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multivirus specific-cytotoxic T lymphocytes after allogeneic hematopoietic stem cell transplantation for B cell malignancies
19th Annual ISCT Meeting
induction overall response (OR)¼85%, partial response (PR)¼65%, very good PR (VGPR)¼20%, stable disease (SD)¼5% and refractory disease (RD)¼10%. Improvement post autograft to PR¼35%, VGPR¼40% and complete response (CR) ¼ 10%. At a median follow-up of 34 (16-45) months, best response achieved is PR¼20%, VGPR¼30% and CR¼40%. In responders best response was achieved post induction in 17%, post autograft in 39% and during maintenance in 44%. Presently 9 (45%) patients continue on maintenance having received a median of 21 (3-45) cycles. Low dose lenalidomide and dexamethasone induction followed by HSCT in untreated myeloma patients is associated with high response rates. Depth of response is improved by HSCT and maintenance treatment. Collection of haematopoietic and dendritic cell precursors is not impaired. 9 PROGRESS TOWARDS THE CGMP PRODUCTION OF PLURIPOTENT STEM CELL DERIVED RED BLOOD CELLS ML Turner1,2, J Mountford1,3, L Forrester2, P de Sousa2,4, A Courtney4, S Parsons5, D Anstee6, H Newlands1, J Pelly1, W Murphy7 1 Scottish National Blood Transfusion Service, Edinburgh, United Kingdom, 2 University of Edinburgh, Edinburgh, United Kingdom, 3University of Glasgow, Glasgow, United Kingdom, 4Roslin Cells, Edinburgh, United Kingdom, 5NHS Blood and Transplant, Bristol, United Kingdom, 6NHS Blood and Transplant, Edinburgh, United Kingdom, 7Irish Blood Transfusion Service, Dublin, Ireland Whilst red cell supply is sufficient, safe and effective in most developed economies, there are problems in many countries in maintaining adequacy of supply, managing the risk of transmission of infectious agents and immune incompatibility between donor and recipient. Human embryonic and induced pluripotent stem cells (hESCs & hiPSC) have unique properties in that they can be maintained indefinitely in culture in an undifferentiated state and yet retain the ability to form all the cells and tissues within the body. They therefore offer a potentially scalable source from which to generate red cells (RBCs) for use in clinical transfusion. We have been working on the generation of hESC to cGMP grade in compliance with UK regulatory requirements for eventual clinical use. We are able to differentiate these to form haematopoietic progenitor cells (HPC) which subsequently result in 95% conversion to erythroid cells with up to 350,000 fold expansion in cell numbers, in a stroma-free, animal product-free suspension based culture system. The culture process consists of 4 different steps and takes up to 30 days. We have also demonstrated that this methodology is similarly effective for hiPSC. The erythroid cells express foetal haemoglobin but enucleation rates remain low at 1-10%. Many challenges exist in taking this product through to clinical trial including improving the rate of enucleation, scale-up and optimisation of culture process, cGMP-translation and cost-reduction in manufacturing and the quality and regulatory challenges of mounting first in man clinical studies.
