Biomedical subjects

Eric Angevin

Publications and source records attributed to Eric Angevin.

15 recordsLinked to original sources

PubMed2005

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.

BACKGROUND: DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome. PATIENTS AND METHODS: Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 x 1014 molecules) or peptides (10 versus 100 mug/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression. RESULTS: The GMP process allowed to harvest about 5 x 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood. CONCLUSION: The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.

Journal Article↗

PubMed2004

IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules.

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-alpha, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DC(GM/IL-4)) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DC(GM/IL-4) capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Analysis of the natural killer mediated immune response in metastatic renal cell carcinoma patients.

Metastatic renal cell carcinomas (MRCC) are considered as immunogeneic tumors on the basis of the clinical responses observed in patients treated by IL-2. However, renal cell carcinoma patients are also characterized by alterations of the immune response that may compromise the immunotherapeutic approaches. In our study, we have studied the phenotype and the functional capacities of peripheral NK cells in a panel of neprectomized metastatic renal cell carcinoma patients. NK cells were harvested by negative immunoselection from fresh peripheral blood samples. In most of MRCC patients analysed (23/28), the expression of NCR (NKp46 and NKp30) was similar to that of donors. Lytic capacities by activated immunoselected NK cells from MRCC patients assessed against K562 and 3 renal tumor cell lines were in the range of that observed in NK cells from normal donors. HLA-I- renal tumor cells UOK23 were killed with a good efficiency, whereas HLA-I renal tumor cells were more resistant. Although LFA-1/ICAM-1 interaction potentiates RCC cell lysis, HLA-I/NKR interaction clearly decreased RCC cell susceptibility to NK cells. In addition, proliferation of NK cells from MRCC patients in response to cytokines was altered.

Aged↗

PubMed2004

Selective accumulation of mature DC-Lamp+ dendritic cells in tumor sites is associated with efficient T-cell-mediated antitumor response and control of metastatic dissemination in melanoma.

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

Adult↗

PubMed2004

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells.

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.

Animals↗

PubMed2004

Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection.

Ideal vaccines should be stable, safe, molecularly defined, and out-of-shelf reagents efficient at triggering effector and memory Ag-specific T cell-based immune responses. Dendritic cell-derived exosomes could be considered as novel peptide-based vaccines because exosomes harbor a discrete set of proteins, bear functional MHC class I and II molecules that can be loaded with synthetic peptides of choice, and are stable reagents that were safely used in pioneering phase I studies. However, we showed in part I that exosomes are efficient to promote primary MHC class I-restricted effector CD8(+) T cell responses only when transferred onto mature DC in vivo. In this work, we bring evidence that among the clinically available reagents, Toll-like receptor 3 and 9 ligands are elective adjuvants capable of triggering efficient MHC-restricted CD8(+) T cell responses when combined to exosomes. Exosome immunogenicity across species allowed to verify the efficacy of good manufactory procedures-manufactured human exosomes admixed with CpG oligonucleotides in prophylactic and therapeutic settings of melanoma in HLA-A2 transgenic mice. CpG adjuvants appear to be ideal adjuvants for exosome-based cancer vaccines.

Adjuvants, Immunologic↗

PubMed2004

Exosome-based immunotherapy.

Exosomes are small membrane vesicles originating from late endosomes and secreted by hematopoietic and epithelial cells in culture. Exosome proteic and lipid composition is unique and might shed some light into exosome biogenesis and function. Exosomes secreted from professional antigen-presenting cells (i.e., B lymphocytes and dendritic cells) are enriched in MHC class I and II complexes, costimulatory molecules, and hsp70-90 chaperones, and have therefore been more extensively studied for their immunomodulatory capacities in vitro and in vivo. This review will present the main biological features pertaining to tumor or DC-derived exosomes, will emphasize their immunostimulatory function, and will discuss their implementation in cancer immunotherapy.

Antigen Presentation↗

PubMed2004

Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects.

