Christiane Geiger

Generation of Th1-Polarizing Dendritic Cells Using the TLR7/8 Agonist CL075

In this paper, we describe a new method for preparation of human dendritic cells (DCs) that secrete bioactive IL-12(p70) using synthetic immunostimulatory compounds as TLR7/8 agonists. Monocyte-derived DCs were generated using a procedure that provided mature cells within 3 d. Several maturation mixtures that contained various cytokines, IFN-γ, different TLR agonists, and PGE2 were compared for impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation. Mixtures that included the TLR7/8 agonists R848 or CL075, combined with the TLR3 agonist polyinosinic:polycytidylic acid, yielded 3-d mature DCs that secreted high levels of IL-12(p70), showed strong chemotaxis to CCR7 ligands, and had a positive costimulatory potential. They also had excellent capacity to activate NK cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and to induce T cell-mediated cytotoxic function. Thereby, mature DCs prepared within 3 d using such maturation mixtures displayed optimal functions required for vaccine development.

Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

BackgroundAntigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.MethodsIn this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.ResultsAlthough they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivt)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivt RNA.ConclusionsThis new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivt RNA as sources of specific antigen.

A generic RNA-pulsed dendritic cell vaccine strategy for renal cell carcinoma

We present a generic dendritic cell (DC) vaccine strategy for patients with renal cell carcinoma (RCC) based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs). Instead of preparing RNA from tumor tissue of each individual RCC patient, we propose to substitute RNA prepared from a well characterized highly immunogenic RCC cell line (RCC-26 tumor cells) as a generic source of TAAs for loading of DCs. We demonstrate here that efficient RNA transfer can be achieved using lipofection of immature DCs, which are subsequently matured with a cytokine cocktail to express high levels of MHC and costimulatory molecules as well as the chemokine receptor CCR7. Neither RNA itself nor the lipid component impacted on the phenotype or the cytokine secretion of mature DCs.Following RNA loading, DCs derived from HLA-A2-positive donors were able to activate effector-memory cytotoxic T lymphocytes (CTLs) specific for a TAA ligand expressed by the RCC-26 cell line. CTL responses to RNA-loaded DCs reached levels comparable to those stimulated directly by the RCC-26 tumor cells. Furthermore, DCs expressing tumor cell RNA primed naïve T cells, yielding T cell lines with cytotoxicity and cytokine secretion after contact with RCC tumor cells. RCC-26 cell lines are available as good manufacturing practice (GMP)-certified reagents enabling this source of RNA to be easily standardized and adapted for clinical testing. In addition, well defined immune monitoring tools, including the use of RNA expressing B cell lines, are available. Thus, this DC vaccine strategy can be directly compared with an ongoing gene therapy trial using genetically-engineered variants of the RCC-26 cell line as vaccines for RCC patients with metastatic disease.

Cell-based vaccines for renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells

Initial vaccine developments for renal cell carcinoma (RCC) have concentrated on cell-based approaches in which tumor cells themselves provide mixtures of unknown tumor-associated antigens as immunizing agents. Antigens derived from autologous tumors can direct responses to molecular composites characteristic of individual tumors, whereas antigens derived from allogeneic tumor cells must be commonly shared by RCC. Three types of cell-based vaccine for RCC have been investigated: isolated tumor cell suspensions, gene modified tumor cells and dendritic cells (DCs) expressing RCC-associated antigens. Approaches using genetic modification of autologous RCC have included ex vivo modification of tumor cells or modification of tumors in vivo. We have used gene-modification of allogeneic tumor cell lines to create generic RCC vaccines. More recently, emphasis has shifted to the use of DCs as cell-based vaccines for RCC. DCs have moved to a position of central interest because of their excellent stimulatory capacity, combined with their ability to process and present antigens to both naive CD4 and CD8 cells. The long impasse in identifying molecular targets for specific immunotherapy of RCC is now rapidly being overcome through the use of tools and information emerging from human genome research. Identification of candidate molecules expressed by RCC using cDNA arrays, combined with protein arrays and identification of peptides presented by MHC molecules, allow specific vaccines to be tailored to the antigenic profile of individual tumors, providing the basis for development of patient-specific vaccines.

Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

BackgroundActive dendritic cell (DC) immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML). Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR) agonists in intensively pretreated patients with AML.MethodsFour different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls.ResultsMaturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C), induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70), showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8+ T cells.ConclusionsOur results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses.

Harnessing innate and adaptive immunity for adoptive cell therapy of renal cell carcinoma

The development of immunotherapies for renal cell carcinoma (RCC) has been the subject of research for several decades. In addition to cytokine therapy, the benefit of various adoptive cell therapies has again come into focus in the past several years. Nevertheless, success in fighting this immunogenic tumor is still disappointing. RCC can attract a multitude of different effector cells of both the innate and adaptive immune system, including natural killer (NK) cells, γδ T cells, NK-like T cells, peptide-specific T cells, dendritic cells (DC), and regulatory T cells (Tregs). Based on intensive research on the biology and function of different immune cells, we now understand that individual cell types do not act in isolation but function within a complex network of intercellular interactions. These interactions play a pivotal role in the efficient activation and function of effector cells, which is a prerequisite for successful tumor elimination. This review provides a current overview of the diversity of effector cells having the capacity to recognize RCC. Aspects of the functions and anti-tumor properties that make them attractive candidates for adoptive cell therapies, as well as experience in clinical application are discussed. Improved knowledge of the biology of this immune network may help us to effectively harness various effector cells, placing us in a better position to develop new therapeutic strategies to successfully fight RCC.

Generation of allo-restricted peptide-specific T cells using RNA-pulsed dendritic cells: A three phase experimental procedure

Designer T cells expressing transgenic T cell receptors (TCR) with anti-tumor specificity offer new treatment options for cancer patients. We developed a three phase procedure to identify T cells of high avidity based on the fact that T cells recognizing peptides presented by allogeneic MHC efficiently kill tumor cells. Autologous dendritic cells (DC) are co-transfected with ivt-RNA encoding an allogeneic MHC molecule and a selected antigen to allow them to express allogeneic MHC-peptide complexes that activate allo-restricted peptide-specific T cells. This approach provides great flexibility for obtaining high-avidity T cells as potential sources of TCR for adoptive T cell therapy.

A novel and effective method to generate human porcine-specific regulatory T cells with high expression of IL-10, TGF-β1 and IL-35

Organ transplantation remains the most effective treatment for patients with late stage organ failure. Transgenic pigs provide an alternative organ donor source to the limited availability of human organs. However, cellular rejection still remains to be the obstacle for xenotransplantation. Superior to other methods, antigen-specific regulatory T cells (Treg) alleviate cellular rejection with fewer side effects. Here we demonstrate the use of a fast method to provide tolerogenic dendritic cells (tolDC) that can be used to generate effective porcine-specific Treg cells (PSTreg). TolDC were produced within three days from human monocytes in medium supplemented with anti-inflammatory cytokines. Treg were generated from naïve CD4+ T cells and induced to become PSTreg by cocultivation with porcine-antigen-loaded tolDC. Results showed that PSTreg exhibited the expected phenotype, CD4+CD25+CD127low/− Foxp3+, and a more activated phenotype. The specificity of PSTreg was demonstrated by suppression of effector T cell (Teff) activation markers of different stages and inhibition of Teff cell proliferation. TolDC and PSTreg exhibited high expression of IL-10 and TGF-β1 at both protein and RNA levels, and PSTreg also highly expressed IL-35 at RNA levels. Upon restimulation, PSTreg retained the activated phenotype and specificity. Taken together, the newly developed procedure allows efficient generation of highly suppressive PSTreg.