S7
Safety. Seven patients (3 ALL, 2 CLL, 2 CLL/Richter’s transformation) were infused with a total of 3 to 9 107 cells/m2 without infusion related toxicity. Persistence. There was an initial rise and then a decline of transduced T-cells in the peripheral blood 1e4 weeks following infusion. However, persistence up to 9 weeks was documented in disease sites including the gastrointestinal tract (patient 2; 55 copies/1000 ng DNA), the bone marrow (patients 1 and 3; 45 and 26 copies/1000 ng DNA respectively) and in a diseased lymph node (patient 5; 32 copies/1000 ng DNA). Anti-Tumor Activity. Transient clinical responses were seen in 3/6 patients with detectable diseaseat time of infusion. One patient received T-cells early post transplant and is currently disease-free. Antiviral Activity. Patient 1 had adenovirus positivity in stool samples which resolved without antiviral treatment correlating with an increase in adenovirus-specific T cells in the peripheral blood. No other patient developed viral infections post CTL. These results support the study of CD19CAR/multivirus specific T cells for treating B-cell malignancies early after HSCT as a strategy to protect patients from both viral infections and relapse. 11 MAPPING AND MODELLING EX VIVO CELL CULTURE PROCESSES DIRECTLY FROM CELL TRACKING DATA RE Nordon1, J Cornwell1,2, J Li1, RP Harvey2, T Schroeder3 1 University of New South Wales, Sydney, Australia, 2Victor Chang Cardiac Research Institute, Sydney, Australia, 3Helmholtz Center, Munich, Germany Cost-effect culture methods for large-scale cell expansion and directed differentiation are central considerations for developing cell therapy products. However, it is not possible to directly quantify the rates of cellular events that govern productivity from bulk culture observations, namely mitosis, apoptosis and differentiation. The goal of this research is to develop live cell imaging tools to gain a deeper understanding of the cell expansion and differentiation process. Initiating cells and their progeny are continuously tracked by live cell imaging to determine how ex vivo manipulations influence probabilistic single cell fates. Our initial focus was to characterise granulocyte/macrophage progenitor and cardiac mesenchymal-like lineage expansion, however a similar approach can be applied to virtually any cell expansion product. A software and hardware platform for live cell analysis was developed to extract kinetic parameters for optimising cell expansion processes including a) semiautomated cell trajectory mapping from time lapse movies b) lineage tree generation and c) statistical methods for analysing and predicting the effect of culture constituents on cell time-to-events. Extrapolation from single cell observations to bulk culture systems is possible using a multitype branching model [1]. We have also developed microwell perfusion bioreactors for high content imaging of non-adherent cell culture processes [2]. Future development of high-content, live-cell imaging hardware for performing factorial design experiments will enable a deeper understanding of the molecular events that drive cell expansion and differentiation. References:
10 INFUSION OF CD19-DIRECTED/MULTIVIRUS SPECIFICCYTOTOXIC T LYMPHOCYTES AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR B CELL MALIGNANCIES CR Cruz1, KP Micklethwaite1, B Savoldo1, S Lam1, S Ku1, RA Krance1, O Diouf1, AM Leen1, RT Kamble1, A Kennedy-Nasser1, J Barrett2, E Shpall3, HE Heslop1, CM Rooney1, MK Brenner1, CM Bollard1, G Dotti1 1 Baylor College of Medicine, Houston, TX, 2National Institutes of Health, Bethesda, MD, 3University of Texas MD Anderson Cancer Center, Houston, TX Allogeneic hematopoietic stem cell transplant (HSCT) may increase long term disease-free survival in patients with high-risk-B-cell malignancies, but is associated with delayed immune reconstitution contributing to viral infections and relapse. We hypothesized that a single T-cell platform mediating both antiviral and antileukemic activity may be beneficial. We prepared CTLs with specificities directed towards EBV/CMV/adenovirus, then engineered them to express chimeric antigen receptors (CAR) targeting CD19 (expressed by B-cell malignancies). Donor-derived antigen presenting cells were transduced with an Ad5f35 vector encoding CMVpp65 transgene to stimulate and expand multivirus specific T cells. After 3 stimulations, multivirus-specific T cells were transduced with a retroviral vector encoding CAR-CD19.28 zeta.
