- Email: [email protected]
A simple rapid car T-cell cytotoxicity and degranulation flow cytometric assay
Poster Abstract Presentations Ò
scale single use stirred-tank ambr 250 bioreactor under agitated conditions. Building on these results, the same platform was used for the growth of CART cells. A range of speeds was tested in order to find the optimal expansion conditions under which T-cells can be grown without losing their functionality. The outcomes from different experiments were compared with respect to cell yield, CAR-T cell fold expansion, viability, metabolite flux and T-cell subpopulations. pH and dissolved oxygen (dO2) concentration were monitored throughout the 7 days expansion allowing a better understanding of the culture requirements. Stirred-tank bioreactors are not commonly used for the manufacture of CAR-T cell products, therefore a further comparison in terms of growth and quality of CAR-T cells achieved in routinely used static and dynamic systems, as for example T-Flasks, G-Rex platform and Rocking Motion bioreactors, was needed. Seeding density, feeding regime and media composition were kept constant across the different platforms in order to be able to compare the outcome. Each condition was tested with different donors in order to assess the inter-donor variability in terms of growth kinetic and starting material. The stirred-tank bioreactor was proven to perform better in terms of fold expansion. The outcome from this extensive comparability study will be used to improve the current manufacturing process and reduce the manufacturing cost of the commercial product. Furthermore, using a stirred tank bioreactor will allow for an easier scaling-up of the process to 1-5 litres bioreactors. These can be used for the initial development of allogeneic CAR-T therapies. 91 POTENT ANTITUMOR EFFICACY OF EX VIVO EXPANDED AND CRYOPRESERVED HUMAN NATURAL KILLER CELLS AGAINST ORTHOTOPIC PANCREATIC CANCER G. Park, B. Yang, E. Kim, H. Kim, Y. Kim, S. Cho, Y. Hwang & B. Min GCLabcell, Yongin-si, Gyeonggi-do, the Republic of Korea Background & Aim: Natural killer (NK) cells are specialized components of the innate immune system that contribute to the first line of defense against viral infections and cancer. Ex vivo expanded NK cells are a promising tool for the use in adoptive immunotherapy. Pancreatic cancer is one of the most aggressive tumor types and severely limits the therapeutic efficacy of both immunotherapeutics and conventional chemotherapeutics because ECM act as a major physical barrier against drug penetration. Methods, Results & Conclusion: To address these challenges, we expanded and cryopreserved allogeneic NK cells. And cryopreserved NK cells were evaluated for their therapeutic efficacy against desmoplastic pancreatic tumors, ultimately aiming to develop easily accessible and mass-producible off-the-shelf cellbased immunotherapeutics. Ex vivoexpanded and cryopreserved cells retained highly pure and activated population of NK cells. In addition, ex vivo expanded and cryopreserved NK cells exhibited not only potent killing activity and cytokine production against various pancreatic cancer cell lines in vitro but also excellent tumor growth suppression in vivo orthotopic pancreatic cancer model. In conclusion, ex vivo expanded and cryopreserved NK cells are a strong candidate for future cell-mediated systemic immunotherapy against pancreatic cancer. 92 IDENTIFICATION OF AN OPTIMAL GENE DELIVERY METHOD FOR CAR-T GENERATION IN A MICRO FACTORY PLATFORM T. Panagopoulou & Q.A. Rafiq Department of Biochemical Engineering, University College London, London, United Kingdom Background & Aim: The approval of the very first chimeric antigen receptor (CAR) - T therapies (Kymriah and Yescarta) by European and American agencies has marked a truly revolutionary period in cancer immuno-therapeutics. Currently, most autologous CAR-T therapies are generated via a centralized manufacturing process whereby the patients’ T cells are isolated, followed by introduction of the genetic material via (lenti) viral transduction, expansion of the transduced T cells and finally re-infusion of the CAR-T cells into the patient. The generation of autologous CAR-T treatments is very laborious and is associated with very high costs. Additionally, the requirement of large batches of clinical grade (lenti) virus is adding further complexity to the overall process. For that reason, efforts are being made to both simplify the manufacturing process and reduce the overall costs. The work described herein is part of a consolidated effort among leading UK universities to generate an on- or near- body manufacturing device that will generate autologous CAR-T cells.
