- Email: [email protected]
Cryopreservation of leukopaks show maintained viability and functionality
S22
Poster Abstracts
and functionality for successful adoptive transfer. Moreover, a xeno-free and serum free media (XSFM) is ideal to reduce cost, limit variability and lower the risk of foreign contaminants. Irvine Scientific (IS) has developed a xeno- free and serum free (XSFM) Tcell expansion media that can be used to activate and expand naïve T-cells. This media supports higher rate of growth expansion as compared to serum containing media and commercially available media. Phenotypic analysis shows the percentage of expanded CD4+ and CD8+ are maintained even after expansion. The XSFM expansion media supports T-cell differentiation potential into major T-cell subsets such as Th1, Th2, and Tregs when introduced to polarization cocktail of rIL-12 and anti-IL-4, rIL-4 and anti-IFNg, and TGF-b1 and retinoic acid respectively. The media also supports continued re-activation and expansion of T-cells during long term culture. Potential uses for large scale cultures have also been demonstrated. In this study, we demonstrate how the XSFM expansion media can be a superior alternative than serum-containing media to expand T-cells on a large scale platform while maintaining T cell phenotypes and polarization potential. 55 CRYOPRESERVATION OF LEUKOPAKS SHOW MAINTAINED VIABILITY AND FUNCTIONALITY W Wang, T Hexom, L-M Nucho, G Eastwood HemaCare Corporation, Van Nuys, California, United States Human cells are critical raw materials for research and manufacturing of cell therapy products. However, the accessibility to freshly procured cells can be limited, creating a crucial need for a suitable alternative to fresh cells that are viable and functional—especially when transporting materials globally. At HemaCare, we have investigated the viability and functionality of lymphocytes, fresh and cryopreserved, from leukopaks procured within our FDA registered cGMP donor collection facility. Fresh leukopaks were evaluated for cell viability via flow cytometry over the course of seven days. Studies found that viability of leukopaks in autologous plasma at room temperature were greater than 80% up to 144 hours post collection. However, the cell counts decreased steadily over time leading us to look at how cryopreservation might help to circumvent these drawbacks in typical leukopak transport. The viability of T-cells, monocytes, B-cells, and NK cells from leukopaks were evaluated from the same donor pre- and post-cryopreservation. Preliminary data for whole leukopaks shows that post-cryopreserved viability averages 97.5% (+/- 1.2 sem), and the distribution of the CD3+, CD4+, and CD8+ populations were 43.4%, 28.7%, and 12.2%, respectively, within the total leukopak. Distribution of B- cells, NK cells, and monocytes shows 8.38%, 12.5%, and 20.4%, respectively. T-cell functionality data was also obtained as this cell type is sensitive to the cryopreservation process. Results of CFSE-labeled T cells functional assays show multiple divisions over 5 days, and high expression of Ki67 and CD25 after 5 day monocyte derived DC stimulation. These preliminary results suggest that cryopreserved leukopaks can serve as an acceptable alternative to fresh leukopaks thus being a valuable and significant option for emerging autologous and allogeneic cell therapies which require apheresis shipments from collection centers to cell therapy processing facilities around the world.
