Increasing vaccine potency through exosome antigen targeting.

While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA "cancer vaccines" using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established which takes advantage of the ability of the factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC). To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations.

Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

Exosomes as novel therapeutic nanodevices.

Exosomes are small vesicles (60 to 100 nm) that are released by many cell types. Their heterogeneous protein and lipid compositions, in addition to their enduring physicochemical features have led to the idea of using these natural vesicles as nanodevices for the development of new therapeutic applications. The first exosome-based nanodevices evaluated in the clinic consisted of autologous dexosomes (patient-specific exosomes released by dendritic cells and loaded with tumor antigen-derived peptides). They were tested in two phase I trials as immunotherapeutic regimens for melanoma and nonsmall-cell lung cancer. These studies revealed that dexosome immunotherapy was feasible, safe and led to the induction of both innate and adaptive immune responses, disease stabilization and long-term survival for several patients. The recent steps made towards transforming exosomes into product candidates for immunotherapy are summarized. In addition, recent developments in the field of exosome research that we believe will lead to improved and/or new therapeutic applications are highlighted. For example, a technology known as exosome display can be utilized to develop genetic vaccines that could induce exosome-mediated immunity without requiring the preparation of patient-derived exosomes.

Exosome Display technology: applications to the development of new diagnostics and therapeutics.

Exosome Display is a novel methodology enabling the manipulation of exosome protein content. This technology stems from the identification of addressing domains that mediate the specific distribution of proteins on exosomes. More particularly, Lactadherin expressed in non-mammary gland tissue has been found to localize to exosomes via binding of its C1C2 domain to exosome lipids. Exosome Display of soluble antigens and extracellular domains of membrane proteins that are not naturally found on exosomes occurs upon fusion of proteins with the Lactadherin C1C2 domain. Exosome Display of native full-length membrane proteins can also be achieved by non-restricted expression or sampling of membrane proteins on exosomes. These novel findings enable us to manipulate exosome composition and tailor exosomes with new desirable properties. The Exosome Display technology is very versatile since soluble, membrane-bound, trans-membrane or multimeric antigens that are not naturally found on exosomes can now be efficiently expressed at their surface in a native conformation. The technology was applied to the generation of antibodies against tumor biomarkers such as HLA/peptide complex. This antibody method called ExoMAb can be used to generate antibodies against any drug target candidates, notably including G-protein coupled receptors. The potential of Exosome Display technology for developing a broad range of novel diagnostics and therapeutics is discussed.

An analysis of variability in the manufacturing of dexosomes: implications for development of an autologous therapy.

Dexosomes are nanometer-size vesicles released by dendritic-cells, possessing much of the cellular machinery required to stimulate an immune response (i.e. MHC Class I and II). The ability of patient-derived dexosomes loaded with tumor antigens to elicit anti-tumor activity is currently being evaluated in clinical trials. Unlike conventional biologics, where variability between lots of product arises mostly from the manufacturing process, an autologous product has inherent variability in the starting material due to heterogeneity in the human population. In an effort to assess the variability arising from the dexosome manufacturing process versus the human starting material, 144 dexosome preparations from normal donors (111) and cancer patients (33) from two Phase I clinical trials were analyzed. A large variability in the quantity of dexosomes (measured as the number of MHC Class II molecules) produced between individual lots was observed ( > 50-fold). An analysis of intra-lot variability shows that the manufacturing process introduces relatively little of this variability. To identify the source(s) of variability arising from the human starting material, distributions of the key parameters involved in dexosome production were established, and a model created. Computer simulations using this model were performed, and compared to the actual data observed. The main conclusion from these simulations is that the number of cells collected per individual and the productivity of these cells of are the principal sources of variability in the production of Class II. The approach described here can be extended to other autologous therapies in general to evaluate control of manufacturing processes. Moreover, this analysis of process variability is directly applicable to production at a commercial scale, since the large scale manufacture of autologous products entails an exact process replication rather than scale-up in volume, as is the case with traditional drugs or biologics.

Dexosomes as a therapeutic cancer vaccine: from bench to bedside.

