US20060024318A1 - Vaccination with immuno-isolated cells producing an immunomodulator - Google Patents

Vaccination with immuno-isolated cells producing an immunomodulator Download PDF

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US20060024318A1
US20060024318A1 US11/058,883 US5888305A US2006024318A1 US 20060024318 A1 US20060024318 A1 US 20060024318A1 US 5888305 A US5888305 A US 5888305A US 2006024318 A1 US2006024318 A1 US 2006024318A1
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Nicolas Mach
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Novimmune SA
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Assigned to NOVIMMUNE S.A. reassignment NOVIMMUNE S.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MACH, NICOLAS
Publication of US20060024318A1 publication Critical patent/US20060024318A1/en
Priority to US11/900,670 priority patent/US20080107686A1/en
Priority to US14/218,171 priority patent/US9814682B2/en
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    • A—HUMAN NECESSITIES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
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    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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    • A—HUMAN NECESSITIES
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a new approach for providing an active adjuvant or immunomodulator, for example in the context of immunisation in humans and animals.
  • an immunomodulator for example GM-CSF (granulocyte-macrophage colony stimulating factor)
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • This system is particularly well adapted to vaccination in that it provides the immunomodulator in an active form, in continuous, non-immunogenic manner in the immediate vicinity of the vaccine antigen(s).
  • the strategy of the invention is perfectly suited for both cancer immunotherapy and vaccination against infectious agents.
  • first generation vaccines comprised only the antigen against which an immune response was desired.
  • second generation of vaccines was developed, wherein the vaccinating composition includes one or more adjuvants as immunomodulators to enhance this immune response.
  • a widely applicable technique used for providing the necessary adjuvant is simply to combine the antigen with the adjuvant in the vaccinating composition.
  • the resulting composition is administered directly to the subject, thereby supplying the antigen and adjuvant in a simultaneous and co-localised manner.
  • the immunomodulator may either be “naked”, or alternatively may be administered in a slow release formulation using pegylated, liposomal microspheres (International patent application WO 98/33520, filed by Bystryn). This strategy is however limited by technical and biochemical difficulties, as well as some degree of systemic release inducing potential toxicities.
  • Borrello et al Human Gene Therapy, 1999 has described a strategy in which the GM-CSF supplying cell is a cell line, K-562, which fails to express HLA class I or II antigens, potentially decreasing the magnitude of the alloresponses generated on repeated immunisations.
  • the K-562 cells engineered to secrete GM-CSF do not express MHC molecules on their surface. They are HLA negative. These cells are however human cancer cells and are highly sensitive to potent rejection mechanisms occurring without the involvement of HLA class I or II proteins. These defence strategies are less specific but very rapid and potent for cellular destruction.
  • ⁇ T cells At least two subtypes of lymphocytes known as ⁇ T cells or natural killer (NK) lymphocytes are known to attack and destroy foreign cells by mechanism independent of HLA class I or II. Regarding K-562 they are known to be very sensitive to NK cells and also to ⁇ T cells (J. Immunotherapy 2000 23:536-548 Schilbach K. et al) and are therefore used as a postive control for NK cell activity.
  • K-562 cells can express MHC class I upon Interferon ⁇ exposure. It is possible that such cytokine could be present or released at the vaccination site during the first or after repeated immunizations. Such MHC class I upregulation will also lead to rapid cell destruction via classical cellular immunity.
  • the source of antigen is usually a whole tumour cell.
  • This cell can be engineered, for example by transfection, to simultaneously produce the necessary adjuvant. Potent, specific and long lasting anti-tumour immune responses have been reported in the mouse model using this technique, relying on retroviral vectors as the gene transfer method for engineering tumour cells delivering GM-CSF.
  • tumour cells have therefore been proposed to circumvent these difficulties.
  • Engineered viruses like adenovirus can infect tumour cells very efficiently and with much simpler procedures. Because adenovirus can infect non-dividing cells, the harvested tumour cells can be infected right away, preventing the long and tedious primary culture step required when using retroviral vectors.
  • the antigenic source is provided by cell-lines derived from other patients with similar type of cancer.
  • the patient is then immunized with repeated injections of irradiated, GM-CSF secreting, allogeneic (from another human being) tumour cells.
  • the present invention provides a new approach overcoming the drawbacks associated with the previous strategies.
  • the invention is based on the immuno-isolation of adjuvant producing cells by a physical barrier such as a capsule.
  • the present invention provides a general protocol for vaccination, named “Maxi-Vax” characterized by the delivery, in close proximity, of antigens and of live cells engineered to secrete immuno-activating cytokines (such as GM-CSF), these cells being immuno-protected by macro- or micro-encapsulation in order to sustain prolonged in situ delivery of the immune activator.
  • cytokines such as GM-CSF
  • a particular case of the invention utilises macro-encapsulation of cells in hollowfibers PES capsules, but other forms of encapsulation, within devices or within gel matrices, are included in the invention.
  • a particular case of the invention concerns the use of cancer cells as the antigen, for the purpose of immunising against cancer-specific antigens and establishing a systemic anti-tumour cell protective immune response.
  • the later case is referred to as “Onco-Maxi-Vax”.
  • Another particular case of the invention applies to vaccination against infectious agents, such as viruses (including HIV and Hepatitis C).
  • infectious agents such as viruses (including HIV and Hepatitis C).
  • FIG. 1 Clinical immunization protocol for Onco-Maxi-Vax
  • the vaccination treatment with Onco-Maxi-Vax comprises repeated immunizations.