56 SCALING UP CLINICAL T CELL EXPANSION IN A XURIÔ CELL EXPANSION SYSTEM C Mölleryd1, C Nunes3, U Eistrand2 1 Research and Applied Markets - Cell Therapy Technologies, GE Healthcare Bio-Sciences AB, Uppsala, Sweden, 2Vecura GMP Facility, Karolinska University Hospital, Stockholm, Sweden, 3Research and Applied Markets Cell Therapy Technologies, GE Healthcare UK Limited UK, Cardiff, United Kingdom Adoptive T cell therapies are the most recent personalized medicine to show signs of therapeutic value to patients, predominantly tumor-infiltrating T cells and genetic modified T cells. Nevertheless, T cell therapies have also been acknowledged to have unique culturing requirements, depending on the type of T cell product. A protocol for expansion of clinically relevant T cells usually requires steps with minimal cell manipulation and an initial stage of cell-to-cell contact for T cell stimulation and further expansion. To facilitate the required interactions, minimal movement of the cell suspension is a necessity, and static culture using flasks or static culture bags placed in an incubator has been applied widely in the field. When trying to adapt the protocol to a more automated and contained process for production of a regulatory compliant cellular immunotherapy, the
number of bioreactor systems available for this application is limited. The Xuri Cell expansion System provides a functionally closed environment, pre-sterilized bioreactor with perfusion capabilitites which is favorable for applications requiring low risk of contamination and ease of use, such as expansion of cells for adoptive T cell therapy. The generation of clinical scale T cells using the Xuri Cell expansion platform will provide an alternative approach that will permit customers to scale-up their cell expansion process in an optimal automated closed environment, negating the need of multiple open systems. 57 TARGETING GLIOMA STEM CELLS USING T CELLS RECOGNIZING THE PLURIPOTENCY FACTOR OCT4 KE Wright1, F Saglio2, S Bose1, H Lang1, C Bollard1, CR Cruz1 1 Children’s National Medical Center, Washington, District of Columbia, United States, 2Baylor College of Medicine, Houston, Texas, United States Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and prognosis remains poor for patients with the disease. Tumor recurrence and therapy resistance is thought to be due to the persistence of glioma stem cells (GSCs). Immune therapies directed against these cells are promising approaches. To specifically eliminate GSCs, we proposed targeting the pluripotency factor Oct4, a transcription factor required by GSCs to maintain stemness and overexpressed in GBM compared to healthy brain tissue. We hypothesized that targeting Oct4 using antigen specific T cells will eliminate GSCs and present a viable therapeutic option for GBM. We investigated the feasibility of generating T cells recognizing Oct4 from the peripheral blood of healthy donors. We stimulated mononuclear cells with autologous dendritic cells pulsed with a 15mer peptide library overlapping by 11 amino acids followed by peptide-pulsed CD3-28 blasts or PHA blasts. T cells were successfully expanded from 15 donors (mean 249 fold expansion +/363, n¼15, range 3.3-1553.7). Ten of these donors demonstrated specificity for Oct4 peptides as determined by IFNg enzyme-linked immunospot (ELISpot) assay (mean 207 +/- 177 spots per 2x105 cells, n¼10, range 29-497.5). Studies of naïve and memory subsets indicated that the Oct4-specific T cell response in healthy donors is derived predominately from the memory compartment. Functionally, Oct4-specific T cells are capable of lysing peptide-pulsed autologous CD3-28 blasts or PHA blasts as determined by Cr51 cytotoxicity assay in two of five Oct4-specific lines (20:1 E:T, 23.1% lysed, +/- 7.3, n¼2). The T cells were predominately effector memory phenotype as evaluated by flow cytometry (mean 75% CCR7-/CD45RA-/CD45RO+, +/- 15%, n¼8). These findings indicate that it may be feasible to generate Oct4-CTL from patients with GBM for use in further studies that will investigate whether Oct4-CTL can eliminate GSCs and prevent tumor recurrence in vivo. 58 CRYOPRESERVATION OF ACTIVATED DC1 MAKES LARGE SCALE DENDRITIC CELL VACCINES FEASIBLE IN CANCER THERAPY E Fitzpatrick, S Xu, J Datta, J Cintolo, B Czerniecki Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Dendritic cell (DCs) vaccines that optimize antigen sensitization by CD4+ and CD8+ T cells induce Th1 cytokine production offer promise for the future of cancer therapeutics; widespread application