- Email: [email protected]
Developing allogeneic immunotherapy with iPSC derived cytotoxic T and NK cells
S48
Poster Abstracts
Data derived from the large cohort showed that highly purified NK cell products can be consistently obtained by using 2-step CliniMACS selection. This work can provide a benchmark for use of CliniMACS selection device. It is possible to derive in-process baseline values and control thresholds to alert, monitor and improve selection efficacy. When used in trend evaluation (e.g., LeveyJennings plot) of the performance on cell processing, early investigational action and root cause analysis of the purity and/or recovery of NK cells below a predetermined threshold (e.g., mean - 2SD) can prompt corrective actions to avoid any compromise of patient safety. 93 TARGETED DENDRITIC CELL THERAPY UTILISING ANTIBODY-DISPLAYING POROUS SILICON NANOPARTICLES LOADED WITH RAPAMYCIN S. Stead1, S. McInnes2, P.T. Coates1, N. Voelcker2 1 School of Medicine, University of Adelaide, Faculty of Health Sciences, Adelaide, South Australia, Australia, 2Future Industries Institute, University of South Australia, Mawson Lakes, South Australia, Australia Summary and background: Porous silicon nanoparticles (pSiNPs) are emerging as an exciting drug delivery vehicle in both clinical and therapeutic settings. pSi offers many advantages as a cell targeting and drug delivery vehicle because it provides a non-toxic, biodegradable and biocompatible implantable material that is fully bioresorbable and is readily functionalised with many different chemistries including biomolecules and polymers to create nanocomposites that are effective drug delivery vehicles. Here, a novel method of targeting the dendritic cell (DC) is explored. DCs are the most potent antigen-presenting and rejection-initiating cell, and key to establishing transplant tolerance. Targeting DCs via the uniquely expressed DC-SIGN is a potential target for DCspecific therapy. Here, we utilised pSiNPs harbouring immunosuppressant rapamycin and displaying monoclonal antibody DC-SIGN to observe their effects on DCs. Methods: DCs were derived from human peripheral blood monocytes and cultured with fluorescein isothiocyanate (FITC)-labelled anti-DC-SIGN monoclonal antibodies conjugated to pSiNPs. Uptake of the functionalised pSiNPs was compared to non-functionalised and isotype conjugated NPs. Cell uptake of FITClabelled NPs was assessed by flow cytometry and transmission electron microscopy. Drug loading of the pSiNPs was confirmed with UV and infrared spectroscopy. The phenotype of DCs co-cultured with rapamycin loaded NPs and free rapamycin were compared looking at surface molecule expression of CD40, CD80, CD83, CD209, MHC Class I and MHC Class II. Results: DC-SIGN antibody displaying pSi nanoparticles favourably targeted and were phagocytised by monocyte-derived and myeloid DC in whole blood in a time- and dose-dependent manner. DC preconditioning with rapamycinloaded nanoparticles, resulted in a maturation resistant phenotype and significantly suppressed allogeneic T-cell proliferation. We describe non-human primate models for in vivo testing and homing of the nanoparticles. Conclusions: DC-SIGN antibody displaying pSiNP provide an effective means for delivering immunosuppressive medications directly to a DC population ex vivo in an increased capacity and in a reduced amount of time compared to a non-targeting nanoparticle. 94 DEVELOPING ALLOGENEIC IMMUNOTHERAPY WITH IPSC DERIVED CYTOTOXIC T AND NK CELLS W. Wang, M. Vodyanyk, X. Zhang, A. Brandl, E. Nuwaysir Cellular Dynamics International Inc. - a Fujifilm company, Madison, Wisconsin, United States Using cutting-edge technologies, Cellular Dynamics International (CDI) has pioneered techniques for developing and manufacturing induced pluripotent stem cells (iPSCs) and differentiating them into functional human cells. In combination with superdonor iPSC lines from common HLA haplotypes, we are exploring the potential of iPSC derived T and NK cells as an alternative source for allogeneic cancer immunotherapy. Foot print free iPSCs can also be created from T cells and differentiated back to T cells providing a possibility for generating large number of a rear T cell population. With feeder-free/serum-free culture conditions, we have developed methods for iPSC differentiation to lymphoid CD34+ precursors and their following differentiation to CD3+ T and CD56+CD3- NK cells with >100 × T or NK cells/iPSC yields during 3–4 weeks. iPSC-derived CD3+ T cells contained CD4+ and CD8+ single positive subsets,