  • the immunization is repeated at two week intervals at different sites, always sub-cutaneous.
  • the total number of vaccination is a minimum of five.
  • FIG. 2 Provides a novel immunity against wild-type tumour challenged by various irradiated, cytokine secreting tumour cells.
  • This figure compares the efficacy of various immunomodulatory molecules in vaccination mouse model. Each block represents the percentage of mice surviving a tumour challenge after vaccination with irradiated tumour cells producing the described molecule.
  • mice vaccinated with irradiated tumour cells secreting GM-CSF are protected. 25% of the mice vaccinated with irradiated tumour cells secreting FLT3-L are protected.
  • FIG. 3 GM-CSF release from encapsulated, non irradiated Renca-GM-CSF secreting cells.
  • This table shows an analysis of GM-CSF secretion outside the capsule at various time points by encapsulated, GM-CSF secreting Renca cells. GM-CSF release is expressed in ng/capsule/24 h.
  • Measurement of in-vitro release of murine GM-CSF from the capsule containing GM-CSF secreting cells is carried out at Days 4, 7, 11, 14 and 21 post loading the cells into the capsule. This is performed with standard monoclonal antibodies against murine GM-CSF in Enzyme-Linked immunoabsorbent Assays (ELISA). (R&D systems). The amounts of protein released as well as the reproducibility from one capsule to the others are assessed.
  • ELISA Enzyme-Linked immunoabsorbent Assays
  • FIG. 4 Survival at 50 days after B16WT challenge.
  • This graph shows the survival at 50 days of mice immunized at day—7 with
  • the graph represents the mean value over 5 mice per group.
  • FIG. 5 Survival of mice after B 16WT challenge.
  • the experimental conditions are identical to the conditions specified in FIG. 4 and exposed in example 4.
  • An immunostimulatory agent or an immuno-activator is an immunomodulator which specifically enhances or amplifies the immune response to an antigen or an immunogen.
  • immunomodulator an immunomodulator which specifically enhances or amplifies the immune response to an antigen or an immunogen.
  • immuno-activator is used synonymously with the term “adjuvant”.
  • immuno-isolated cells are not attacked or destroyed by the immune response of the host, because they are undetectable by the immune system, which prevents any immune response against them and because they are physically protected against any immune response.
  • the present invention relates to encapsulated cells producing and secreting an immunomodulator (preferably GM-CSF), for use in therapy and in vaccination (preferably with cancer cells).
  • an immunomodulator preferably GM-CSF
  • the encapsulated cells are engineered to produce the immunomodulator, although the invention also encompasses the use of cells and cell-lines which naturally produce the immunomodulator. It also relates to pharmaceutical compositions, vaccines and kits which can be used in this context. It finally relates to processes for vaccinating or treating patients.
  • the present invention relates to cells which produce an immunomodulatory agent and which are physically immuno-isolated.
  • the immunomodulatory agent produced is preferably a protein synthesized by the cells, but it can also be for example a cell-component such as a lipid, or an exogenous molecule further transformed by the cell, for example antigens processed by antigen-presenting cells or metabolites.
  • the immunomodulatory agent is immunostimulatory. Antigens are frequently too weak to trigger a significant immune response and some molecules involved in this response are known to enhance or amplify it.
  • An immunostimulatory agent may act in attracting Antigen-presenting cells, for example dendritic cells. It may also act in stimulating the activities of CD4 or CD8 T-cells.
  • Particularly potent immunostimulatory agents which are preferred in the context of the present application, belong to the cytokine family.
  • Preferred cytokines are IL-3, IL-4, IL-9, IL-1, IL-2, IL-7 (interleukine), transmembrane receptors of IFN ⁇ , SCF (Stem Cell Factor) soluble or membranous, FL (Flt3 Ligand), G-CSF and GM-CSF (Granulocyte and Granulocyte-Macrophage Stimulating Factor), and combinations thereof.
  • Particularly preferred immunostimulatory agents are FL and GM-CSF.
  • Preferred cytokines are human cytokines, for example human GM-CSF.
  • GM-CSF is particularly recommended as immunostimulatory agent because it has been identified as the most potent cytokine for activating systemic antitumor immunity (Dranoff et al, 1993).
  • immunomodulatory agents In order to induce an adequate immune response, it can be very advantageous to combine several immunomodulatory agents in order to stimulate different pathways.
  • a preferred combination is the association of GM-CSF and FL.
  • Other combinations of 2 or more immunomodulatory agents are also envisaged by the present application.
  • an immuno-isolated cell of the invention can fulfil other additional functions. It can for example play a role in the detection of the magnitude or the localisation of the expected immune response.
  • Another major additional function is to provide the antigenic agent.
  • the same cell can produce both the antigenic agent triggering the response and the immunostimulatory agent enhancing the response. This is particularly advantageous in the case of antigen-based vaccination, or cell-based vaccination where the antigens are secreted or released from the cell membrane. This approach ensures that antigenic and immunostimulatory agents are co-localised.
  • the immuno-isolated cells of the invention are possibly chosen among cancerous cells expressing TAA (tumour associated-antigens) which preferably are lineage specific and tumour-specific.
  • TAA tumor associated-antigens
  • the produced immunomodulatory agent is preferably soluble in order to be secreted and released.
  • the antigenic agents like TAA.
  • the source of immunomodulator i.e. the immuno-isolated cells of the invention are dissociated from the source of antigenic agent.
  • a first cell or group of cells constitutes the antigen source and a second, immuno-isolated cell or group of cells constitutes the adjuvant source.