is inhibited by laborious manufacturing. Cryopreservation of mature type I polarized DCs alleviates exhaustive production practices and increase world-wide accessibility. Methods: Freshly matured DCs (DC1s) and DCs cryopreserved mature (cryoDC1s), rapidly maturated, employed to activate cells into DC1s by a 2-day culture using IFN-g and LPS. Populations were compared for viability, recovery and phenotypic maturity. Functionality was determined by cytokine secretion, ability to stimulate CD4+ and CD8+ T cells by allosensitization and antigen-specific sensitization of CD8+ T cells. Results: Significant differences were not seen in viability (p¼0.481), and recovery (p¼0.122). Initial IL-12 p70 production was 44.76 ng/mL (DC1s) and 29.94 ng/mL (cryoDC1s) with no significant differences. IL-12 p70 continued for of 30 hours, peak secretion between 8 and 12 hours. Key cytokines in promoting a Th1 cell mediate response (TNF-a, RANTES, MIP1-a and MIP1-b) secreted noticeable levels; moreover populations were not significantly different. CD80, 83, and 86 demonstrate DC maturation, expression revealed no significant difference. Functional abilities were induced by CD4+
Poster Abstracts
and functionality for successful adoptive transfer. Moreover, a xeno-free and serum free media (XSFM) is ideal to reduce cost, limit variability and lower the risk of foreign contaminants. Irvine Scientific (IS) has developed a xeno- free and serum free (XSFM) Tcell expansion media that can be used to activate and expand naïve T-cells. This media supports higher rate of growth expansion as compared to serum containing media and commercially available media. Phenotypic analysis shows the percentage of expanded CD4+ and CD8+ are maintained even after expansion. The XSFM expansion media supports T-cell differentiation potential into major T-cell subsets such as Th1, Th2, and Tregs when introduced to polarization cocktail of rIL-12 and anti-IL-4, rIL-4 and anti-IFNg, and TGF-b1 and retinoic acid respectively. The media also supports continued re-activation and expansion of T-cells during long term culture. Potential uses for large scale cultures have also been demonstrated. In this study, we demonstrate how the XSFM expansion media can be a superior alternative than serum-containing media to expand T-cells on a large scale platform while maintaining T cell phenotypes and polarization potential. 55 CRYOPRESERVATION OF LEUKOPAKS SHOW MAINTAINED VIABILITY AND FUNCTIONALITY W Wang, T Hexom, L-M Nucho, G Eastwood HemaCare Corporation, Van Nuys, California, United States Human cells are critical raw materials for research and manufacturing of cell therapy products. However, the accessibility to freshly procured cells can be limited, creating a crucial need for a suitable alternative to fresh cells that are viable and functional—especially when transporting materials globally. At HemaCare, we have investigated the viability and functionality of lymphocytes, fresh and cryopreserved, from leukopaks procured within our FDA registered cGMP donor collection facility. Fresh leukopaks were evaluated for cell viability via flow cytometry over the course of seven days. Studies found that viability of leukopaks in autologous plasma at room temperature were greater than 80% up to 144 hours post collection. However, the cell counts decreased steadily over time leading us to look at how cryopreservation might help to circumvent these drawbacks in typical leukopak transport. The viability of T-cells, monocytes, B-cells, and NK cells from leukopaks were evaluated from the same donor pre- and post-cryopreservation. Preliminary data for whole leukopaks shows that post-cryopreserved viability averages 97.5% (+/- 1.2 sem), and the distribution of the CD3+, CD4+, and CD8+ populations were 43.4%, 28.7%, and 12.2%, respectively, within the total leukopak. Distribution of B- cells, NK cells, and monocytes shows 8.38%, 12.5%, and 20.4%, respectively. T-cell functionality data was also obtained as this cell type is sensitive to the cryopreservation process. Results of CFSE-labeled T cells functional assays show multiple divisions over 5 days, and high expression of Ki67 and CD25 after 5 day monocyte derived DC stimulation. These preliminary results suggest that cryopreserved leukopaks can serve as an acceptable alternative to fresh leukopaks thus being a valuable and significant option for emerging autologous and allogeneic cell therapies which require apheresis shipments from collection centers to cell therapy processing facilities around the world.