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.

Animals↗

PubMed2002

Dendritic cells for NK/LAK activation: rationale for multicellular immunotherapy in neuroblastoma patients.

Natural killer (NK)/lymphokine-activated killer (LAK) cell-based immunotherapy could be beneficial against major histocompatibility complex class I-negative tumor residual disease such as neuroblastoma (NB), provided that interleukin 2 (IL-2) or surrogate nontoxic NK cell stimulatory factors could sustain NK cell activation and survival in vivo. Here we show that human monocyte-derived dendritic cells (MD-DCs) promote potent NK/LAK effector functions and long-term survival, circumventing the need for IL-2. This study demonstrates (1) the feasibility of differentiating granulocyte colony-stimulating factor-mobilized hematopoietic peripheral blood stem cells (PBSCs) into high numbers of functional MD-DCs and NK/LAK cells in a series of 12 children with stage 4 neuroblastoma (NB); (2) potent DC-mediated NK cell activation in autologous settings; (3) the reciprocal capacity of NK/LAK cells to turn immature DCs into maturing cells electively capable of triggering NK cell functions; and (4) the unique capacity of maturing DCs to sustain NK cell survival, superior to that achieved in IL-2. These data show a reciprocal interaction between DCs and NK/LAK cells, leading to the amplification of NK cell effector functions, and support the implementation of DC/NK cell-based immunotherapy for purging the graft and/or controlling minimal residual disease after autologous stem cell transplantation.

Cell Survival↗

PubMed2002

Malignant effusions and immunogenic tumour-derived exosomes.

BACKGROUND: Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supernatants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells. METHODS: We isolated exosomes by ultracentrifugation on sucrose and D(2)O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes. FINDINGS: Malignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules. INTERPRETATION: Exosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.

Adenocarcinoma↗

PubMed2002

In vivo-targeted gene delivery using antibody-based nonviral vector.

Tissue-specific gene transfer remains one of the main challenges to deliver genes into designated and/or disseminated cells. We have previously shown successful gene transfer with a nonviral gene delivery system based on the simple chemical conjugation of plasmid DNA with antibody. However, this approach was hampered by low efficiency due to the poor translocation rate of DNA to the nucleus. To improve this approach, we have modified our vector by introducing noncovalent binding between the antibody and DNA, allowing the possibility to introduce different important molecules. The noncovalent association was achieved with neutravidin and biotinylated components: (1) biotinylated antibodies; (2) a biotinylated hemagglutinin fusogenic peptide of influenza virus to favor endosomal escape; and (3) biotinylated histone H1 to compact, protect, and associate DNA to the complex. We report here that this delivery system can be internalized by tumor cells targeted by a specific monoclonal antibody, permits the protection of the transfected DNA, and allows its subsequent transfer into the nucleus after escape from the endosomal compartment. We also demonstrate that, in vitro, gene transfer with this vector showed much higher reporter activity in cells (15 vs. 0.5%) and a stronger production of murine interleukin 2 as compared with our previous vector. In vivo, a single intravenous injection of the vector containing an antibody directed to the G250 renal cell carcinoma-associated antigen led to beta-galactosidase expression in engrafted tumor bearing G250 but not in G250-negative tumor or in other tissues. Altogether, these results indicate that our antibody-based vector is suitable to promote gene delivery in vitro and in vivo in tumor cells.

Animals↗

PubMedDate not supplied

Exosomes as a tumor vaccine: enhancing potency through direct loading of antigenic peptides.

