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Background & Aim Mesenchymal stromal cells (MSC) are clinically applied for their immunomodulatory and regenerative capacities. Despite promising results, comparison of the many different trials is hampered by the heterogeneity in MSC manufacturing protocols. Understanding and delineation of the impact of different production methods by harmonized QC-tests and in-process controls in this respect, will be crucial to optimize MSC therapy for use in larger groups of patients. Methods, Results & Conclusion In our center, MSC are generated from bone marrow mononuclear cells (BM-MNC) using ficoll density centrifugation and subsequent expansion steps in T-flasks. After passaging 3 times, MSC are harvested and cryopreserved in a typical product size of 300 × 106 cells. Previously, changes in our clinical MSC manufacturing protocol involved changes in antibiotics and providers of raw materials such as ficoll and albumin. For GMP-conform change of control validation, comparability studies were performed and >30 real-time manufacturing procedures before and after these changes were compared for classical release criteria such as identity, purity and potency as well as by in-process tests and controls. Notwithstanding the seemingly small changes, the change in ficoll e.g. modulated the BM-MNC subsets composition of the initial MSC culture. Moreover, changing the antibiotics-regimen significantly decreased the population doubling level and decreased the drug product yield at second passage. Despite these differences, identity and purity of the drug products were comparable for both manufacturing protocols. In conclusion, even minor changes in a clinical manufacturing protocol can have a significant impact on MSC production and possibly also on its therapeutic characteristics. Our results indicate that for proper comparisons between trial results, detailed harmonization of production methods but also verification of this by standardized QC testing is needed. This will be the key for optimizing and and proper comparison of cell therapies.

288 ENHANCED CELL RECOVERY RESULTING IN HIGHER YIELDS OF MSC WHEN USING THE COBE 2991 FOR CELL DENSITY SEPARATION OF BONE MARROW MATERIAL: A VALIDATION STUDY BETWEEN TWO MSC PRODUCTION CENTERS Tden Bleker-Grijsen, L Carlee, E Steeneveld, I Lommerse, H Roelofs, J Zwaginga, C Voermans, D Thijssen-Timmer Laboratory for Cell Therapy, Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, University of Amsterdam, Amsterdam, Netherlands, Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, Netherlands

Treatment of patients with relapsed or persistent acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL) after allo-SCT with donor lymphocyte infusions (DLI) is frequently unsuccessful, probably due to the limited immunogenicity of these malignant B cells, and the lack of specificity of the T cells. In this study, we generated a broadly applicable stimulation and isolation procedure for the induction of leukemia-reactive T cells under good manufacturing practice (GMP) conditions. First, B-cell leukemias were modified into professional antigen presenting cells (APC). We previously demonstrated that CD40 crosslinking was required for the production of appropriate malignant B cell-APCs. CD40L expressing mouse fibroblasts were potent activators of CD40, but not GMP approved. CD40L trimers and CD40 antibodies crosslinked to plates or beads only minimally triggered CD40 on malignant B cells. As previously demonstrated, activated T cells briefly express CD40L. We first determined the dynamics and kinetics of CD40L expression on peripheral blood (PB) donor T cells after stimulation with CD3/28 Dynal T cell expansion-beads added in a ratio of 1 bead/10 PBMC. The kinetics were highly variable, with the optimal surface expression of CD40L on a median of 20% of the T cells (range 10–35%, n=6) being detectable between day 1 and 5. We next investigated the capacity of these activated T cells to stimulate malignant B cells in PB from patients with ALL or CLL containing 60–95% leukemic cells. We developed a two-step strategy using special CD3/28 Dynal isolation beads applicable not only for T cell activation, but also for T cell isolation. Using these beads, the T cells were depleted from the PB of the patient, directly irradiated and added back to the B cell cultures. After 3–7 days all B cells displayed a good APC phenotype with expression of CD80, CD86, and CD83 in the patient samples containing >5% T cells. If insufficient T cell numbers were present, donor T cells could be used as source of CD40L. Leukemia-reactive T cells could be reproducibly generated after two stimulations of fully HLA matched donor T cells with the leukemic APC under mild stimulatory conditions, followed by isolation of the responding T cells based on their production of interferon gamma (IFNg). Next, we investigated whether the leukemia-reactive T cells were the result of the successful induction of a primary immune response or that recognition of mimicry epitopes by previously activated T cells was underlying the activity. We separated unmodified donor T cells into CD45 RO+ memory cells, CD45RA+/CD27+ naive cells, and CD45RA+/CD27− effector cells by MACS CD45RO depletion, followed by FACS sorting on CD27, and stimulated these fractions with malignant APCs. Whereas high frequencies of IFNg producing T cells (5–15%) with cytotoxic activity against the primary leukemia (20–50% lysis) were induced from the naive T cell population, no leukemia-reactive T cells could be isolated from the memory or effector T cell populations (n=3). In conclusion, T cells stimulated with CD3/28 Dynal beads have a transient expression of CD40L and can be used as an alternative source of CD40L to generate good APC of malignant B cells under GMP conditions. These malignant B cell APCs were capable of inducing efficient priming of primary anti-leukemic immune responses by activating naive donor T cells.