expressed TCR α/β, T cell-specific (CD5, CD27), NK-associated and activation markers (CD94, CD161, CD69). When transferred to CD3 activation cultures, iPSC-derived T cells could further be expanded to produce homogeneous proliferating T cell cultures composed of irregularly shaped blasts morphologically similar to primary activated T cells. Further in vitro studies of iPSC-derived T cells demonstrated their Type 1 cytokine secretion profile (IL2, IFNγ TNF), cytotoxic identity (perforin and granzyme B secretion, target cell cytolysis), and iPSC-derived T cells expressing anti-CD19 CAR demonstrated lytic activity against CD19+ targets. Our studies revealed a remarkable potential of iPSCs to be a source of allogeneic cytotoxic T and NK cells generated with a defined culture condition building on CDI’s industrial human cell production platform. Current studies are in progress to demonstrate in vivo function of iPSC-derived CAR T cells for tumor cell rejection. 95 INFUSION OF DONOR LYMPHOCYTES SPECIFICALLY DIRECTED TO MULTIPLE TUMOR ANTIGENS FOR THE TREATMENT OF HIGH RISK PATIENTS AFTER HCT K.M. Williams1, M. Grant1, M. Ismail1, F. Hoq1, M. Martin-Manso1, J. Hoover1, K. Mintz1, A. Namata1, E. Williams1, C. Barese1, S. Albihani1, R. Cruz1, H. Lang1, P. Hanley1, S. Gottschalk2, S. McCurdy3, R. Jones3, C. Bollard1 1 CETI, Children’s National Medical Center, Washington, District of Columbia, United States, 2Texas Children’s Hospital, Houston, Texas, United States, 3Johns Hopkins Hospital, Baltimore, Maryland, United States Background: Relapse of hematologic malignancies after hematopoietic cell transplantation (HCT) confers high mortality because of progressive disease and graftvs.-host disease (GVHD) after immune suppression withdrawal and/or donor lymphocyte infusions. Hence, we hypothesized that tumor-associated antigenspecific T cells (TAA-L), would be specific for multiple tumor-associated antigens diminishing immune escape, and could safely decrease cancer burden in vivo. Methods: We expanded lymphocytes reactive to TAA, including WT1, PRAME, Survivin, which are over-expressed and immunogenic in acute leukemias and lymphomas. On a prospective study: NCT002203902, HCT donor lymphocytes were expanded in the presence of donor-derived antigen-presenting cells with TAA pepmixes. Eligible patients received TAA-L infusions at sequential dose levels: up to 4.0 e7/m2 per dose. Results: Twenty-three donor-derived TAA-L products were generated. The TAA-L products were diverse: predominantly CD4s, CD8s, gdTs, or NKs and lacked alloreactivity in vitro. TAA-L products contained T cells that were predominantly effector memory phenotype, with >30% expressing the activation marker CD69, with <15% of T-cells expressing exhaustion markers (PD1, TIM3, LAG3). Specificity of TAA-L for TAA was evaluated by ELIspot assay with greatest response to PRAME and by interferon gamma capture showing TAAspecific activity in CD4+, CD8+, and CD56+/CD3+ cells. Ten HCT recipients (age range 9–70 years) with relapsed Hodgkin’s disease (n = 2), B cell ALL (n = 3), or AML (n = 5) received 1–2 doses of TAA-L. All patients had relapsed 2–12 months after HCT. Post infusion, there were no adverse events attributed to TAA-L and no GVHD. One patient withdrew prior to completion of followup due to progressive disease in the setting of steroids for sepsis. Responses of evaluable patients (n = 9) were: progressive disease (n = 2), mixed response (n = 1), stable disease (n = 2), CR (n = 3) and PR (n = 1) with only one patient receiving adjunct therapy after TAA-L. Immunosequencing of T cell receptors detected evidence of multiple TCRs derived from the infused TAA-L within 4–5 weeks of infusion. Conclusion: This unique immunotherapeutic has been well tolerated without causing GVHD. Despite aggressive and multiply relapsed disease, 7/9 evaluable patients have demonstrated evidence of disease control after TAA-L infusion, suggesting that TAA-L may have efficacy in relapsed high-risk hematological malignancies after HCT. 96 CORD BLOOD NATURAL KILLER CELLS EXPRESSING A DOMINANT NEGATIVE TGF-B RECEPTOR: IMPLICATIONS FOR ADOPTIVE IMMUNOTHERAPY E. Yvon1, R.A. Burga2, A. Powell2, R. Fernandes2, T. Nguyen3, M. Abdel-Baki4, C. Barese5, C. Cruz2, C. Bollard2 1 Bellicum Pharmaceuticals, Houston, Texas, United States, 2Sheikh Zayed Institute, Children’s National Health System, Washington, District of Columbia, United States, 3MD Anderson Cancer Center, Houston, Texas,
