Poly I: C-activated dendritic cells that were generated in CellGro for use in cancer immunotherapy trials

<p>Abstract</p> <p>Background</p> <p>For clinical applications, dendritic cells (DCs) need to be generated using GMP-approved reagents. In this study, we tested the characteristics of DCs generated in two clinical grade culture media and activated by three maturation stimuli, Poly I: C, LPS and the mixture of proinflammatory cytokines in order to identify the optimal combination of culture media and activation stimulus for the clinical use.</p> <p>Method</p> <p>We tested DCs generation using two GMP-certified culture media, CellGro and RPMI+5% human AB serum and evaluated DCs morphology, viability and capapability to mature. We tested three maturation stimuli, PolyI:C, LPS and the mixture of proinflammatory cytokines consisting of IL-1, IL-6, TNF and prostaglandin E2. We evaluated the capacity of activated DCs to induce antigen-specific T cells and regulatory T lymphocytes.</p> <p>Results</p> <p>Cell culture in CellGro resulted in a higher yield of immature DCs resulting from increased number of adherent monocytes. DCs that were generated in CellGro and activated using Poly I:C were the most efficient in expanding antigen-specific T cells compared to the DCs that were generated in other media and activated using LPS or the cocktail of proinflammatory cytokines. A comparison of all tested combinations revealed that DCs that were generated in CellGro and activated using Poly I:C induced low numbers of regulatory T cells.</p> <p>Conclusion</p> <p>In this study, we identified monocyte-derived DCs that were generated in CellGro and activated using Poly I:C as the most potent clinical-grade DCs for the induction of antigen-specific T cells.</p

Immunogenic cell death and its application in imunotherapy protocols

Treatment of tumors by protocols based on combination of surgery, radiotherapy and systemic chemotherapy resulted in the improved prognosis of many human cancers. Despite the continuous introduction of new drugs and further improvements of chemotherapy protocols, it's likely that at some point chemotherapy will reach its limits and clinical efficacy will plateau. Immunotherapy has emerged as another treatment modality with the potential to contribute to further improvements in the survival. The products of advanced cellular terapies must be generated using GMP-approved reagents and number of studies have addressed the need to generate large numbers of DCs for clinical trials according to regulatory authorities and to current legislation. The rising knowledge about dendritic cells (DCs) in the immune response against tumors and the ability to prepaire DCs in vitro leaded to the establishment of immunotherapy protocols. Breakthrough studies that identified markers of immunogenic tumor cell death after chemotherapy treatment challenge the long-time perception of chemotherapy and immunotherapy as opposing and incompatible treatment modalities. My PhD thesis is a contributiont to this topic. In the first paper we identified monocyte-derived DCs that were generated in CellGro and activated using PolyI:C as the..

Preparation of anti-cancer vaccine / / immunotherapy for ovarian cancer under conditions of good manufacturing practice

Generation of clinical-grade dendritic cell-based vaccine for immunotherapy of ovarian cancer Introduction: Dendritic cells (DCs) the most potent antigen presenting cells. Recent technological advances allow for generation of large numbers of DCs from peripheral blood monocytes. Administration of activated DCs loaded with tumor antigens is thus an attractive approach for immunotherapy of cancer. Prerequisite for the initiation of clinical trials in cancer immunotherapy is the development of protocols for DC-based vaccine generation according to Good Manufacturing Practice (GMP) conditions. Aim of the study: Development of protocol for the generation of clinical grade vaccine based on activated DCs loaded with killed tumor cells for use in the immunotherapy of ovarian cancer. Materials and methods: Immature DCs were generated from peripheral blood monocytes of healthy donors. We tested Cell Gro and RPMI+5% pooled human serum (5% PHS) as clinical grade culture media. Immature DCs were then activated by three distinct stimuli (Poly I:C, LPS and cocktail of proinflammatory cytokines (TNF, IL-1 and IL-6)). Activated DCs were evaluated for their phenotypic and functional characteristics and for their capacity to activate antigen specific and/or regulatory T cells. Results: Culture of monocytes in Cell Gro yielded..

Preparation of anti-cancer vaccine / / immunotherapy for ovarian cancer under conditions of good manufacturing practice

Generation of clinical-grade dendritic cell-based vaccine for immunotherapy of ovarian cancer Introduction: Dendritic cells (DCs) the most potent antigen presenting cells. Recent technological advances allow for generation of large numbers of DCs from peripheral blood monocytes. Administration of activated DCs loaded with tumor antigens is thus an attractive approach for immunotherapy of cancer. Prerequisite for the initiation of clinical trials in cancer immunotherapy is the development of protocols for DC-based vaccine generation according to Good Manufacturing Practice (GMP) conditions. Aim of the study: Development of protocol for the generation of clinical grade vaccine based on activated DCs loaded with killed tumor cells for use in the immunotherapy of ovarian cancer. Materials and methods: Immature DCs were generated from peripheral blood monocytes of healthy donors. We tested Cell Gro and RPMI+5% pooled human serum (5% PHS) as clinical grade culture media. Immature DCs were then activated by three distinct stimuli (Poly I:C, LPS and cocktail of proinflammatory cytokines (TNF, IL-1 and IL-6)). Activated DCs were evaluated for their phenotypic and functional characteristics and for their capacity to activate antigen specific and/or regulatory T cells. Results: Culture of monocytes in Cell Gro yielded..

Studies of CD44 glycoprotein activation by using the specific monoclonal antibody.

Acute myeloid leukemia (AML) is an heterogenous leukemia characterized by the blockage of myeloid differentiation at different stages. The ligation of CD44 with specifics monoclonal antibodies (MAbs) (A3D8 and H90), triggers terminal differentiation of leukemic blasts in AML-M1/2 to AML-M5 subtypes. The CD44 surface antigen is the most important hyaluronic acid receptor and is involved in cell adhesion and migration. To investigate the mechanism of myeloid differentiation due to CD44 specific monoclonal antibodies, and to confirmed our hypothesis on involvement of cleavage of intracellular domain of CD44 (ICD) and migration of ICD in the nucleus, we have used the THP-1 (AML-M5 subtype) cell line and the CHO cell line transfected with a CD44 molecule tagged in the C-terminal with the Myc Tag (CD44-Myc). In the first step we have purified ascite H90 MAb by affinity Protein G chromatography, followed by SDS PAGE electrophoresis to analyse the purity of MAb. In second step, we have performed differentiation assays of THP-1 to test the efficacity of the MAb. THP-1 and CHO cells have been selected for the highest CD44-Myc Tag expression after G418 antibiotic (geneticin) selection using FACS analysis. To analyse ICD Tagged (ICD-Myc) localisation in cells, we have first performed analysis of nuclear and cytoplasmic..

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine

Classification of current anticancer immunotherapies.

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into passive and active based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches. Oncotarget 2014 Dec 20; 5(24):12472-508