S35
The specific primary aim of this study was to identify the optimal methodology for CAR gene delivery within a micro factory setting. Methods, Results & Conclusion: The gene delivery methods that were explored were both genome-integrative and non-integrative, were both viral and non-viral and included: 1) lentiviral vector delivery, 2) mRNA delivery and 3) minicircle-based Sleeping Beautytransposon/transposase delivery. The efficiency of CAR-T cell generation was assessed via flow cytometric analysis. 93 A SIMPLE RAPID CAR T-CELL CYTOTOXICITY AND DEGRANULATION FLOW CYTOMETRIC ASSAY L.M. Brownrigg12, D.B. Tan1, E. Bosio3,4 & M. Sturm1,5 1 Cell and Tissue Therapies WA, Royal Perth Hospital, Perth, Western Australia, Australia, 2School of Medicine, University of Western Australia, Perth, Western Australia, Australia, 3Centre for Clinical Research in Emergency Medicine, School of Medicine, University of Western Australia, Perth, Western Australia, Australia, 4Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia, 5School of Biomedical Sciences, University of Western Australia, Perth, Western Australia, Australia Background & Aim: The gold standard for determining cell-cell cytotoxicity has historically been the chromium-release assay. Other less-hazardous, nonradioactive options are now available and preferable to chromium assays from an occupational health and safety perspective. Flow cytometry allows information in addition to cytotoxicity to be gained from a single assay. The following assay panel requires only an entry-level 8-colour flow cytometer. Methods, Results & Conclusion: An assay was developed for anti-CD19 CAR T-cells using CD19+ (Nalm-6) or CD19- (Jurkat) target cell lines freshly stained with Violet Proliferation Dye 450 (VPD450) and detectable in the V_450/50 channel, allowing discrimination between violet target cells and non-violet effector cells. Target cell lines are treated by incubation with or without anti-CD19 CAR T-cells in standard tissue culture media without any pre-stimulation or additional interleukins or cytokines. Target cells and effector cells can then be analysed separately from the same sample via efficient gating strategies. CAR T-cells appear VPD450-negative and fluoresce green in the B_530/30 channel, either via EGFP or FITC-labelling. CD107a is a marker of T-cell degranulation that is exposed to the T-cell surface when activated cytotoxic Tcells secrete perforins and granzymes against targets. CAR T-cell activation is determined via binding of a PE-labelled anti-CD107a antibody present in the cell culture media during the incubation period, and only binds CAR T-cells if they are actively degranulating. Apoptosis and necrosis of target cell populations are measured to determine overall cytotoxicity. Apoptosis is determined via Annexin V staining, and necrosis via Fixed Viability Stain 780 (FVS780) staining. As expected, CD19+ target viability decreases upon co-culture with CAR Tcells. CAR T-cells are activated upon exposure to CD19+ target cells. Neither target cell viability nor CAR T-cell activation changes when CD19- target cells are co-cultured with CAR T-cells. Results are comparable to those previously published. This flexible assay allows for safer determination of cytotoxicity than via traditional chromium assays, with additional CAR T-cell activation data available from the same samples, saving time and money. The panel is designed to be accessible for laboratories with basic flow cytometry capability, and may be expanded to include more cytokines of interest in a higher specification cytometer. 94 VIABLE MANUFACTURE OF CELL THERAPIES THROUGHTHE INTEGRATION OF MULTIPLE UNIT PROCESSES ONTO A COUNTER-FLOW CENTRIFUGATION DEVICE A. Klarer & D. Smith Innovation and Engineering, Hitachi Chemical Advanced Therapeutic Solutions, Allendale, New Jersey, United States Background & Aim: With growing proof of efficacy in varied indications, regenerative medicine has reached a positive inflection point in the investment of time and money by established industry leaders and disruptive startups. With rapid growth comes the need to devote resources to the engineering challenges that currently prevent the quick and cost-effective manufacture of therapies that maintain a consistent, high-level of quality and, in turn, can support commercial manufacturing. This is especially true when looking at patient specific cell therapies that require rapid change over of equipment and benefit little from traditional sterile barriers (i.e. filters and heat inactivation).