Poster Abstracts
Data derived from the large cohort showed that highly purified NK cell products can be consistently obtained by using 2-step CliniMACS selection. This work can provide a benchmark for use of CliniMACS selection device. It is possible to derive in-process baseline values and control thresholds to alert, monitor and improve selection efficacy. When used in trend evaluation (e.g., LeveyJennings plot) of the performance on cell processing, early investigational action and root cause analysis of the purity and/or recovery of NK cells below a predetermined threshold (e.g., mean - 2SD) can prompt corrective actions to avoid any compromise of patient safety. 93 TARGETED DENDRITIC CELL THERAPY UTILISING ANTIBODY-DISPLAYING POROUS SILICON NANOPARTICLES LOADED WITH RAPAMYCIN S. Stead1, S. McInnes2, P.T. Coates1, N. Voelcker2 1 School of Medicine, University of Adelaide, Faculty of Health Sciences, Adelaide, South Australia, Australia, 2Future Industries Institute, University of South Australia, Mawson Lakes, South Australia, Australia Summary and background: Porous silicon nanoparticles (pSiNPs) are emerging as an exciting drug delivery vehicle in both clinical and therapeutic settings. pSi offers many advantages as a cell targeting and drug delivery vehicle because it provides a non-toxic, biodegradable and biocompatible implantable material that is fully bioresorbable and is readily functionalised with many different chemistries including biomolecules and polymers to create nanocomposites that are effective drug delivery vehicles. Here, a novel method of targeting the dendritic cell (DC) is explored. DCs are the most potent antigen-presenting and rejection-initiating cell, and key to establishing transplant tolerance. Targeting DCs via the uniquely expressed DC-SIGN is a potential target for DCspecific therapy. Here, we utilised pSiNPs harbouring immunosuppressant rapamycin and displaying monoclonal antibody DC-SIGN to observe their effects on DCs. Methods: DCs were derived from human peripheral blood monocytes and cultured with fluorescein isothiocyanate (FITC)-labelled anti-DC-SIGN monoclonal antibodies conjugated to pSiNPs. Uptake of the functionalised pSiNPs was compared to non-functionalised and isotype conjugated NPs. Cell uptake of FITClabelled NPs was assessed by flow cytometry and transmission electron microscopy. Drug loading of the pSiNPs was confirmed with UV and infrared spectroscopy. The phenotype of DCs co-cultured with rapamycin loaded NPs and free rapamycin were compared looking at surface molecule expression of CD40, CD80, CD83, CD209, MHC Class I and MHC Class II. Results: DC-SIGN antibody displaying pSi nanoparticles favourably targeted and were phagocytised by monocyte-derived and myeloid DC in whole blood in a time- and dose-dependent manner. DC preconditioning with rapamycinloaded nanoparticles, resulted in a maturation resistant phenotype and significantly suppressed allogeneic T-cell proliferation. We describe non-human primate models for in vivo testing and homing of the nanoparticles. Conclusions: DC-SIGN antibody displaying pSiNP provide an effective means for delivering immunosuppressive medications directly to a DC population ex vivo in an increased capacity and in a reduced amount of time compared to a non-targeting nanoparticle. 94 DEVELOPING ALLOGENEIC IMMUNOTHERAPY WITH IPSC DERIVED CYTOTOXIC T AND NK CELLS W. Wang, M. Vodyanyk, X. Zhang, A. Brandl, E. Nuwaysir Cellular Dynamics International Inc. - a Fujifilm company, Madison, Wisconsin, United States Using cutting-edge technologies, Cellular Dynamics International (CDI) has pioneered techniques for developing and manufacturing induced pluripotent stem cells (iPSCs) and differentiating them into functional human cells. In combination with superdonor iPSC lines from common HLA haplotypes, we are exploring the potential of iPSC derived T and NK cells as an alternative source for allogeneic cancer immunotherapy. Foot print free iPSCs can also be created from T cells and differentiated back to T cells providing a possibility for generating large number of a rear T cell population. With feeder-free/serum-free culture conditions, we have developed methods for iPSC differentiation to lymphoid CD34+ precursors and their following differentiation to CD3+ T and CD56+CD3- NK cells with >100 × T or NK cells/iPSC yields during 3–4 weeks. iPSC-derived CD3+ T cells contained CD4+ and CD8+ single positive subsets,