By CD40 crosslinking in the presence of cytokines leukemic cells can be modified into good antigen presenting cells (APC) expressing costimulatory molecules CD40, CD80, CD86, and CD83. Previously, primary alloreactive T cell responses from HLA-matched donors have been generated using these leukemic-APC as stimulator cells against acute and chronic myeloid leukemia (AML&CML), and acute and chronic lymphocytic leukemias (ALL&CLL). However, the likelihood of generating a good immune response is highly unpredictable and long-term culture in the presence of high dose IL-2 is needed to enrich for leukemia-reactive T cells. Since the length of the in-vitro culture period has been shown to be inversely correlated with the potential of cells to survive and expand in-vivo, we developed a method facilitating early activation, detection and rapid isolation of leukemia-reactive T cells based on their interferon-gamma (IFNg) secretion using the cytokine capture assay (Miltenyi). In order to enrich for leukemia-reactive T cells and to synchronize the production of IFNg, T cells were first stimulated with the leukemic APC with addition of low dose IL-2 (10 IU/mL) at day 7, resulting in re-entry of the majority of the T cells into a quiescent state after 14 days of culture. Then, the cells were specifically restimulated resulting in synchronized production of IFNg and allowing efficient isolation. Using this method we were able to isolate T cell populations containing a high frequency of leukemia-reactive T cells against CLL, ALL, AML, and CML in 11 donor/patient pairs. Using a CFSE-based cytotoxicity assay (Jedema, Blood2004; 103: 2677) as read-out we were able to demonstrate 20–80% lysis of the primary leukemic blasts by the IFNg+ T cells at very low E/T ratios (3/1-0.3/1) in the majority of the responses, whereas the IFNg- fractions induced only 5–30% lysis. Single cell sorting of the IFNg producing T cells revealed that 15–30% of the T cell clones was capable of exerting minor antigen specific cytotoxic activity against the patient cells in an HLA-restricted fashion. However, in individual cases despite minor antigen disparities between donor and patient no specific anti-leukemia immune response could be detected. Prior to exposure to the leukemic-APC in-vivo activated T cells were observed in the responder T cell population of these donors that contained a high frequency of regulatory T cells defined as CD4+/CD25+, CD4+/CD152+, and CD8+/CD28−. We hypothesized that these regulatory T cells might actively inhibit the induction of an anti-leukemic T cell response. Therefore, in a donor/CLL patient pair, in which we were not able to induce a cytotoxic immune response against the CLL-APC we removed the in-vivo activated T cells from the responder material prior to the initial activation with the CLL-APC. Whereas no cytotoxic activity could be isolated from unmodified responder material (only 1/288 clones was cytotoxic), the IFNg+ T cells isolated from the response induced after depletion of the in-vivo activated T cells was capable of exerting massive cytotoxicity against both the primary CLL (55%) and the CLL-APC (70%). Single cell cloning of this response revealed that 35/129 T cell clones (>25%, 26 CD8+, 9 CD4+) exerted HLA-restricted CLL-specific cytotoxicity. From these results we conclude that the likelihood of generating a primary anti-leukemic immune response is not only determined by the frequency of precursor CTLs, but also by the frequency of inhibitory regulatory T cells at the onset of the immune response.