[1] R. E. Nordon, K. H. Ko, R. Odell, and T. Schroeder, “Multi-type branching models to describe cell differentiation programs,” Journal of Theoretical Biology, vol. 277, pp. 7e18, May 21 2011. [2] H. Y. Chen, J. J. Li, H. Zhang, M. S. Li, G. Rosengarten, and R. E. Nordon, “Microwell perfusion array for high-throughput, long-term imaging of clonal growth,” Biomicrofluidics, vol. 5, Dec 2011. 12 REVISIONS TO ISBT 128 NOMENCLATURE FOR CELLULAR THERAPY I Slaper-Cortenbach1, P Distler2, P Ashford2 1 University Medical Center Utrecht, Utrecht, Netherlands, 2ICCBBA, San Bernardino, CA ISBT 128 terminology has been widely adopted by cellular therapy facilities globally and its use has been an accreditation requirement by AABB, FACT and JACIE for several years. Accredited facilities are now required to put in place an implementation plan for the full use of ISBT 128 coding and labeling in addition to terminology, and many facilities are moving forward with this implementation. Responding to regulator concern about the use of TC (“therapeutic cell”) terminology, the Cellular Therapy Coding and Labeling Advisory Group (CTCLAG) re-evaluated existing terminology. As a result of this review,
induction overall response (OR)¼85%, partial response (PR)¼65%, very good PR (VGPR)¼20%, stable disease (SD)¼5% and refractory disease (RD)¼10%. Improvement post autograft to PR¼35%, VGPR¼40% and complete response (CR) ¼ 10%. At a median follow-up of 34 (16-45) months, best response achieved is PR¼20%, VGPR¼30% and CR¼40%. In responders best response was achieved post induction in 17%, post autograft in 39% and during maintenance in 44%. Presently 9 (45%) patients continue on maintenance having received a median of 21 (3-45) cycles. Low dose lenalidomide and dexamethasone induction followed by HSCT in untreated myeloma patients is associated with high response rates. Depth of response is improved by HSCT and maintenance treatment. Collection of haematopoietic and dendritic cell precursors is not impaired. 9 PROGRESS TOWARDS THE CGMP PRODUCTION OF PLURIPOTENT STEM CELL DERIVED RED BLOOD CELLS ML Turner1,2, J Mountford1,3, L Forrester2, P de Sousa2,4, A Courtney4, S Parsons5, D Anstee6, H Newlands1, J Pelly1, W Murphy7 1 Scottish National Blood Transfusion Service, Edinburgh, United Kingdom, 2 University of Edinburgh, Edinburgh, United Kingdom, 3University of Glasgow, Glasgow, United Kingdom, 4Roslin Cells, Edinburgh, United Kingdom, 5NHS Blood and Transplant, Bristol, United Kingdom, 6NHS Blood and Transplant, Edinburgh, United Kingdom, 7Irish Blood Transfusion Service, Dublin, Ireland Whilst red cell supply is sufficient, safe and effective in most developed economies, there are problems in many countries in maintaining adequacy of supply, managing the risk of transmission of infectious agents and immune incompatibility between donor and recipient. Human embryonic and induced pluripotent stem cells (hESCs & hiPSC) have unique properties in that they can be maintained indefinitely in culture in an undifferentiated state and yet retain the ability to form all the cells and tissues within the body. They therefore offer a potentially scalable source from which to generate red cells (RBCs) for use in clinical transfusion. We have been working on the generation of hESC to cGMP grade in compliance with UK regulatory requirements for eventual clinical use. We are able to differentiate these to form haematopoietic progenitor cells (HPC) which subsequently result in 95% conversion to erythroid cells with up to 350,000 fold expansion in cell numbers, in a stroma-free, animal product-free suspension based culture system. The culture process consists of 4 different steps and takes up to 30 days. We have also demonstrated that this methodology is similarly effective for hiPSC. The erythroid cells express foetal haemoglobin but enucleation rates remain low at 1-10%. Many challenges exist in taking this product through to clinical trial including improving the rate of enucleation, scale-up and optimisation of culture process, cGMP-translation and cost-reduction in manufacturing and the quality and regulatory challenges of mounting first in man clinical studies.
S7
Safety. Seven patients (3 ALL, 2 CLL, 2 CLL/Richter’s transformation) were infused with a total of 3 to 9 107 cells/m2 without infusion related toxicity. Persistence. There was an initial rise and then a decline of transduced T-cells in the peripheral blood 1e4 weeks following infusion. However, persistence up to 9 weeks was documented in disease sites including the gastrointestinal tract (patient 2; 55 copies/1000 ng DNA), the bone marrow (patients 1 and 3; 45 and 26 copies/1000 ng DNA respectively) and in a diseased lymph node (patient 5; 32 copies/1000 ng DNA). Anti-Tumor Activity. Transient clinical responses were seen in 3/6 patients with detectable diseaseat time of infusion. One patient received T-cells early post transplant and is currently disease-free. Antiviral Activity. Patient 1 had adenovirus positivity in stool samples which resolved without antiviral treatment correlating with an increase in adenovirus-specific T cells in the peripheral blood. No other patient developed viral infections post CTL. These results support the study of CD19CAR/multivirus specific T cells for treating B-cell malignancies early after HSCT as a strategy to protect patients from both viral infections and relapse. 