expressed TCR α/β, T cell-specific (CD5, CD27), NK-associated and activation markers (CD94, CD161, CD69). When transferred to CD3 activation cultures, iPSC-derived T cells could further be expanded to produce homogeneous proliferating T cell cultures composed of irregularly shaped blasts morphologically similar to primary activated T cells. Further in vitro studies of iPSC-derived T cells demonstrated their Type 1 cytokine secretion profile (IL2, IFNγ TNF), cytotoxic identity (perforin and granzyme B secretion, target cell cytolysis), and iPSC-derived T cells expressing anti-CD19 CAR demonstrated lytic activity against CD19+ targets. Our studies revealed a remarkable potential of iPSCs to be a source of allogeneic cytotoxic T and NK cells generated with a defined culture condition building on CDI’s industrial human cell production platform. Current studies are in progress to demonstrate in vivo function of iPSC-derived CAR T cells for tumor cell rejection. 95 INFUSION OF DONOR LYMPHOCYTES SPECIFICALLY DIRECTED TO MULTIPLE TUMOR ANTIGENS FOR THE TREATMENT OF HIGH RISK PATIENTS AFTER HCT K.M. Williams1, M. Grant1, M. Ismail1, F. Hoq1, M. Martin-Manso1, J. Hoover1, K. Mintz1, A. Namata1, E. Williams1, C. Barese1, S. Albihani1, R. Cruz1, H. Lang1, P. Hanley1, S. Gottschalk2, S. McCurdy3, R. Jones3, C. Bollard1 1 CETI, Children’s National Medical Center, Washington, District of Columbia, United States, 2Texas Children’s Hospital, Houston, Texas, United States, 3Johns Hopkins Hospital, Baltimore, Maryland, United States Background: Relapse of hematologic malignancies after hematopoietic cell transplantation (HCT) confers high mortality because of progressive disease and graftvs.-host disease (GVHD) after immune suppression withdrawal and/or donor lymphocyte infusions. Hence, we hypothesized that tumor-associated antigenspecific T cells (TAA-L), would be specific for multiple tumor-associated antigens diminishing immune escape, and could safely decrease cancer burden in vivo. Methods: We expanded lymphocytes reactive to TAA, including WT1, PRAME, Survivin, which are over-expressed and immunogenic in acute leukemias and lymphomas. On a prospective study: NCT002203902, HCT donor lymphocytes were expanded in the presence of donor-derived antigen-presenting cells with TAA pepmixes. Eligible patients received TAA-L infusions at sequential dose levels: up to 4.0 e7/m2 per dose. Results: Twenty-three donor-derived TAA-L products were generated. The TAA-L products were diverse: predominantly CD4s, CD8s, gdTs, or NKs and lacked alloreactivity in vitro. TAA-L products contained T cells that were predominantly effector memory phenotype, with >30% expressing the activation marker CD69, with <15% of T-cells expressing exhaustion markers (PD1, TIM3, LAG3). Specificity of TAA-L for TAA was evaluated by ELIspot assay with greatest response to PRAME and by interferon gamma capture showing TAAspecific activity in CD4+, CD8+, and CD56+/CD3+ cells. Ten HCT recipients (age range 9–70 years) with relapsed Hodgkin’s disease (n = 2), B cell ALL (n = 3), or AML (n = 5) received 1–2 doses of TAA-L. All patients had relapsed 2–12 months after HCT. Post infusion, there were no adverse events attributed to TAA-L and no GVHD. One patient withdrew prior to completion of followup due to progressive disease in the setting of steroids for sepsis. Responses of evaluable patients (n = 9) were: progressive disease (n = 2), mixed response (n = 1), stable disease (n = 2), CR (n = 3) and PR (n = 1) with only one patient receiving adjunct therapy after TAA-L. Immunosequencing of T cell receptors detected evidence of multiple TCRs derived from the infused TAA-L within 4–5 weeks of infusion. Conclusion: This unique immunotherapeutic has been well tolerated without causing GVHD. Despite aggressive and multiply relapsed disease, 7/9 evaluable patients have demonstrated evidence of disease control after TAA-L infusion, suggesting that TAA-L may have efficacy in relapsed high-risk hematological malignancies after HCT. 96 CORD BLOOD NATURAL KILLER CELLS EXPRESSING A DOMINANT NEGATIVE TGF-B RECEPTOR: IMPLICATIONS FOR ADOPTIVE IMMUNOTHERAPY E. Yvon1, R.A. Burga2, A. Powell2, R. Fernandes2, T. Nguyen3, M. Abdel-Baki4, C. Barese5, C. Cruz2, C. Bollard2 1 Bellicum Pharmaceuticals, Houston, Texas, United States, 2Sheikh Zayed Institute, Children’s National Health System, Washington, District of Columbia, United States, 3MD Anderson Cancer Center, Houston, Texas,