  • a preferred way to immuno-isolate cells is to provide a physical barrier “hiding” them from the general immune system. This goal can be achieved by a barrier device.
  • barrier devices are suitable, in particular microcapsules and macrocapsules.
  • the actions of encasing a cell or population of cells in such a barrier are referred to herein as microencapsulation and macroencapsulation, respectively.
  • Immuno-isolation overcomes the significant disadvantages associated with the implantation of free cells.
  • the use of free cells generally requires immuno-suppressing drugs in order to protect them against the immune system of the host.
  • barrier devices around grafted cells obviate the need for immunosuppressive therapy.
  • the cells can be retrieved readily after a while, if need be. This last property allows a switchable release of the immunomodulatory agent. By retrieving the device, the release of the agent is stopped, which prevents unwanted presence of a molecule after the end of the immunization process.
  • Capsules of the invention can be specifically engineered to facilitate their withdrawal, for example by incorporation of a string or other means to ease their retrieval.
  • Immuno-isolation also overcomes the significant disadvantages associated with the use of HLA-negative cells such as K-562 cell line which fails to express HLA class I or II antigens. Since the encapsulated cells are entirely protected against the immune system, they are not destroyed by innate or cellular immunity, whereas K-562 cells are involved in innate immunity rejection. The capacity of encapsulated cells to survive, to secrete protein for a prolonged period of time and to allow multiple immunizations is directly linked to the physical barrier of the capsule. Moreover, the amount of GM-CSF release into the patient after capsule implantation is not likely to differ from one individual to another depending on his or her innate immunity or immunosuppression. In contrast, the stability of GM-CSF release is likely to vary significantly not only in any given patient at the first and subsequent immunizations but also from one patient to another, with the injection of GM-CSF secreting K-562 cells.
  • the main property of the barrier device is to separate living cells from the immune system of the host by a synthetic, selectively permeable, non-immunogenic membrane.
  • the cells of the invention are living and preferably provide the chosen immunomodulatory agent on a long-term basis.
  • experimental results showed that encapsulated cells, engineered to secrete GM-CSF, do release GM-CSF outside the capsule for at least fifteen days. For this purpose, they must be supplied with all the factors necessary for their survival, their growth and the production of the immunomodulator of interest.
  • the device In order to allow free exchange of nutriments, proteins, oxygen and biotherapeutic substances between exterior and interior, the device preferably is selectively permeable. Small molecules can transit via pores, especially molecules necessary for the survival of the cells, whereas high-molecular-weight substances such as immunocytes or antibodies are excluded. It moreover excludes inflammatory cells and thereby protects the encapsulated cells from tissue rejection.
  • immunomodulatory agents produced by the cells of the invention can be delivered through the pores into the external medium.
  • the diameter of the pores is preferably chosen in a range such that small molecules or proteins and immunomodulators are allowed to cross the barrier and that bigger ones like immunoglobulins are not, in order for the device to retain its immuno-protective property.
  • microcapsules For the manufacture of microcapsules, a mixture of cells and sodium alginate are extruded, and solidified into beads generating a matrix which allows free exchange of proteins, nutriments and oxygen between encapsulated cells and the host. Advantages of this device include great surface to volume ratio and ease of implantation.
  • the macroencapsulation makes use of preformed macrocapsules, initially empty units that are loaded with a matrix and all the cells needed for treatment.
  • the matrix is preferably a polymer, for example polyvinyl alcohol or a biopolymer like alginate.
  • the matrix ensures a good ordering of the cells inside the capsule, specifically a homogenous distribution, and prevents agglutination at the walls.
  • Macrocapsules are more durable and rugged than microcapsules, they contain internal reinforcements, can be tested for seal integrity before implantation and can be designed to be refillable in the body. They can also be retrieved simply.
  • Preferred polymers for the capsule are thermoplastic polyethersulfone (PES) hollow fibers (OD:720 ⁇ m; ID:524 ⁇ m, molecular weight cut-offs: 32 and 80 kDa; Akzo Nobel Faster A G, Wupperthal, Germany) and AN-69 polymer (acrylonitrile and sodium metallysulphonate anionic copolymer, Hospal R&D Int, Meyzieu, France).
  • PES thermoplastic polyethersulfone
  • the macrocapsules can have various sizes ranging from few micrometers to three to four centimetres. Depending on the size of the capsule and the size of the cells, as many as 200 000 cells can be loaded into a 1 cm device.
  • Particularly preferred devices of the invention are microcapsules and macrocapsules.
  • cells of the invention produce a immunomodulatory agent. Either the cells naturally produce the agent, or this goal is achieved by modifying the cells. In a preferred case, cells are genetically modified to express the immunomodulatory agent. This is particularly convenient for many reasons.
  • the use of cells of the invention is not limited to those naturally producing it. It is particularly advantageous that the agents are not limited to those naturally occurring. As it is known that mutated proteins sometimes exert improved activities, it is very advantageous to use this modified version of the protein instead of the wild-type one. It is also sometimes convenient to clone the soluble version of a membranous protein, in order to achieve its secretion.
  • cells of the invention by genetically modifying cells of the invention, it is also possible to control the expression level of the immunomodulatory agent.
  • a particularly attractive situation is the overexpression of the agent by cloning its sequence under the control of a promoter known to be very strong in the used cell.
  • the modified cells become engineered factories producing high levels of immunomodulatory agent.
  • the promoter can be chosen according to its activity such as to have a controlled level of immunomodulator expression.