scale single use stirred-tank ambr 250 bioreactor under agitated conditions. Building on these results, the same platform was used for the growth of CART cells. A range of speeds was tested in order to find the optimal expansion conditions under which T-cells can be grown without losing their functionality. The outcomes from different experiments were compared with respect to cell yield, CAR-T cell fold expansion, viability, metabolite flux and T-cell subpopulations. pH and dissolved oxygen (dO2) concentration were monitored throughout the 7 days expansion allowing a better understanding of the culture requirements. Stirred-tank bioreactors are not commonly used for the manufacture of CAR-T cell products, therefore a further comparison in terms of growth and quality of CAR-T cells achieved in routinely used static and dynamic systems, as for example T-Flasks, G-Rex platform and Rocking Motion bioreactors, was needed. Seeding density, feeding regime and media composition were kept constant across the different platforms in order to be able to compare the outcome. Each condition was tested with different donors in order to assess the inter-donor variability in terms of growth kinetic and starting material. The stirred-tank bioreactor was proven to perform better in terms of fold expansion. The outcome from this extensive comparability study will be used to improve the current manufacturing process and reduce the manufacturing cost of the commercial product. Furthermore, using a stirred tank bioreactor will allow for an easier scaling-up of the process to 1-5 litres bioreactors. These can be used for the initial development of allogeneic CAR-T therapies. 91 POTENT ANTITUMOR EFFICACY OF EX VIVO EXPANDED AND CRYOPRESERVED HUMAN NATURAL KILLER CELLS AGAINST ORTHOTOPIC PANCREATIC CANCER G. Park, B. Yang, E. Kim, H. Kim, Y. Kim, S. Cho, Y. Hwang & B. Min GCLabcell, Yongin-si, Gyeonggi-do, the Republic of Korea Background & Aim: Natural killer (NK) cells are specialized components of the innate immune system that contribute to the first line of defense against viral infections and cancer. Ex vivo expanded NK cells are a promising tool for the use in adoptive immunotherapy. Pancreatic cancer is one of the most aggressive tumor types and severely limits the therapeutic efficacy of both immunotherapeutics and conventional chemotherapeutics because ECM act as a major physical barrier against drug penetration. Methods, Results & Conclusion: To address these challenges, we expanded and cryopreserved allogeneic NK cells. And cryopreserved NK cells were evaluated for their therapeutic efficacy against desmoplastic pancreatic tumors, ultimately aiming to develop easily accessible and mass-producible off-the-shelf cellbased immunotherapeutics. Ex vivoexpanded and cryopreserved cells retained highly pure and activated population of NK cells. In addition, ex vivo expanded and cryopreserved NK cells exhibited not only potent killing activity and cytokine production against various pancreatic cancer cell lines in vitro but also excellent tumor growth suppression in vivo orthotopic pancreatic cancer model. In conclusion, ex vivo expanded and cryopreserved NK cells are a strong candidate for future cell-mediated systemic immunotherapy against pancreatic cancer. 92 IDENTIFICATION OF AN OPTIMAL GENE DELIVERY METHOD FOR CAR-T GENERATION IN A MICRO FACTORY PLATFORM T. Panagopoulou & Q.A. Rafiq Department of Biochemical Engineering, University College London, London, United Kingdom Background & Aim: The approval of the very first chimeric antigen receptor (CAR) - T therapies (Kymriah and Yescarta) by European and American agencies has marked a truly revolutionary period in cancer immuno-therapeutics. Currently, most autologous CAR-T therapies are generated via a centralized manufacturing process whereby the patients’ T cells are isolated, followed by introduction of the genetic material via (lenti) viral transduction, expansion of the transduced T cells and finally re-infusion of the CAR-T cells into the patient. The generation of autologous CAR-T treatments is very laborious and is associated with very high costs. Additionally, the requirement of large batches of clinical grade (lenti) virus is adding further complexity to the overall process. For that reason, efforts are being made to both simplify the manufacturing process and reduce the overall costs. The work described herein is part of a consolidated effort among leading UK universities to generate an on- or near- body manufacturing device that will generate autologous CAR-T cells.