11 MAPPING AND MODELLING EX VIVO CELL CULTURE PROCESSES DIRECTLY FROM CELL TRACKING DATA RE Nordon1, J Cornwell1,2, J Li1, RP Harvey2, T Schroeder3 1 University of New South Wales, Sydney, Australia, 2Victor Chang Cardiac Research Institute, Sydney, Australia, 3Helmholtz Center, Munich, Germany Cost-effect culture methods for large-scale cell expansion and directed differentiation are central considerations for developing cell therapy products. However, it is not possible to directly quantify the rates of cellular events that govern productivity from bulk culture observations, namely mitosis, apoptosis and differentiation. The goal of this research is to develop live cell imaging tools to gain a deeper understanding of the cell expansion and differentiation process. Initiating cells and their progeny are continuously tracked by live cell imaging to determine how ex vivo manipulations influence probabilistic single cell fates. Our initial focus was to characterise granulocyte/macrophage progenitor and cardiac mesenchymal-like lineage expansion, however a similar approach can be applied to virtually any cell expansion product. A software and hardware platform for live cell analysis was developed to extract kinetic parameters for optimising cell expansion processes including a) semiautomated cell trajectory mapping from time lapse movies b) lineage tree generation and c) statistical methods for analysing and predicting the effect of culture constituents on cell time-to-events. Extrapolation from single cell observations to bulk culture systems is possible using a multitype branching model [1]. We have also developed microwell perfusion bioreactors for high content imaging of non-adherent cell culture processes [2]. Future development of high-content, live-cell imaging hardware for performing factorial design experiments will enable a deeper understanding of the molecular events that drive cell expansion and differentiation. References:
10 INFUSION OF CD19-DIRECTED/MULTIVIRUS SPECIFICCYTOTOXIC T LYMPHOCYTES AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR B CELL MALIGNANCIES CR Cruz1, KP Micklethwaite1, B Savoldo1, S Lam1, S Ku1, RA Krance1, O Diouf1, AM Leen1, RT Kamble1, A Kennedy-Nasser1, J Barrett2, E Shpall3, HE Heslop1, CM Rooney1, MK Brenner1, CM Bollard1, G Dotti1 1 Baylor College of Medicine, Houston, TX, 2National Institutes of Health, Bethesda, MD, 3University of Texas MD Anderson Cancer Center, Houston, TX Allogeneic hematopoietic stem cell transplant (HSCT) may increase long term disease-free survival in patients with high-risk-B-cell malignancies, but is associated with delayed immune reconstitution contributing to viral infections and relapse. We hypothesized that a single T-cell platform mediating both antiviral and antileukemic activity may be beneficial. We prepared CTLs with specificities directed towards EBV/CMV/adenovirus, then engineered them to express chimeric antigen receptors (CAR) targeting CD19 (expressed by B-cell malignancies). Donor-derived antigen presenting cells were transduced with an Ad5f35 vector encoding CMVpp65 transgene to stimulate and expand multivirus specific T cells. After 3 stimulations, multivirus-specific T cells were transduced with a retroviral vector encoding CAR-CD19.28 zeta.
[1] R. E. Nordon, K. H. Ko, R. Odell, and T. Schroeder, “Multi-type branching models to describe cell differentiation programs,” Journal of Theoretical Biology, vol. 277, pp. 7e18, May 21 2011. [2] H. Y. Chen, J. J. Li, H. Zhang, M. S. Li, G. Rosengarten, and R. E. Nordon, “Microwell perfusion array for high-throughput, long-term imaging of clonal growth,” Biomicrofluidics, vol. 5, Dec 2011. 12 REVISIONS TO ISBT 128 NOMENCLATURE FOR CELLULAR THERAPY I Slaper-Cortenbach1, P Distler2, P Ashford2 1 University Medical Center Utrecht, Utrecht, Netherlands, 2ICCBBA, San Bernardino, CA ISBT 128 terminology has been widely adopted by cellular therapy facilities globally and its use has been an accreditation requirement by AABB, FACT and JACIE for several years. Accredited facilities are now required to put in place an implementation plan for the full use of ISBT 128 coding and labeling in addition to terminology, and many facilities are moving forward with this implementation. Responding to regulator concern about the use of TC (“therapeutic cell”) terminology, the Cellular Therapy Coding and Labeling Advisory Group (CTCLAG) re-evaluated existing terminology. As a result of this review,