Kevin Nishimoto

Generation of immunogenic dendritic cells from human embryonic stem cells without serum and feeder cells.

AIM Dendritic cell (DC)-based vaccines have a potential utility for use in the treatment of malignancy. Human embryonic stem cells (hESCs) may provide a more cost-effective and reliable source of DCs for immunotherapy purposes, providing on-demand access for patients. METHOD We developed a protocol to generate DCs from hESCs in vitro in the absence of serum and feeder cells. This protocol uses growth factors bone morphogenetic protein-4, granulocyte macrophage-colony stimulating factor (GM-CSF), stem cell factor and VEGF in serum-free media to generate hESC-derived monocytic cells. These cells are further differentiated to hESC-derived immature DCs with GM-CSF and IL-4, and matured to hESC-derived mature DCs with a maturation cocktail consisting of GM-CSF, TNF-alpha, IL-1beta, IFN-gamma and PGE2. RESULTS This study demonstrates the applicability of our defined differentiation process in generating functional hESC-derived DCs from multiple hESC lines. We show that hESC-derived immature DCs phagocytose, process, and present antigen upon maturation. hESC-derived mature DCs express the maturation marker CD83, produce Th1-directing cytokine IL-12p70, migrate in response to chemokine, and activate both viral and tumor antigen-specific T-cell responses. CONCLUSION We developed a chemically defined system to generate unlimited numbers of DCs from hESCs. Our results demonstrate that hESC-derived DCs generated from this process are immunogenic and have the potential to be used for DC immunotherapy.

Gangliosides as targets for immunotherapy for pancreatic adenocarcinoma

Pancreatic adenocarcinoma cells express gangliosides and sialyl Lewis (sLe) antigens. It is not known whether these carbohydrate antigens can be targeted by immunotherapy. The authors measured the expression of GM2 and sLe antigens on the surface of pancreatic carcinoma cells and the serum levels of total gangliosides, GM2, and antiganglioside antibodies in patients with pancreatic carcinoma.

Differentiation of dendritic cells from human embryonic stem cells.

Improving our understanding of the interactions between human dendritic cells (DCs) and T cells may contribute to the development of therapeutic strategies for a variety of immune-mediated disorders. The possibility of using DCs themselves as tools to manipulate immune responses opens even greater therapeutic avenues. Current methods of generating human DCs are both inadequate and susceptible to high levels of variability between individuals. DCs differentiated from human embryonic stem cells (hESCs) could provide a more reliable, consistent solution. DCs have now successfully been differentiated from hESCs and more recently this has been repeated using protocols that avoid the inclusion of animal products, an important modification for clinical use. We have developed a novel method for the generation of DCs from hESCs in the absence of animal products that does not necessitate a separate embryoid body (EB) generation step. The technique involves the use of four growth factors and their successive removal from culture, resulting in accumulation of DCs with phenotypic, morphological, and immunostimulatory properties comparable to those of classical human monocyte-derived DCs. In addition to the application of hESC-derived DCs in basic research and novel approaches to cancer immunotherapy, they may also play a central role in the field of regenerative medicine. Tolerogenic DCs differentiated from hESCs may be used to persuade the immune system of the recipients of cell replacement therapy to tolerate allogeneic tissues differentiated from the same hESC line. Such an approach may help to address the immunological barriers that threaten to derail the clinical application of hESCs.

Immune responses and long-term disease recurrence status after telomerase-based dendritic cell immunotherapy in patients with acute myeloid leukemia

Telomerase activity in leukemic blasts frequently is increased among patients with high‐risk acute myeloid leukemia (AML). In the current study, the authors evaluated the feasibility, safety, immunogenicity, and therapeutic potential of human telomerase reverse transcriptase (hTERT)‐expressing autologous dendritic cells (hTERT‐DCs) in adult patients with AML.

Rapamycin conditioning of dendritic cells differentiated from human ES cells promotes a tolerogenic phenotype.

While human embryonic stem cells (hESCs) may one day facilitate the treatment of degenerative diseases requiring cell replacement therapy, the success of regenerative medicine is predicated on overcoming the rejection of replacement tissues. Given the role played by dendritic cells (DCs) in the establishment of immunological tolerance, we have proposed that DC, rendered tolerogenic during their differentiation from hESC, might predispose recipients to accept replacement tissues. As a first step towards this goal, we demonstrate that DC differentiated from H1 hESCs (H1-DCs) are particularly responsive to the immunosuppressive agent rapamycin compared to monocyte-derived DC (moDC). While rapamycin had only modest impact on the phenotype and function of moDC, H1-DC failed to upregulate CD40 upon maturation and displayed reduced immunostimulatory capacity. Furthermore, coculture of naïve allogeneic T cells with rapamycin-treated H1-DC promoted an increased appearance of CD25hi Foxp3+ regulatory T cells, compared to moDC. Our findings suggest that conditioning of hESC-derived DC with rapamycin favours a tolerogenic phenotype.