Next-generation dendritic cells for immunotherapy of acute myeloid leukemia

Post-remission therapy of patients with acute myeloid leukemia (AML) is critical for the elimination of minimal residual disease (MRD) and a prerequisite for achieving cure. Cellular immunotherapy is a highly effective treatment option as demonstrated by the low relapse rate after allogeneic stem cell transplantation (SCT). However, many patients are not eligible for this treatment. Therapeutic vaccination with autologous dendritic cells (DCs) is a promising strategy to induce anti-cancer immune responses. We have developed a GMP-compliant protocol for the generation of next-generation DCs. A short 3-day differentiation period is combined with a novel maturation cocktail including a TLR7/8 agonist, resulting in DCs characterized by a positive co-stimulatory profile, high production of IL-12p70, polarization of T helper cells into Th1 and efficient stimulation of cytotoxic T lymphocytes and NK cells. In a current proof-of-concept Phase I/II clinical trial we evaluate next-generation DCs as post-remission therapy for AML patients with a non-favorable risk profile (NCT01734304). Standard exclusion criteria apply, and patients have to be ineligible for allogeneic SCT. DCs are generated from patients´ monocytes and loaded with RNA encoding the leukemia-associated antigens WT1, PRAME or CMVpp65 as an adjuvant and surrogate antigen. Patients are vaccinated intradermally with 5x106 DCs of each of the three different batches up to 10 times within 26 weeks. Primary endpoints are feasibility and safety, and secondary endpoints include immune responses and disease control with a particular focus on MRD conversion. Phase I will include 6 patients and Phase II another 14 patients. So far, three patients have been enrolled and two of them have been vaccinated for at least six times each. DCs fulfilled all quality criteria (phenotype, viability, sterility, cell count, purity), and after thawing maintained their positive co-stimulatory profile as well as their capacity to secrete high amounts of IL-12p70. DCs expressed all three antigens and were able to induce a selective T cell response in vitro, suggesting proper antigen processing and presentation. In both vaccinated patients delayed type hypersensitivity reactions developed. Apart from erythema and itching at the injection site, no higher grade adverse events have been observed. As of yet, both patients are relapse-free and MRD-negative. Up-to-date clinical and immunomonitoring data including evaluation of T and NK cell activation and specific T cell responses will be presented.

P55. Dendritic cell vaccination for postremission therapy in AML.

Cellular immunotherapy is a highly effective treatment option for patients with acute myeloid leukaemia (AML) as shown by the low relapse rate after allogeneic stem cell transplantation. However, many patients are not eligible for this treatment. This has lead to the development of various immunotherapeutic approaches that aim at inducing autologous cellular and humoral immune responses against AML and specifically against residual leukaemic stem cells (LSCs). Dendritic cells (DCs) are important regulators of the human immune response. We have developed a three-day DC manufacturing protocol that starts with peripheral blood monocytes, e.g. from AML patients in remission following intensive chemotherapy. By using a cytokine cocktail containing a synthetic TLR7/8 agonist, the resulting DCs develop improved immunogenicity. For healthy donors as well as for AML patients, we were able to show that these DCs display a positive costimulatory profile, secrete high levels of IL-12p70, show chemotaxis to CCR7 ligands, and activate NK cells. After loading the DCs with mRNA, they effectively induce antigen-specific T cell responses with a strong type-1 polarization. Due to these properties, this DC type seems highly suitable for application in cancer immunotherapy. We have recently initiated a phase I/II clinical trial for the application of these DCs in the setting of AML postremission strategy. WT1 and PRAME were chosen as leukaemia-associated antigens due to their overexpression on leukaemic blasts and specifically on cells that are enriched for LSCs. DCs transfected with mRNA encoding CMV-pp65 are included into the vaccine as an adjuvant as well as a surrogate antigen. 20 patients with a non-favourable risk profile or with confirmed minimal residual disease (MRD), but who are not eligible for allogeneic stem cell transplantation, will be included. The primary objective of this study is to evaluate feasibility and safety of this immunotherapeutic approach. Important secondary endpoints are immune responses to the applied antigens and MRD control. First results of this study will be presented.