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Maturation of dendritic cells from CD14+ monocytes in an automated functionally closed hollow fiber bioreactor system
20th Annual ISCT Meeting
In order to ensure a standardized and consistent Drug Product freezing and to minimize batch-to-batch differences in cell recovery and viability postthaw, TxCell scientists tested the CoolCellÒ container, a passive freezing device, as an alternative to the classical controlled rate freezer. Results showed that by using a CoolCell freezing device, Ag-Tregs can be successfully cryopreserved and recovered in a standardized way acceptable to the processing and manufacturing of cell therapies. TxCell now intends to use the CoolCellÒ device in its phase IIb clinical study with its lead AgTreg cell product candidate, OvasaveÒ. Use of the CoolCellÒ passive freezing device for cell therapy manufacturing of Ag-Treg represents a new standardized method of cell therapy product cryopreservation. The ability to develop and store functional Treg in a cost-effective and reproducible manner is an important milestone in the ultimate use of these cells in the clinic.
79 VALIDATION OF A NOVEL PORTABLE FREEZING DEVICE IN THE OPTIMAL FREEZING OF PERIPHERAL BLOOD MONONUCLEAR CELLS FOR POTENTIAL CELL THERAPY USE 1 2 1 1 M Thompson , Q Tang , B Schryver , R Ehrhardt 1 BioCision, Larkspur, California, United States, 2Surgery, UCSF, San Francisco, California, United States Peripheral blood mononuclear cells (PBMCs) are used in fighting diseases such as leukemia, cancer and infectious disease. They are also used as a starting materials for the isolation of T cells, NK cells, monocytes and dendritic cells for therapeutic applications. High viability of PBMCs is essential for efficient immunotherapy and consistent trial results. Therefore there is a need to isolate and preserve PBMCs in a standardized, reproducible way. Researchers at USCF are currently investigating the cryopreservation of PBMCs with the intention of isolating regulatory T lymphocytes (Tregs) for autologous cell-based immunotherapy treatment in patients with autoimmune diseases or transplantation recipients. In the past, to successfully freeze PBMCs, controlled-rate freezers were used. However they are expensive, difficult to operate, prone to malfunction and logistically difficult to ensure at all cell therapy collection sites. Using the CoolCellÒ container, a passive freezing device, as an alternative to the electronically controlled rate freezer, the researchers showed that PBMCs could be cryopreserved and recovered with high viability and recovery as seen using controlled-rate freezers. The incorporation of the CoolCell passive freezing device in cell therapy research will overcome the hurdles of multiple clinical site collections due to its portability, lack of necessary maintenance and ease of use. The ability to develop and store PBMCs and potentially functional Treg in a cost-effective and reproducible manner is an important milestone in the ultimate use of these cells in the clinic. 80 MATURATION OF DENDRITIC CELLS FROM CD14+ MONOCYTES IN AN AUTOMATED FUNCTIONALLY CLOSED HOLLOW FIBER BIOREACTOR SYSTEM T Startz, K Nguyen, R Peters, B Nankervis, M Jones, R Kilian, N Frank, B Vang, D Hill Terumo BCT, Lakewood, Colorado, United States Dendritic cells make up a scarce population of immune cells that process antigen material in the peripheral blood, acting as a messenger between the innate and active immunity. Their rarity in the human body and difficulty to isolate has been a major obstacle in researching their genesis and development. It is only recently that their therapeutic value to present foreign and self antigens to T-cells has been recognized due to advancements with in vitro cell culture techniques. The large number needed for cellular therapy and their relatively short life span in the mature state has shown a necessity to derive these cells from a more abundant source and be delivered in an expedited manner to the patient. CD14+ monocytes have been shown to differentiate into mature dendritic cells with addition of GM-CSF, IL-4, TNFa and IL-1b to culture media. In the Quantum System we have been able to transform a large percentage of CD14 positive monocytes into mature dendritic cells in a functionally closed environment that reduces the time and resources needed to produce a therapeutic dose while reducing the risk of contamination by eliminating open steps. Mature DC phenotype was confirmed by flow cytometry as Lymphocyte lineage (CD3, CD14, CD16, CD19, CD20 AND
S29
CD56) negative and positive for HLA-DR, CD83, CD86, CD197 and CD209. Dendritic Cell functionality was shown by the antigen uptake of Alexa Flour 488 labeled Dextran and increased production of IL-6 and IL-12 p70 cytokines.