  • cells may be used which naturally contain the gene for the immunomodulatory agent, said gene being transcriptionally silent in that particular cell. Transcription can be activated by insertion of appropriate regulatory sequences, for example by homologus recombination. Inducible regulatory sequences which respond to specific stimuli such as substances, light, etc. . . . may also be used.
  • immunomodulatory agent-secreting cells secrete more than 10 ng/10 6 cells/24 hr of immunomodulatory agent.
  • Preferred cells of the invention 10 6 secrete a quantity of immunomodulatory agent equal or superior to 100 ng/10 6 cells/24 hr, preferably more than 500 ng/10 6 cells/24 hr. If needed, several capsules may be implanted simultaneously.
  • a cell of the invention secretes more than 10 ⁇ 10 ⁇ 5 g of immunomodulatory agent per 24 hr, preferably more than 100 ⁇ 10 ⁇ 15 g/24 hr of immunomodulatory agent.
  • a cell of the invention secretes for example between 80 and 960 ⁇ 10 ⁇ 15 g/24 hr, preferably more than 500 ⁇ 10 ⁇ 15 g/24 hr of immunomodulatory agent.
  • Retroviruses are well suited in the context of the present invention because they can be engineered to introduce a gene coding for the immunomodulatory agent into the genome of host cell.
  • cells of the invention are not limited to cells naturally producing an immunomodulatory agent of interest. All sorts of cells can be used, particularly preferred are cells which are easy to transduce or transfect, and to culture and propagate. It is not necessary to use tumour cells as the by-stander immunomodulator producer. Based on the medical literature, different cell types can been used to produce for example cytokines. These include immortalized non-tumoral fibroblasts, myoblasts or tumour cells. Cells of the invention are advantageously endothelial cells or fibroblasts.
  • Particularly preferred cells are in general immortal cell lines. It is thus possible to genetically modify a cell line just once and to use cells from it for all applications of the present invention. Because the cells are immuno-isolated, they are not endangered by the immune system of the host, but they also do not endanger the other cells in their vicinity.
  • Cells of the invention are preferably human cells.
  • the immuno-isolation of the cells is thus very advantageous over some prior systems because the sources of immunomodulatory agent can be considered as “universal” and not limited to a unique individual.
  • Cells of the invention are living. This ensures that the immunomodulator is continuously produced, for at least several days and even more if needed.
  • encapsulated cells engineered to secrete a protein have been implanted in patients for weeks or months. This long-lasting release overcomes the common problem of short half-life of immunomodulators.
  • cells of the invention are implanted for a few days, preferably less than 12 days, for example between 4 and 10 days, for example between 5 to 7 days. Implantation of encapsulated cells for such a short period will not lead to marked fibrosis induced by the release of the immunomodulatory agent. Inflammation, at the vaccination site, around the capsule, will not induce a decrease of cell viability within the capsule and therefore it will not prevent the production and release of the immunomodulatory agent in that time frame.
  • the cell-containing capsule is irradiated, for example by X-Rays, before implantation.
  • this irradiation ensures that, even if disruption of the capsule occurs, enclosed cells are not capable of propagation.
  • this irradiation ensures that the secretion of the immunomodulatory agent will stop after around 10 days, due to irradiation-induced cell death. This may be advantageous, should the secreted immunomodulatory agent generate a violent inflammatory response.
  • subcutaneous implantation of GM-CSF-secreting cells, encapsulated or not could induce cutaneous necrosis if implanted during a period exceeding 15 days to 1 month. Irradiation of the capsule or cells before implantation does not prevent subsequent retrieval of the capsule.
  • Onco-Maxi-Vax is a therapeutic product (therapeutic vaccine) made of two components that are physically in close proximity during the immunization.
  • the first component of the Onco-Maxi-Vax system is the source of tumour antigens, this antigenic load is made of irradiated cells harvested from the patient to be treated, this component is specific for each patient.
  • the source of antigen is made of each patient's own tumour cells. These are harvested surgically or endoscopically from the patient, digested in order to obtain a cell-suspension and then irradiated at 10000 Rad before storage in aliquots in liquid nitrogen. Irradiation is a safety measure in order to prevent any growth of tumour cells that will be re-injected to the patients. This procedure has already been performed safely.
  • This component of the Onco-Maxi-Vax system is unique to each patient, as it will be harvested from each individual and used in combination with the second component of the vaccine.
  • the second component of the Onco-Maxi-Vax vaccine is common (or “universal”) to all patients, it is the immunomodulator provider. It is composed either of a large capsule (macro-encapsulation) or small capsules (micro-encapsulation) that contain living cells.
  • the capsule(s) is (are) required to immuno-isolate allogeneic human cells.
  • the capsule(s) is (are) semi-permeable. It allows the survival of the cells inside by nutrient migrations and it prevents cells exposure to an environment which would normally destroy them.
  • the cells to be encapsulated are genetically engineered to secrete immunostimulatory molecules. It should be seen as an immuno-isolated bio-reactor that produces and releases, at the site of vaccination, strong immuno-stimulatory signals. So far Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is probably the most potent immunostimulatory molecule for anti-tumor immune response. Because of biochemical properties GM-CSF needs to be produced locally at the vaccine site in order to obtain a sustained release of the protein for at least five to seven days. Initially the encapsulated cells are engineered to secrete GM-CSF only. Depending on synergistic studies, other immunomodulatory molecules can be added without difficulty (other cytokines such as IL-12, IL-15, IL-4, Interferon gamma, chemokines or dendritic growth factors).