Modification of human embryonic stem cell-derived dendritic cells with mRNA for efficient antigen presentation and enhanced potency

AIM Dendritic cell (DC)-based vaccines are designed to exploit the intrinsic capacity of these highly effective antigen presenting cells to prime and boost antigen-specific T-cell immune responses. Successful development of DC-based vaccines will be dependent on the ability to utilize and harness the full potential of these potent immune stimulatory cells. Recent advances to generate DCs derived from human embryonic stem cells (hESCs) that are suitable for clinical use represent an alternative strategy from conventional approaches of using patient-specific DCs. Although the differentiation of hESC-derived DCs in serum-free defined conditions has been established, the stimulatory potential of these hESC-derived DCs have not been fully evaluated. METHODS hESC-derived DCs were differentiated in serum-free defined culture conditions. The delivery of antigen into hESC-derived DCs was investigated using mRNA transfection and replication-deficient adenoviral vector transduction. hESC-derived DCs modified with antigen were evaluated for their capacity to stimulate antigen-specific T-cell responses with known HLA matching. Since IL-12 is a key cytokine that drives T-cell function, further enhancement of DC potency was evaluated by transfecting mRNA encoding the IL-12p70 protein into hESC-derived DCs. RESULTS The transfection of mRNA into hESC-derived DCs was effective for heterologous protein expression. The efficiency of adenoviral vector transduction into hESC-derived DCs was poor. These mRNA-transfected DCs were capable of stimulating human telomerase reverse transcriptase antigen-specific T cells composed of varying degrees of HLA matching. In addition, we observed the transfection of mRNA encoding IL-12p70 enhanced the T-cell stimulation potency of hESC-derived DCs. CONCLUSION These data provide support for the development and modification of hESC-derived DCs with mRNA as a potential strategy for the induction of T-cell-mediated immunity.

276. Long-Term Relapse-Free Survival of Patients with Acute Myeloid Leukemia (AML) Receiving a Telomerase-Engineered Dendritic Cell Immunotherapy

There are few treatment options for patients with intermediate and high risk AML, and remission and relapse rates are dismal especially in patients ≥60 years old. A Phase 2 clinical trial was conducted in subjects with AML to assess a dendritic cell immunotherapy (AST-VAC1) engineered to express a modified form of telomerase that is processed through both the MHC Class I and II antigen presentation pathways. AST-VAC1 was prepared from leukapheresis collections from 33 subjects and were transfected with an mRNA encoding near full length telomerase (hTERT) modified with a lysosomal sorting signal, LAMP-1, which enhances presentation to both CD4+ helper and CD8+ cytotoxic T cells. hTERT is essential for maintaining the extended proliferative lifespan of tumor cells. AML patients were enrolled if they were in complete remission (CR1 or CR2) with intermediate or high risk cytogenetics. AST-VAC1 was prepared after induction therapy and before or after completion of consolidation cycles. AST-VAC1 containing 1 × 107 cells was administered as 6 weekly followed by 6 biweekly intradermal injections. If AST-VAC1 doses were available, patients were eligible to receive additional monthly boosts. Twenty one patients (median age: 55) in complete remission (16 CR1 and 3 CR2) or early relapse (2) received at least 3 injections of AST-VAC1. Only one Grade 3 or 4 adverse event, (idiopathic thrombocytopenia), possibly related to the immunotherapy was observed. The majority of adverse events were transient including headache, fatigue, erythema and rash. The two patients who were vaccinated during early relapse progressed rapidly and did not receive the full dosing regimen of AST-VAC1. Of the 19 patients that were in CR, 13 received all 12 doses of AST-VAC1. Fifty-eight percent (11/19) developed T cell immune responses to hTERT as assessed by ELISpot analysis. Eleven of 19 patients (median follow-up 52 mos.) were in remission as of last follow-up; seven developed detectable cellular immune responses to hTERT. Of the 19 CR patients, 7 were ≥ 60 years old at the time of AST-VAC1 immunotherapy. Four of 7 patients ≥ 60 years old remained relapse free 52-59 months post AST-VAC1 immunotherapy with all four developing immune responses to hTERT. The three patients that received AST-VAC1 while in CR2 were in remission as of their last follow-up of 24, 50 and 59 months with two having hTERT immune responses. The median duration of complete remission was greater than that observed in historical controls especially for patients ≥ 60 years old where relapse-free survival at 4 years is typically 5-20%. The results suggest that immunotherapy with AST-VAC1 is safe, can stimulate immune responses to telomerase, and may extend relapse-free survival even in patients with high risk AML.

222. AST-VAC2: An Embryonic Stem Cell-Derived Dendritic Cell Cancer Immunotherapy

AST-VAC2 is a cancer immunotherapy product comprising embryonic stem cell-derived dendritic cells electroporated with an mRNA encoding a telomerase/lysosome-associated membrane protein 1 (LAMP hTERT) chimeric tumor antigen. Previous studies have shown that AST-VAC2 can elicit a telomerase-specific T-cell response from partially HLA-matched donor peripheral blood mononuclear cells. A related product (AST-VAC1), an autologous dendritic cell/LAMP hTERT cancer immunotherapy, has been tested in clinical trials on patients with prostate cancer or acute myeloid leukemia. The AST-VAC1 treatment was well tolerated and generated anti-telomerase immune responses. AST-VAC2 is being developed as an off-the-shelf allogeneic cancer vaccine for clinical testing in partially HLA matched patients with non-small cell lung carcinoma. The AST-VAC2 product is derived from the H1 human embryonic stem cell line. The stem cells are differentiated into mature dendritic cells in a multi-step process via embryoid bodies, prepared growth surfaces and specific growth factors. The differentiation process requires 34 days in culture. The cells are then electroporated with the LAMP hTERT mRNA and irradiated prior to cryopreservation. In preparation for clinical testing the manufacturing process has been scaled up from T-flasks to large surface area cell stacks. This has required a number of novel process modifications including large scale embryoid body filtration and flow-through electroporation. The final product will be rendered incapable of replication by gamma irradiation. The radiation dose required for halting replication while still preserving antigen presentation was determined using a combination colony formation/potency assay. It is anticipated that the proposed Phase 1/2a clinical trial will enroll approximately 30 patients and will include both a dose escalation and a broadening of inclusion criteria to permit an assessment of safety, toxicity and immunogenicity in patients with advanced disease. Asterias has recently partnered with Cancer Research United Kingdom (CRUK) and the University of Southampton for the GMP manufacturing and initial clinical testing of AST-VAC2 in the UK.