81 TOOLS FOR OPTIMIZATION OF ADOPTIVE CELLULAR THERAPY L Brix1, D Pan2, C Halgreen1, H Pedersen1, P Wallace2 1 Immudex, Copenhagen, Denmark, 2Roswell Park Cancer Institute, Buffalo, New York, United States Cell-based therapies using lymphocytes are promising approaches for immunotherapy. The transfusion of T lymphocytes - adoptive cell therapy - is an effective treatment for viral infections in immune-compromised patients, and has induced regression of cancer in early-stage clinical trials. Adoptive T cell therapy can be optimized in several ways: - Patient stratification - only those that benefit from therapy are treated, - Measurement of T-cell immunity pre and post treatment - to evaluate if the desired immune response has been induced and additional therapy is needed, - Quantitative quality control of the cellular product prior to infusion to ensure consistent cell number and quality MHC Multimer assays can be used to stratify patients, measure T-cell immunity, and perform quality control on the cellular infusion products. We used Dextramers to measure CMV-specific T-cell responses in transplant patients. Multiple adoptive cell therapy trials are investigating the benefit of transferring CMV-specific T cells to transplant patients to avoid reactivation of CMV. We used the Dextramer CMV assay, to quantitate CMV-specific T cells in whole blood and processed cell samples. The reconstitution of CMV immunity was successfully followed in >90 transplant patients and it was shown that induction of CMV-specific immunity upon adoptive transfer of CMV-specific T cells could be reliably measured. Furthermore we showed that CMV Dextramers can be used to characterize cellular products with respect to CMV-specific T-cell composition upon in vitro expansion. Our results demonstrate that the CMV Dextramer assay is a valuable tool that may improve CMV-specific adoptive cellular therapy. The Dextramer assay allows Stratification e Identification of patients with low CMV-specific immunity, Immune monitoring e Determination of the induced cellular response in patients, - Quality control - QC of manufactured cellular products. 82 CELLULAR IMMUNOTHERAPY WITH THE CONTINUOUSLY GROWING NK-92 CELL LINE AS AN ALTERNATIVE TO DONOR DERIVED BLOOD NK-CELLS H Klingemann1,2, B Simon1 1 Conkwest Inc, Cambridge, Massachusetts, United States, 2Tufts University Medical School, Boston, Massachusetts, United States Introduction: Infusions with cytokine-activated NK cells obtained from blood of MHC mismatched donors have shown some promising results especially in patients with AML. There is also some evidence that infusions of KIR receptor mismatched NK cells as part of a stem cell transplant result in lower relapse rates. Methods: Expanding NK cells form donors requires them to first undergo leukapheresis with subsequent removal of CD3+ lymphocytes (to prevent GvHD). In contrast, the continuously growing NK cell line NK-92 can be easily expanded to clinical scale in bioreactors. The broad cytotoxicity of NK92 is due to the lack of most of the KIR receptors while expressing a range of activating receptors. Results: We report here results from concluded and ongoing clinical phase I studies with NK-92 cells in patients with advanced cancer, that confirm their safety profile. Anti-tumor responses were seen in some patients with advanced hematological malignancies and solid tumors. NK-92 cells also provide a platform for further tumor targeted engineering. A variant has been generated that expresses a high affinity FcgIIIRA receptor that can augment ADCC of monoclonal antibodies. NK-92 cells have also been engineered to express CARs to make them targeted to melanoma, myeloma, leukemia and brain cancer. Tracking studies show that NK-92 CAR home to the tumor sites and video-lapse studies confirm that they are able to do “serial killing”. Conclusion: The human clinical grade NK-92 cell line has advantages over peripheral blood NK cells as a cell therapy product for cancer patients. NK-92 cells are now in phase II studies as an ‘off the shelf’ tumor targeted local and systemic cell therapy.