  • the capsule used in macro-encapsulation is retrievable and the cells that it contains are irradiated prior to implantation. Previous experiments have shown that irradiation of GM-CSF producer cells does not prevent the production and the release of the protein.
  • the two components of the Onco-Maxi-Vax vaccine are placed under the patient's skin in close proximity or in contact. They are implanted at sites distant from the primary tumour or metastasis in order to perform the vaccination in an immunologically un-disturbed location.
  • the capsule is removed, for example using an especially designed string attached to it.
  • the autologous irradiated tumour cells injected as a component of the Onco-Maxi-Vax are progressively processed and removed by the patient's immune system, via naturally occurring mechanisms.
  • the vaccination treatment with Onco-Maxi-Vax comprises repeated immunizations at two to three week intervals at different sites, usually sub-cutaneous.
  • the total number of vaccinations, although adjustable is preferably a minimum of five.
  • the dose of autologous cells is adjusted to the amount of cells harvested from the patients. It is recommended to have around 10 7 to 10 8 cells per immunization. When this dosage cannot be repeated 5 times, the dose of autologous tumour cells is reduced accordingly.
  • the ideal number of cells to be encapsulated is dependent on the amount of immunomodulatory agent such as GM-CSF that is required at the vaccination site.
  • GM-CSF producing autologous cells were releasing between 80 to 960 ng/10 6 cells/24 hr. Release between 500 to 1000 ng/24 hr is considered a reasonable goal.
  • immunomodulatory agent such as GM-CSF-secreting cells secrete more than 10 ng/10 6 cells/24 hr of immunomodulatory agent.
  • Preferred cells of the invention secrete a quantity of GM-CSF equal or superior to 100 ng/10 6 cells/24 hr, preferably more than 500 ng/10 6 cells/24 hr. If needed, several capsules may be implanted simultaneously.
  • the total quantity of delivered immunomodulatory agent, especially GM-CSF must be at least 1 microgram per 24 hours at the vaccination site.
  • a cell of the invention secretes more than 10 ⁇ 10 ⁇ 15 g of immunomodulatory agent per 24 hr, preferably more than 100 ⁇ 10 ⁇ 15 g/24 hr of immunomodulatory agent.
  • a cell of the invention secretes for example between 80 and 960 ⁇ 10 ⁇ 15 g/24 hr, preferably more than 500 ⁇ 10 ⁇ 15 g/24 hr of immunomodulatory agent.
  • Capsule according to the invention may contain between 2 ⁇ 10 5 cells and 2, 3, 4 or 5 ⁇ 10 7 cells, preferably more than 10 7 cells per capsule, for example 2 ⁇ 10 7 cells per capsule.
  • tumour cells are identical for every patient. Only in patients with tumour cells that can be harvested from a solid primary tumour, a metastasis or from fluid containing tumour cells (pleural, peritoneal, bone marrow or blood) is it possible to generate the full vaccine product.
  • Cancers that are likely to have metastases that can be harvested are dependant on the location of the metastasis. For technical reasons it is more difficult to harvest bone metastases than other localizations. These cancers may be for example:
  • tumours could be preferably treated with Phase I Onco-Maxi-Vax after the specified treatment:
  • Toxicity and feasibility are recorded during the immunization period and also during the follow-up period by bi-weekly visit at the Oncology center and evaluation by an oncologist.
  • Serological tumour markers such as CA 153, CA19-9, CEA, AFP, NSE, CA 125, are monitored when elevated prior to vaccination.
  • tumour evaluation by bi-dimensional measurement may not be the best evaluation method to assess potential efficacy of immunization treatment.
  • Destruction of the tumour cells can be very efficient and replaced with fibrous or inflammatory cells without detectable changes in size on radiological examination.
  • Metabolic activity as assess by PET scan may be of relevance in this setting.
  • the analysis of post immunization tumour lesion is of great interest for immunological analysis such as the characterization of the potential tumour antigen targeted by the treatment.
  • cells of the invention are used in the context of immunisation against various infectious diseases.
  • This product of the invention can thus be named “IA-Maxi Vax” (infectious agent).
  • IA-Maxi-Vax is a therapeutic product (vaccine) made of two components that are physically in close proximity during the immunization.
  • One component represents the source of antigen(s).
  • the antigen comprises one or more components from the infectious agents. Therefore all patients with a specific infection will be treated with the same product.
  • Many known antigen components have been described in infectious diseases caused by viral, bacterial or parasitic pathogens and are used currently for immunization strategies. These antigens can be used as inactivated pathogens, infectious agent's lysates, protein extracts, recombinant proteins, peptides, DNA or other forms. In some conditions depending on the infectious agent or the host medical condition immunization is weak or non-protective leading to significant morbidity or mortality.
  • HIV is an example of failure to naturally eradicate a viral pathogen.
  • Immunosuppression after bone marrow transplantation is an example where the usually benign CMV infection can be life-threatening.
  • the first component of the vaccine comprises a combination of the antigen or a pool of antigens.
  • Encapsulated, bystander cells of the invention that release locally, at the vaccination site, a very potent immunomodulatory signal constitute the second component of the vaccine.
  • the second component of the vaccine is the same (“universal”) for all patients. It is made of a semi-permeable capsule (macro or micro) that contains live cells. The capsule is required to immuno-isolate allogeneic human cells. It allows the survival of the cells inside by nutrients migrations and it prevents cells exposure to the environment that would normally destroy them.