S35
The specific primary aim of this study was to identify the optimal methodology for CAR gene delivery within a micro factory setting. Methods, Results & Conclusion: The gene delivery methods that were explored were both genome-integrative and non-integrative, were both viral and non-viral and included: 1) lentiviral vector delivery, 2) mRNA delivery and 3) minicircle-based Sleeping Beautytransposon/transposase delivery. The efficiency of CAR-T cell generation was assessed via flow cytometric analysis. 93 A SIMPLE RAPID CAR T-CELL CYTOTOXICITY AND DEGRANULATION FLOW CYTOMETRIC ASSAY L.M. Brownrigg12, D.B. Tan1, E. Bosio3,4 & M. Sturm1,5 1 Cell and Tissue Therapies WA, Royal Perth Hospital, Perth, Western Australia, Australia, 2School of Medicine, University of Western Australia, Perth, Western Australia, Australia, 3Centre for Clinical Research in Emergency Medicine, School of Medicine, University of Western Australia, Perth, Western Australia, Australia, 4Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia, 5School of Biomedical Sciences, University of Western Australia, Perth, Western Australia, Australia Background & Aim: The gold standard for determining cell-cell cytotoxicity has historically been the chromium-release assay. Other less-hazardous, nonradioactive options are now available and preferable to chromium assays from an occupational health and safety perspective. Flow cytometry allows information in addition to cytotoxicity to be gained from a single assay. The following assay panel requires only an entry-level 8-colour flow cytometer. Methods, Results & Conclusion: An assay was developed for anti-CD19 CAR T-cells using CD19+ (Nalm-6) or CD19- (Jurkat) target cell lines freshly stained with Violet Proliferation Dye 450 (VPD450) and detectable in the V_450/50 channel, allowing discrimination between violet target cells and non-violet effector cells. Target cell lines are treated by incubation with or without anti-CD19 CAR T-cells in standard tissue culture media without any pre-stimulation or additional interleukins or cytokines. Target cells and effector cells can then be analysed separately from the same sample via efficient gating strategies. CAR T-cells appear VPD450-negative and fluoresce green in the B_530/30 channel, either via EGFP or FITC-labelling. CD107a is a marker of T-cell degranulation that is exposed to the T-cell surface when activated cytotoxic Tcells secrete perforins and granzymes against targets. CAR T-cell activation is determined via binding of a PE-labelled anti-CD107a antibody present in the cell culture media during the incubation period, and only binds CAR T-cells if they are actively degranulating. Apoptosis and necrosis of target cell populations are measured to determine overall cytotoxicity. Apoptosis is determined via Annexin V staining, and necrosis via Fixed Viability Stain 780 (FVS780) staining. As expected, CD19+ target viability decreases upon co-culture with CAR Tcells. CAR T-cells are activated upon exposure to CD19+ target cells. Neither target cell viability nor CAR T-cell activation changes when CD19- target cells are co-cultured with CAR T-cells. Results are comparable to those previously published. This flexible assay allows for safer determination of cytotoxicity than via traditional chromium assays, with additional CAR T-cell activation data available from the same samples, saving time and money. The panel is designed to be accessible for laboratories with basic flow cytometry capability, and may be expanded to include more cytokines of interest in a higher specification cytometer. 94 VIABLE MANUFACTURE OF CELL THERAPIES THROUGHTHE INTEGRATION OF MULTIPLE UNIT PROCESSES ONTO A COUNTER-FLOW CENTRIFUGATION DEVICE A. Klarer & D. Smith Innovation and Engineering, Hitachi Chemical Advanced Therapeutic Solutions, Allendale, New Jersey, United States Background & Aim: With growing proof of efficacy in varied indications, regenerative medicine has reached a positive inflection point in the investment of time and money by established industry leaders and disruptive startups. With rapid growth comes the need to devote resources to the engineering challenges that currently prevent the quick and cost-effective manufacture of therapies that maintain a consistent, high-level of quality and, in turn, can support commercial manufacturing. This is especially true when looking at patient specific cell therapies that require rapid change over of equipment and benefit little from traditional sterile barriers (i.e. filters and heat inactivation).