Exosomes released from dendritic cells, now referred as dexosomes, have recently been extensively characterized. Preclinical studies in mice have shown that, when properly loaded with tumor antigens, dexosomes can elicit a strong antitumor activity. Before dexosomes could be used in humans as a therapeutic vaccine, extensive development work had to be performed to meet the present regulatory requirements. First a manufacturing process amenable to cGMP for isolating and purifying dexosomes was established. Methods for loading the Major Histocompatibility Complex (MHC) molecules class II and I in a quantitative and reproducible way were developed. The most challenging task was the establishment of a quality control method for accessing the biological activity of individual lots. Such a method must remain relatively simple and reflect the mechanism of action of dexosomes. This was accomplished by measuring the transfer of a MHC class II superantigen complex to an antigen presenting cell that was MHC class II negative. More than 100 separate dexosome lots were prepared from blood cells of healthy volunteers to evaluate the variability of the manufacturing process. The analysis of the data showed that the main source of variability was related to the heterogeneity of the human population and not to the manufacturing process. These studies allowed to perform two phase I clinical trials. A total of 24 cancer patients received Dex therapy. Dexosome production from cells of cancer patient was found equivalent to that of normal volunteer. No adverse events related to this therapy were reported. Evidence of dexosome bioactivity was observed.

Dendritic cell-derived exosomes in cancer immunotherapy: exploiting nature's antigen delivery pathway.

Dendritic cells release large quantities of exosomes, known as dexosomes. These dexosomes are heat-stable, small vesicles (60-90 nm in diameter) made up of a lipid bilayer displaying an enrichment in sphingomyelin and a decrease in phosphatidylcholine content with no measurable asymmetry. They incorporate a characteristic set of proteins, including a large quantity of tetraspanins such as CD9 and CD81, all the known antigen presenting molecules (major histocompatibility complex class I and II, CD1 a, b, c and d) and the costimulatory molecule CD86. The function of dexosomes is to transfer antigen-loaded major histocompatibility complex class I and II molecules, and other associated molecules, to naive dendritic cells, potentially leading to the amplification of the cellular immune response. In preclinical mouse models, antigen-loaded dexosomes elicit strong antitumor activity. Human dexosomes can be prepared ex vivo relatively easily from dendritic cells derived from monocytes isolated by leukapheresis of healthy individuals or cancer patients. The feasibility of using dexosomes as a cancer therapeutic vaccine has been tested in two Phase I clinical studies in melanoma and lung cancer patients, respectively. These studies demonstrate that dexosomes can be prepared from cancer patient blood cells and be safely administered. Clinical observations suggested that dexosomes can stimulate both the adaptive (T-cells) and innate (natural killer cells) cellular immune responses. This review focuses on the perspective of using dexosomes in cancer immunotherapy. Concepts for using the exosome pathway in other possible pharmacologic applications are also discussed.

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.

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer.

BACKGROUND: There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). METHODS: This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. RESULTS: Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1-2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52-665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. CONCLUSION: Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors.

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.

Production and characterization of clinical grade exosomes derived from dendritic cells.

We describe methods for the production, purification, and characterization of clinical grade (cGMP) exosomes derived from antigen presenting cells (APCs). Exosomes have been shown to have immunotherapeutic properties through their presentation of biologically relevant antigens [Nat. Med. 4 (1998) 594] and are being developed as an alternative to cellular therapies. Exosomes are 50-90-nm-diameter vesicles secreted from multivesicular bodies (MVBs) found in a variety of both hematopoietic and tumor cells. These particles contain antigen presenting molecules (MHC class I, MHC class II, and CD1), tetraspan molecules (CD9, CD63, CD81), adhesion molecules (CD11b and CD54), and costimulatory molecules (CD86); hence, providing them the necessary machinery required for generating a potent immune response [J. Biol. Chem. 273 (1998) 20121; J. Cell. Sci. 113 (2000) 3365; J. Immunol. Methods 247 (2001) 163; J. Immunol. 166 (2001) 7309]. Exosomes from monocyte-derived dendritic cells (MDDCs) were rapidly purified (e.g. 4-6 h of a 2-3 l culture) based on their unique size and density. Ultrafiltration of the clarified supernatant through a 500-kDa membrane and ultracentrifugation into a 30% sucrose/deuterium oxide (D2O) (98%) cushion (density 1.210 g/cm3) reduced the volume and protein concentration approximately 200- and 1000-fold, respectively. The percentage recovery of exosomes ranged from 40% to 50% based on the exosome MHC class II concentration of the starting clarified supernatant. This methodology was extended to a miniscale process with comparable results. Conversely, the classical differential centrifugation technique is a more lengthy and variable process resulting in exosomes being contaminated with media proteins and containing only 5-25% of the starting exosome MHC class II concentration; hence, making it difficult for their use in clinical development. Lastly, we developed the following quality control assays to standardize the exosome vaccine: quantity (concentration of MHC class II) and protein characterization (FACS). The combination of a rapid and reproducible purification method and quality control assays for exosomes has allowed for its evaluation as a cancer vaccine in clinical trials [Proc. Am. Soc. Oncol. 21 (2002) 11a].