  • the cells to be encapsulated are genetically engineered to secrete immunostimulatory molecules. So far Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is probably one of the most potent immunostimulatory molecule for boosting the immune response. GM-CSF has been shown to increase immunity against various parasitic or viral agents. Because of bio-chemical properties GM-CSF needs to be produced locally at the vaccine site in order to obtain a sustained release of the protein for at least five to seven days. Initially the encapsulated cells are engineered to secrete GM-CSF only. Depending on synergistic studies, other immunomodulatory molecules could easily be added (other cytokine such as IL-12, IL-15, IL-4, Interferon gamma, chemokines or dendritic cells growth factors)
  • cytokine such as IL-12, IL-15, IL-4, Interferon gamma, chemokines or dendritic cells growth factors
  • the capsule is retrievable and the cells that it contains are irradiated prior to implantation. Previous experiment have shown that irradiation of GM-CSF producer cells does not prevent the production and the release of the protein.
  • the two components of the infectious-agent vaccine need to be placed under the patients skin in close proximity or contact. It may be beneficial to have sequential administration of the two components, the capsule being implanted first, followed by the antigenic stimulus.
  • the capsule is removed, for example using an especially designed string attached to it.
  • the vaccination treatment with “IA-Maxi-Vax” comprises repeated immunizations at two to three week intervals at different sites, always sub-cutaneous.
  • the total number of vaccination is dependant of the protocol and dosages and must be adjusted in each particular case.
  • the present invention relates to pharmaceutical compositions, vaccines and kits comprising cells which produce an immunomodulatory agent and which are physically immuno-isolated.
  • a pharmaceutical composition according to the present invention comprises immuno-isolated cells producing an immunomodulator combined with a physiologically acceptable carrier.
  • the density of cells of the invention to incorporate in the composition must be defined for each different case. In particular, the density of cells to incorporate is function of the immunomodulator dose required.
  • suitable physiologically acceptable carriers would consist of a device that would fulfil the following needs: non-toxic (local and systemic), efficient immuno-isolation, permeable allowing sustained release of the immuno-modulator.
  • a macro or microcapsule made of PES or AH-69 fulfil these requirements as well as the products developed by Theracytes Inc.
  • a vaccine composition of the present invention comprises immuno-isolated cells producing an immunomodulator combined with an antigenic component.
  • This second component of the vaccine represents the molecule against which an immunization reaction is expected.
  • the first one secretes the immunomodulator necessary for enhancing the immunization process.
  • the antigenic component can be represented by a protein, for example a viral protein which is known to be immunogeneic, or by a whole cell expressing antigens at its surface, or by an extract containing several antigenic substances.
  • the antigenic component is preferably a tumour cell (therapeutic vaccine). Tumour cells are known to express at their surface tumor-associated antigens against which an immune response is desired.
  • tumour cells When tumour cells are used, as in the cancer context, the tumour cells are preferably irradiated before incorporation into a composition. Irradiation is a safety measure in order to prevent any growth of tumour cells that will be re-injected to the patients. In addition to safety, irradiation may be a very good way to allow the release of potentially useful antigenic determinants.
  • the antigenic component is preferably from the agent against which an immune response is desired, for example from a virus.
  • Preferred viruses are Hepetitis C and HIV.
  • kits comprising immuno-isolated cells producing an immunomodulator combined with an antigenic component.
  • the first component of the kit secretes the immunomodulator necessary for enhancing the immunization process against the second antigenic component.
  • the antigenic component of a kit according to the present invention preferably comprises a cell producing, secreting or releasing an antigen.
  • the antigenic component can be a molecule, for example a protein, or a cellular extract.
  • the antigenic component of the kit is preferably a tumour cell.
  • the same safety measures are preferably taken by irradiating the tumour cell before use.
  • the kit will generally be used for implantation into human body. For this reason, many constraints are imposed on the properties of the kit. In particular, this kit must be designed to be as small as possible. It must also be biocompatible. A kit according to the invention can be placed for a time as long as several weeks or months. The kit must be safe throughout the duration of this period. In particular, in numerous uses of the kit, the encapsulation device needs to be sealed.
  • the invention encompasses uses of immuno-isolated cells producing an immunomodulatory agent, and uses of pharmaceutical compositions, vaccines and kits as described above in the therapeutic domain as well as in the vaccination field.
  • the invention relates in particular to a process for vaccinating a subject in need of such a treatment.
  • This process comprises the administration of a composition comprising cells of the invention.
  • the subject is preferably a human patient, but animals are also possible.
  • a subject can be in need of vaccination for different reasons, because a preventive immunization is preferable, like for benign disease, or is necessary for example in the case of severe epidemics.
  • the immune response is raised against a particular antigenic component.
  • the process of the invention comprises also the step of administrating the antigenic component to the subject.
  • a patient can be preventively vaccinated because he will be in contact with the antigenic component sooner or later. He can also be therapeutically immunized by the process of the invention because he is already in contact with the antigenic component but not able to generate an adequate immune response by himself.
  • the process comprises the administration of cells of the invention and of an antigenic component
  • both administrations can be made simultaneously, or they can be made separately, or sequentially. It can be very advantageous to temporally dissociate the administrations.
  • cells of the invention thanks to their barrier device, have a long-lasting effect.
  • antigenic components injected are likely to be processed and eliminated very rapidly by the host immune system. In such a case, when administrations are dissociated, the administration of antigenic component can be repeated whereas there is a single administration of cells of the invention.
  • the antigenic and the immunomodulatory agents should preferably be co-localised in order to produced an optimised effect.