In order to ensure a standardized and consistent Drug Product freezing and to minimize batch-to-batch differences in cell recovery and viability postthaw, TxCell scientists tested the CoolCellÒ container, a passive freezing device, as an alternative to the classical controlled rate freezer. Results showed that by using a CoolCell freezing device, Ag-Tregs can be successfully cryopreserved and recovered in a standardized way acceptable to the processing and manufacturing of cell therapies. TxCell now intends to use the CoolCellÒ device in its phase IIb clinical study with its lead AgTreg cell product candidate, OvasaveÒ. Use of the CoolCellÒ passive freezing device for cell therapy manufacturing of Ag-Treg represents a new standardized method of cell therapy product cryopreservation. The ability to develop and store functional Treg in a cost-effective and reproducible manner is an important milestone in the ultimate use of these cells in the clinic.
79 VALIDATION OF A NOVEL PORTABLE FREEZING DEVICE IN THE OPTIMAL FREEZING OF PERIPHERAL BLOOD MONONUCLEAR CELLS FOR POTENTIAL CELL THERAPY USE 1 2 1 1 M Thompson , Q Tang , B Schryver , R Ehrhardt 1 BioCision, Larkspur, California, United States, 2Surgery, UCSF, San Francisco, California, United States Peripheral blood mononuclear cells (PBMCs) are used in fighting diseases such as leukemia, cancer and infectious disease. They are also used as a starting materials for the isolation of T cells, NK cells, monocytes and dendritic cells for therapeutic applications. High viability of PBMCs is essential for efficient immunotherapy and consistent trial results. Therefore there is a need to isolate and preserve PBMCs in a standardized, reproducible way. Researchers at USCF are currently investigating the cryopreservation of PBMCs with the intention of isolating regulatory T lymphocytes (Tregs) for autologous cell-based immunotherapy treatment in patients with autoimmune diseases or transplantation recipients. In the past, to successfully freeze PBMCs, controlled-rate freezers were used. However they are expensive, difficult to operate, prone to malfunction and logistically difficult to ensure at all cell therapy collection sites. Using the CoolCellÒ container, a passive freezing device, as an alternative to the electronically controlled rate freezer, the researchers showed that PBMCs could be cryopreserved and recovered with high viability and recovery as seen using controlled-rate freezers. The incorporation of the CoolCell passive freezing device in cell therapy research will overcome the hurdles of multiple clinical site collections due to its portability, lack of necessary maintenance and ease of use. The ability to develop and store PBMCs and potentially functional Treg in a cost-effective and reproducible manner is an important milestone in the ultimate use of these cells in the clinic. 80 MATURATION OF DENDRITIC CELLS FROM CD14+ MONOCYTES IN AN AUTOMATED FUNCTIONALLY CLOSED HOLLOW FIBER BIOREACTOR SYSTEM T Startz, K Nguyen, R Peters, B Nankervis, M Jones, R Kilian, N Frank, B Vang, D Hill Terumo BCT, Lakewood, Colorado, United States Dendritic cells make up a scarce population of immune cells that process antigen material in the peripheral blood, acting as a messenger between the innate and active immunity. Their rarity in the human body and difficulty to isolate has been a major obstacle in researching their genesis and development. It is only recently that their therapeutic value to present foreign and self antigens to T-cells has been recognized due to advancements with in vitro cell culture techniques. The large number needed for cellular therapy and their relatively short life span in the mature state has shown a necessity to derive these cells from a more abundant source and be delivered in an expedited manner to the patient. CD14+ monocytes have been shown to differentiate into mature dendritic cells with addition of GM-CSF, IL-4, TNFa and IL-1b to culture media. In the Quantum System we have been able to transform a large percentage of CD14 positive monocytes into mature dendritic cells in a functionally closed environment that reduces the time and resources needed to produce a therapeutic dose while reducing the risk of contamination by eliminating open steps. Mature DC phenotype was confirmed by flow cytometry as Lymphocyte lineage (CD3, CD14, CD16, CD19, CD20 AND
S29
CD56) negative and positive for HLA-DR, CD83, CD86, CD197 and CD209. Dendritic Cell functionality was shown by the antigen uptake of Alexa Flour 488 labeled Dextran and increased production of IL-6 and IL-12 p70 cytokines.