Exosomes secreted by dendritic cells (DCs) contain MHC-I, MHC-II, and other accessory molecules required for antigen presentation to T cells. Previous studies have shown that exosome MHC-I "indirectly" loaded by adding peptides to DC cultures are immunogenic. However, analysis of peptide binding was not performed to link T-cell-stimulating activity with the amount of MHC-I/peptide complexes on the exosomes. In this study, we measured peptide binding to MHC-I under different loading conditions and tested the exosomes' potencies in T-cell activation assays. We demonstrate that MHC-I on purified exosomes can be directly loaded with peptide at much greater levels than indirect loading. The direct loading method performed in mildly acidic conditions was effective even in the absence of exogenous beta2m. This increase in peptide binding greatly enhanced exosome potency, allowing us to further study the biologic activity of exosomes in vitro. In the presence of antigen-presenting cells (APC), exosomes directly loaded with the HLA-A2 restricted MART1 tumor peptide stimulated an HLA-A2/MART1 specific T-cell line. The T cells responded to exosomes using HLA-A2neg APC, demonstrating transfer of functional MHC-I/peptide complexes and not peptide alone to APC. MHC-II molecules, which are abundantly expressed on DC exosomes, were also functionally loaded under the same conditions as MHC-I. This feature allows for delivery of multiple peptide antigens that can stimulate both CD8+ cytotoxic T cells as well CD4+ T helper cells critical for an effective antitumor response. The optimized loading conditions and the ability to transfer both MHC-I and MHC-II antigens to APC have led to the development of exosomes as an "acellular" immunotherapy approach currently being tested in clinical trials.

Animals↗

PubMedDate not supplied

Dendritic cells (DC) promote natural killer (NK) cell functions: dynamics of the human DC/NK cell cross talk.

Dendritic cells (DC) were originally found critical in the setting of cognate immune responses. We first demonstrated that DC can also induce mouse NK cell activation and NK cell dependent-antitumor effects in mice. Here we analyzed the dynamics between DC and NK cells in human in vitro model systems. In the absence of LPS, DC do not trigger resting NK cells. Conversely, in the presence of LPS, resting bulk NK cells interacting with DC acquire CD25 and CD69 surface expression, produce high levels of IFN-gamma and lyse DAUDI cells. On activated IL-2 dependent NK cell lines, regardless of their differentiation stage, DC maintain or enhance NK cell proliferation and effector functions in the absence of exogenous cytokines. While IL-12, IL-15 and IL-18 are not critical, a direct cell-to-cell contact is mandatory for NK activation by DC and required for optimal proliferation. These data imply that DC also modulate human NK cell innate effector functions.

Antigens, CD↗

PubMedDate not supplied

[Exosomes and anti-tumour immunotherapy].

Exosomes are 60 to 90 nm membrane vesicles originating from late endosomes and secreted from most hematopoietic and epithelial cells in vitro. B cell derived-exosome antigenicity was first reported in 1996 in MHC class II restricted CD4+ T lymphocytes. In 1998, we reported that dendritic cell derived-exosomes are immunogenic in mice leading to tumor rejection. These findings have renewed the interest in exosomes. The current challenge consists in understanding the mechanisms and the physiological relevance of exosomes that could contribute to the design of the optimal exosome based-vaccination. Here, we will focus on the biological features pertaining to dendritic cell- and tumor cell derived-exosomes and will discuss their potential clinical implementation.

Animals↗

PubMedDate not supplied

[NK cell-based immunotherapy: new prospects and involvement of dendritic cells].

Dendritic cells (DC) were initially described as antigen presenting cells able to prime naive T cells. In fact, dendritic cells are also able to activate natural killer (NK) cells. The crosstalk between DC and NK is now regarded as a main stage in the initiation of innate and adaptative immune responses. Moreover, DC mediated-NK cell activation is particularly efficient since DC promote both effector functions and survival or proliferation of NK cells. Furthermore, recent publications in the field of NK cell biology underscore a role for NK cells in anti-tumor effects. NK cell-based immunotherapy should be reconsidered Here, we will report how pioneering immunotherapy trials based on natural cytotoxicity and progress in NK cell biology can promote NK cells in anti-tumor immune responses. Then, we will discuss the biological features of the DC/NK crosstalk and its implementations in immunotherapy protocols.

Cell Communication↗