracy, and reproducibility. Ideally, during early clinical development a relationship can be confirmed between an analytical assay, surrogate measure of biological activity, and clinical outcome. FDA has published a guidance document on potency tests for biological cellular therapy products with the recommendation to identify a potency assay for implementation prior to Phase III human clinical studies 2. Since cellular products are notorious for having a limited stability profile, the data collected from the use of a potency assay may be extremely useful in this regard. Potency assay data may turn out to be a reliable indicator of final product quality, thus providing an accurate assessment of product stability. 2 P a g e

3 Development of an Immunopotency Assay for MSC Characterization MSC-mediated immune inhibition, a prominent desirable feature of MSC in transplantation medicine, is a complex networking of ligand/receptor interactions among leukocyte subsets and MSCs. Thus, it is difficult to prioritize exactly which factors and subsets to analyze in an MSC immunopotency assay. As the starting point we focused our in vitro immunopotency assay (IPA) on CD4+ T helper lymphocytes and the degree to which their proliferation is suppressed by human bone marrow-derived MSCs. Assays focused on CD4+ T cells will serve as a foundation with which to build and expand our analyses to other subsets of factors. Indeed, in the development of our relatively simple base assay, we found that seemingly minor details can become complex issues. Therefore, the principle of keeping it simple has allowed us to better manage the hands-on issues of assay application and interpretation. Since T cells proliferate within the context of a cellular milieu it is important to maintain some semblance of leukocyte composition as it relates to the native in vivo environment. Therefore, we evaluated a single population of lymphocytes within the total peripheral blood leukocyte (PBL) population, and not a purified subset, with which to analyze MSC suppression. In this assay we use PBLs from healthy donors in co-culture with MSC lines. PBL samples are labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE), and added to the respective wells of a 48-well plate with titrated numbers of irradiated MSCs to test different PBL:MSC ratios. The T cells within the PBL milieu are stimulated with anti-cd3 and anti-cd28 monoclonal antibodies to allow for effective polyclonal stimulation and proliferation. After four days in culture, CD4+ T cell proliferation are analyzed by flow cytometry by staining the harvested cells with an allophycocyanin (APC)-labeled anti-cd4 antibody. Cells with proliferative responses are gated against a non-stimulated controls, and the degree of differential proliferation is assessed (Figure 1). Quality Control Procedure IPA We have developed a quality control procedure (QCP) for this assay at our cell production facility. One statistical measure is an IC50 for each MSC line analyzed. Importantly, IC50 analysis is not possible for lines that do not suppress proliferation. As a result, we are currently developing a more appropriate statistical methodology that is all-inclusive for the suppressive strength of any MSC line independent of suppressive capabilities. Even though our cellular proliferation assay is highly reproducible in our laboratory, a potency assay should be reproducible for use in different laboratories. Thus, we are in the process of developing a more practical, simplified assay. For example, supernatants from each MSC proliferation assay have been collected to examine the level of soluble IL-2R (scd25), which has been demonstrated by others to correlate with T cell proliferation. In this way, we demonstrate that there is a strong correlation between the IPA results and sil-2r levels, as IC50s are relatively similar to those achieved by IPA and correlation coefficients approach an R 2 of 1. We are further investigating the extent to which these two assays are interchangeable. Perhaps one of the most intriguing modes of suppression by MSCs is their ability to induce an increase in adaptive CD4+Foxp3+ regulatory T (Treg) cells, that is mediated by factors such as indoleamine 2,3-dioxygenase (IDO), TGF-B, or HLA-G5. Here at the UW-Madison PACT 3 P a g e

4 Center, we have also been able to reproducibly demonstrate MSC-mediated increases in CD4+ CD127 lo CD25 hi Foxp3+ T-reg cells in vitro, using a modified version of our IPA. PACT Support Standardization of MSC Production and Characterization Methodologies The UW-PACT team has utilized the IPA to assess whether different aspects of the MSC culturing process affect the immunopotency of generated cell lines. Our first application of the assay was to assess differences in suppressive capabilities of MSCs grown in different types of cell culture media. We found that MSCs grown in fetal bovine serum (FBS) containing media were more effective at suppressing T-cell proliferation than those grown in two commercially available serum free media. These differential levels of MSC-mediated suppression were further verified by measuring sil-2r levels. As a result of these studies, it has been determined that components in cell culture media can directly affect MSC suppression capabilities in particular the presence of FBS. Importantly, based on results of our IPA MSC banks developed at the UW-PACT Center will be grown in media containing 10% FBS. Multi-center PACT MSC Characterization Project Currently, MSCs are being generated at several PACT sites for a wide range of indications, and using different production methodologies. The MSC culture methodologies between PACT sites vary in terms of cell plating density, culture media formulation, frequency of media changes, and number of cell passages. Our current focus is to determine whether these differences affect MSC phenotypic characteristics as measured by cell surface marker expression, as well as evaluate relative immunosuppressive properties using a variety of immunopotency assays. The goal of this project is to compare the in vitro immunomodulatory capabilities of MSCs generated by the different PACT Centers. Each PACT Center is providing the UW-PACT Center with clinical-grade MSCs generated in their facility using their specified production method for evaluation. These cells will undergo extensive evaluation to include: the CFSE-based IPA, in accordance with UW s QCP; multiplex analysis on the supernatants generated from the IPA; and T-reg analysis as assessed by CD4+CD127 lo CD25 hi percentages by flow cytometry. We have also developed a qualified quality control procedure (QCP) at UW-Madison PACT to characterize MSCs using multi-color flow cytometry. In accordance with our QCP, MSC lines will be analyzed for their expression of CD105, CD90, CD73, CD54, HLA Class I and II, CD19, CD14, CD45, and CD34. This assay is performed using a 10-color flow on an LSRII Becton- Dickinson cytometer equipped with 4 lasers. Percent positivity and mean fluorescence indices (MFIs) for each cell line will be calculated and documented for each surface marker. We believe that these QCP assays will provide reproducible and reliable comparability results on MSCs generated at the various PACT Centers and thus facilitate the global characterization of PACT-generated MSC banks which should have a positive impact on acceptance of these different MSC banks for clinical evaluation. Perhaps more importantly; however, is that each PACT Center may use these data to determine whether these assays correlate with future clinical outcomes. 4 P a g e

5 Figure 1: Right: Dot plots of PBL:MSC co-cultures after a four-day proliferation period. Shown are PBL:MSC ratios of 1-0, 1-1, and Proliferation is gated against a non-stimulated control. Left: The full range of ratios analyzed in an IPA. Shown are suppression/proliferation results typically seen for our Reference Standard Passage 5 MSCs, using PBLs from the healthy donors of LeukoPak 3 (LPK3) and LeukoPak 2 (LPK2). References: 1 Nauta AJ, Fibbe WE., Immunomodulatory properties of mesenchymal stromal cells, Blood 2007, Nov 15; 110 (10): Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products, FDA, January P a g e