81 TOOLS FOR OPTIMIZATION OF ADOPTIVE CELLULAR THERAPY L Brix1, D Pan2, C Halgreen1, H Pedersen1, P Wallace2 1 Immudex, Copenhagen, Denmark, 2Roswell Park Cancer Institute, Buffalo, New York, United States Cell-based therapies using lymphocytes are promising approaches for immunotherapy. The transfusion of T lymphocytes - adoptive cell therapy - is an effective treatment for viral infections in immune-compromised patients, and has induced regression of cancer in early-stage clinical trials. Adoptive T cell therapy can be optimized in several ways: - Patient stratification - only those that benefit from therapy are treated, - Measurement of T-cell immunity pre and post treatment - to evaluate if the desired immune response has been induced and additional therapy is needed, - Quantitative quality control of the cellular product prior to infusion to ensure consistent cell number and quality MHC Multimer assays can be used to stratify patients, measure T-cell immunity, and perform quality control on the cellular infusion products. We used Dextramers to measure CMV-specific T-cell responses in transplant patients. Multiple adoptive cell therapy trials are investigating the benefit of transferring CMV-specific T cells to transplant patients to avoid reactivation of CMV. We used the Dextramer CMV assay, to quantitate CMV-specific T cells in whole blood and processed cell samples. The reconstitution of CMV immunity was successfully followed in >90 transplant patients and it was shown that induction of CMV-specific immunity upon adoptive transfer of CMV-specific T cells could be reliably measured. Furthermore we showed that CMV Dextramers can be used to characterize cellular products with respect to CMV-specific T-cell composition upon in vitro expansion. Our results demonstrate that the CMV Dextramer assay is a valuable tool that may improve CMV-specific adoptive cellular therapy. The Dextramer assay allows Stratification e Identification of patients with low CMV-specific immunity, Immune monitoring e Determination of the induced cellular response in patients, - Quality control - QC of manufactured cellular products. 82 CELLULAR IMMUNOTHERAPY WITH THE CONTINUOUSLY GROWING NK-92 CELL LINE AS AN ALTERNATIVE TO DONOR DERIVED BLOOD NK-CELLS H Klingemann1,2, B Simon1 1 Conkwest Inc, Cambridge, Massachusetts, United States, 2Tufts University Medical School, Boston, Massachusetts, United States Introduction: Infusions with cytokine-activated NK cells obtained from blood of MHC mismatched donors have shown some promising results especially in patients with AML. There is also some evidence that infusions of KIR receptor mismatched NK cells as part of a stem cell transplant result in lower relapse rates. Methods: Expanding NK cells form donors requires them to first undergo leukapheresis with subsequent removal of CD3+ lymphocytes (to prevent GvHD). In contrast, the continuously growing NK cell line NK-92 can be easily expanded to clinical scale in bioreactors. The broad cytotoxicity of NK92 is due to the lack of most of the KIR receptors while expressing a range of activating receptors. Results: We report here results from concluded and ongoing clinical phase I studies with NK-92 cells in patients with advanced cancer, that confirm their safety profile. Anti-tumor responses were seen in some patients with advanced hematological malignancies and solid tumors. NK-92 cells also provide a platform for further tumor targeted engineering. A variant has been generated that expresses a high affinity FcgIIIRA receptor that can augment ADCC of monoclonal antibodies. NK-92 cells have also been engineered to express CARs to make them targeted to melanoma, myeloma, leukemia and brain cancer. Tracking studies show that NK-92 CAR home to the tumor sites and video-lapse studies confirm that they are able to do “serial killing”. Conclusion: The human clinical grade NK-92 cell line has advantages over peripheral blood NK cells as a cell therapy product for cancer patients. NK-92 cells are now in phase II studies as an ‘off the shelf’ tumor targeted local and systemic cell therapy.