56 SCALING UP CLINICAL T CELL EXPANSION IN A XURIÔ CELL EXPANSION SYSTEM C Mölleryd1, C Nunes3, U Eistrand2 1 Research and Applied Markets - Cell Therapy Technologies, GE Healthcare Bio-Sciences AB, Uppsala, Sweden, 2Vecura GMP Facility, Karolinska University Hospital, Stockholm, Sweden, 3Research and Applied Markets Cell Therapy Technologies, GE Healthcare UK Limited UK, Cardiff, United Kingdom Adoptive T cell therapies are the most recent personalized medicine to show signs of therapeutic value to patients, predominantly tumor-infiltrating T cells and genetic modified T cells. Nevertheless, T cell therapies have also been acknowledged to have unique culturing requirements, depending on the type of T cell product. A protocol for expansion of clinically relevant T cells usually requires steps with minimal cell manipulation and an initial stage of cell-to-cell contact for T cell stimulation and further expansion. To facilitate the required interactions, minimal movement of the cell suspension is a necessity, and static culture using flasks or static culture bags placed in an incubator has been applied widely in the field. When trying to adapt the protocol to a more automated and contained process for production of a regulatory compliant cellular immunotherapy, the
number of bioreactor systems available for this application is limited. The Xuri Cell expansion System provides a functionally closed environment, pre-sterilized bioreactor with perfusion capabilitites which is favorable for applications requiring low risk of contamination and ease of use, such as expansion of cells for adoptive T cell therapy. The generation of clinical scale T cells using the Xuri Cell expansion platform will provide an alternative approach that will permit customers to scale-up their cell expansion process in an optimal automated closed environment, negating the need of multiple open systems. 57 TARGETING GLIOMA STEM CELLS USING T CELLS RECOGNIZING THE PLURIPOTENCY FACTOR OCT4 KE Wright1, F Saglio2, S Bose1, H Lang1, C Bollard1, CR Cruz1 1 Children’s National Medical Center, Washington, District of Columbia, United States, 2Baylor College of Medicine, Houston, Texas, United States Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and prognosis remains poor for patients with the disease. Tumor recurrence and therapy resistance is thought to be due to the persistence of glioma stem cells (GSCs). Immune therapies directed against these cells are promising approaches. To specifically eliminate GSCs, we proposed targeting the pluripotency factor Oct4, a transcription factor required by GSCs to maintain stemness and overexpressed in GBM compared to healthy brain tissue. We hypothesized that targeting Oct4 using antigen specific T cells will eliminate GSCs and present a viable therapeutic option for GBM. We investigated the feasibility of generating T cells recognizing Oct4 from the peripheral blood of healthy donors. We stimulated mononuclear cells with autologous dendritic cells pulsed with a 15mer peptide library overlapping by 11 amino acids followed by peptide-pulsed CD3-28 blasts or PHA blasts. T cells were successfully expanded from 15 donors (mean 249 fold expansion +/363, n¼15, range 3.3-1553.7). Ten of these donors demonstrated specificity for Oct4 peptides as determined by IFNg enzyme-linked immunospot (ELISpot) assay (mean 207 +/- 177 spots per 2x105 cells, n¼10, range 29-497.5). Studies of naïve and memory subsets indicated that the Oct4-specific T cell response in healthy donors is derived predominately from the memory compartment. Functionally, Oct4-specific T cells are capable of lysing peptide-pulsed autologous CD3-28 blasts or PHA blasts as determined by Cr51 cytotoxicity assay in two of five Oct4-specific lines (20:1 E:T, 23.1% lysed, +/- 7.3, n¼2). The T cells were predominately effector memory phenotype as evaluated by flow cytometry (mean 75% CCR7-/CD45RA-/CD45RO+, +/- 15%, n¼8). These findings indicate that it may be feasible to generate Oct4-CTL from patients with GBM for use in further studies that will investigate whether Oct4-CTL can eliminate GSCs and prevent tumor recurrence in vivo. 58 CRYOPRESERVATION OF ACTIVATED DC1 MAKES LARGE SCALE DENDRITIC CELL VACCINES FEASIBLE IN CANCER THERAPY E Fitzpatrick, S Xu, J Datta, J Cintolo, B Czerniecki Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Dendritic cell (DCs) vaccines that optimize antigen sensitization by CD4+ and CD8+ T cells induce Th1 cytokine production offer promise for the future of cancer therapeutics; widespread application is inhibited by laborious manufacturing. Cryopreservation of mature type I polarized DCs alleviates exhaustive production practices and increase world-wide accessibility. Methods: Freshly matured DCs (DC1s) and DCs cryopreserved mature (cryoDC1s), rapidly maturated, employed to activate cells into DC1s by a 2-day culture using IFN-g and LPS. Populations were compared for viability, recovery and phenotypic maturity. Functionality was determined by cytokine secretion, ability to stimulate CD4+ and CD8+ T cells by allosensitization and antigen-specific sensitization of CD8+ T cells. Results: Significant differences were not seen in viability (p¼0.481), and recovery (p¼0.122). Initial IL-12 p70 production was 44.76 ng/mL (DC1s) and 29.94 ng/mL (cryoDC1s) with no significant differences. IL-12 p70 continued for of 30 hours, peak secretion between 8 and 12 hours. Key cytokines in promoting a Th1 cell mediate response (TNF-a, RANTES, MIP1-a and MIP1-b) secreted noticeable levels; moreover populations were not significantly different. CD80, 83, and 86 demonstrate DC maturation, expression revealed no significant difference. Functional abilities were induced by CD4+