  • cells of the invention are irradiated before administration.
  • the immuno-isolation consists in a capsule, it is preferred to irradiate the capsule. This irradiation step is preferably done shortly prior administration, but may occur a few days before.
  • the administration is repeated several times, preferably the administrations of cells of the invention as well as antigenic component are repeated.
  • the administration is repeated more than twice, preferably between 3 and 6 times, preferably 5 times.
  • the immuno-isolation device of the cells producing the immunomodulator avoids these problems. It represents a technical advantage over the systems developed in the prior art.
  • the antigenic component is preferably a whole tumour cell, advantageously irradiated. Because it is known that some antigens are present on many tumours from the same lineage, the used tumour cells may be an allogeneic ones. However, the tumour cells are preferably autologous with respect to the patient.
  • the tumour cells which are the source of antigenic component
  • the immuno-isolated cells which are the source of immunomodulatory component
  • the administration mode of cells of the invention is chosen to be the most efficient.
  • the mode is adapted to the desired localisation for the cells and the antigenic component.
  • a preferred localisation is sub-cutaneous because this region is rich in dendritic cells.
  • the administration can be made intradermally. Other localisations likely to favour the expected immune response are also preferred.
  • the immunomodulatory secreting cells are loaded in a capsule, which is to be implanted.
  • the capsule is removed after 2 to 7 days, preferably after 5 to 7 days.
  • the invention also relates to a process for treating a patient suffering from cancer. Particularly well suited cancers and states are described in detail above.
  • This process comprises the administration of a composition containing immuno-isolated cells producing an immunomodulatory agent.
  • the immuno-isolated cells may be autologous or allogeneic, in particular, they may be autologous tumour cells.
  • the administered composition may be injected, ingested, implanted, applied, or any other administration means.
  • the composition may comprises any pharmaceutical additive necessary for the survival of the cells and for the success of the administration or implantation.
  • the composition may also contain an antigenic component which is generally irradiated whole tumour cells. It is preferably administered at a site distant from the tumour location, where no previous immune response is supposed to have taken place. However, the composition may be administrated in the vicinity of tumour cells of the patient to be treated.
  • immuno-isolated cells producing or secreting an immunomodulatory agent, as described above, are used in therapy, particularly in cancer therapy. These cells are also possibly used in the field of vaccination.
  • Another use which is perfectly adapted to the cells of the invention is in the manufacture of an adjuvant.
  • the function of the adjuvant is to enhance an immune response which is considered too weak. If so, the immunomodulatory agent produced by the immuno-isolated cells of the invention is an immunostimulator.
  • Another use is in the manufacture of a medicament for the prevention or the treatment of cancer.
  • the characteristics of such an application are already well documented above.
  • Kits comprising cells of the invention are described above.
  • the present application also envisages the use of at least one of these kits for the manufacture of a vaccine.
  • This example shows the ability of different cell lines to secrete human GM-CSF with and without being irradiated.
  • GM-CSF cDNA Human GM-CSF cDNA was cloned into a retroviral vector (MFG) as described in the literature (Danos Olivier and Mulligan Richard 1988 PNAS Vol. 85 p6460-64; Jaffe Elizabeth et al. 1993 Cancer research Vol. 53 p2221-26).
  • MFG retroviral vector
  • the human GM-CSF is inserted in-frame into the retroviral vector.
  • the MFG-hGM-CSF construct was sequenced in order to ensure correct in-frame cloning.
  • transient transfection technique with lipofectamine MFG-hGM-CSF construct was transfected into 293-gpg cells as described in the literature (Ory Daniel et al. 1996 PNAS Vol. 93 p11400-06). With adequate selection, these cells produce pseudotyped retroviral particle containing the hGM-CSF gene. These viral particles are replication defective but can infect a wide range of mammalian cells.
  • the supernatant of transfected 293-gpg cells is used to infect dividing cells. This is performed with polybrene and no selection is performed. Therefore the whole cell population is used for subsequent analysis.
  • the infected cells are then tested for their ability to secrete human GM-CSF.
  • a classical ELISA is performed to measure the amount of GM-CSF in the supernatant of the various cell type tested.
  • Murine and human cell lines were tested for their ability to secrete human GM-CSF at various time points and also after irradiation 3500 rads.
  • the different tested cell lines are the following:
  • Non-transfected cells are also tested as negative control.
  • the Results are expressed in ng of h-GM-CSF by 10 6 cells at 48 hours no irradiation irradiation Renca wt 0 0 B16-wt 0 0 Renca h-GM-CSF 23400 9300 B16-h-GM-CSF 37650 Cell line A 0.9 Cell line A h-GM-CSF 18600 14900 Cell line B 1.2 Cell line B h-GM-CSF 6300 2950 Media only 0 0
  • Murine cancer cell lines and human fibroblast cell lines are able to secrete large amount of human GM-CSF for prolonged period of time in-vitro. No decrease has been observed in the production with time (at least three weeks after infection, for non-irradiated cells).
  • This example concerns the reproduction, using Onco-Maxi Vax, of the vaccination efficacy observed in the classical setting, when GM-CSF is produced by the irradiated tumour cells, in both wild-type mice and GM-CSF deficient mice. It also enables documentation of any new toxicity related to the use of the capsule, its manipulation or the cells it contains.
  • characterization of the response by standard techniques is carried out.
  • the negative control group Vaccination with irradiated, unmanipulated wild-type B16 melanoma cells (B16 WT).
  • the positive control group The standard technique used in the laboratory: Vaccination with irradiated, GM-CSF secreting B 16 melanoma cells. (B16-GM)
  • the investigational group Vaccination with irradiated, un-manipulated B16 melanoma cells (B16 WT) in close contact with a sub-cutaneoulsy implanted capsule containing cells releasing murine GM-CSF.
  • the vaccine combines irradiated wild type B16 melanoma cells injected in close contact to macrocapsule made of PES (polyethersulfone). This capsule contains 200 000 Renca cells retrovirally engineered to secrete GM-CSF. The encapsulated cells are mixed with a collagen-based matrix.
  • the B16 GM and the Renca GM cells are generated using the same transfection technique. Briefly, the murine GM-CSF cDNA was inserted into the MFG retroviral vector. Retroviral particles containing the mGM-CSF cDNA were obtained after lipofectamine transfection of 293-CPG cells. This infectious, non-replicative retroviral particles were harvested, centrifuged, concentrated and used to infect B 16 and the Renca cells respectively. The amount of GM-CSF released by the B16-GM and the Renca-GM is measured by standard Elisa technique.
  • Cells Culture in DMEM media with 10% inactivated calf serum +penicillin and streptomycin. Harvest from cell culture plates is performed one to two hours before injection. B16 WT and B16 GM adherent cells are washed with PBS x1, detached with Trypsin EDTA (Life tech) then washed ⁇ 3 in HBSS. Cells are then counted and resuspended in HBSS at the described concentration.
  • Renca GM and Renca Wt are the cells loaded into the capsules. These cells are cultured in the same DMEM media as above.
  • B16-WT or B16 GM are harvested, resuspended at the concentration of 2 ⁇ 10 6 /ml and irradiated at 3500 rad. Capsules are implanted subcutaneously on the abdomen in the appropriate groups after irradiation (3500 rad).
  • Vaccination is performed sub-cutaneously with 1 ⁇ 10 6 cells into 500 ul on the abdomen. Groups with the capsule and the B16 cells co-injection, the cells are injected in close contact with the capsule. Superficial anesthesia is used to ensure reproducibility of the procedure.
  • B16 WT are harvested from culture dishes and resuspended at a concentration of 1 ⁇ 10 6 /ml. Injection of 5 ⁇ 10 5 cells in 500 ul is performed on the upper-back.
  • mice free of tumour at day 80 have long-lasting specific anti-tumour immunity as long as the control groups gave the results that allow validation of the experiments.
  • mice with implanted capsule containing increasing numbers of irradiated GM secreting cells The effect of empty capsule is also analysed. This is performed by observation of the animal for any local or systemic toxicity. Serum level of GM-CSF are assessed by ELISA. Histological analysis is performed on the vaccination site. This toxicity evaluation is performed with 2 mice per group.
  • This example concerns the preparation of Onco-Maxi Vax, for the vaccination of a human patient.
  • the protocol gives detailed information regarding the preparation of the antigenic load, the generation of the immuno-isolated cytokine provider and the immunization with the two components from the Onco-Maxi-Vax.
  • tumour mass primary lesion or metastasis
  • An standard pathological examination is performed on a portion of the mass in order to confirm the malignant nature of the harvested material. It is then processed in order to obtain a single cell suspension. This is performed by both mechanical and enzymatic methods.
  • the tumour mass is first cut in smaller pieces using dissecting microscope, then the tumor is put into a sterile bag with a sterile solution containing various enzymes (collagenase).
  • the bag is inserted into a cell blender (Stomacher Lab System) that will process the product into a cell suspension.
  • the combination of enzymatic and mechanical activities at 37° C. for few hours allows the efficient dissociation of the extra-cellular matrix of the tumour and turn it into single cell suspension. This is performed in serum free solution.
  • the cells are then washed three time with HBSS using a refrigerated centrifuge (Sorvall) 4° C., 5 minutes, 700 rpm, and resuspended in HBSS. Cells are then counted using Trypan blue (Fluka) solution and a Neubauer chamber.
  • a refrigerated centrifuge Sorvall 4° C., 5 minutes, 700 rpm, and resuspended in HBSS.
  • Cells are then counted using Trypan blue (Fluka) solution and a Neubauer chamber.
  • the cells are resuspended at a chosen concentration, irradiated at 10000 rads in an irradiator devoted for clinical use, aliquoted and frozen in freezing media containing 10% DMSO.
  • the cells to be introduced into the capsules are allogeneic (obtained from a human cell line).
  • a human cell line obtained from a human cell line.
  • cell lines that have already been approved in clinical protocols such as immortalized fibroblasts or myoblasts. These cells are first stably transfected with human GM-CSF cDNA.
  • retroviral and electroporation Two methods of transfection can be used: retroviral and electroporation.
  • retroviral transfection hGM-CSF cDNA is inserted in-frame into the MFG retroviral vector and transcription is driven by the LTR of the virus.
  • the plasmid does not contain any selection marker or antibiotic resistance gene.
  • hGM-CSF cDNA is under the CMV promoter and the plasmid contains a selective marker (such as an antibiotic resistance gene).
  • the invention therefore includes the use of different types of cells for transfection and of different GM-CSF plasmids. This leaves more flexibility with respects to local health department regulations.
  • the cytokine producing cells is cultured in serum free media at 37° C. with 5% CO2 using standard techniques. Harvesting is performed as follow: The supernatant of confluent, adherent cells in a 10 cm culture plate is removed and the cells are washed once with 5 ml of autoclaved Phosphate buffered Saline (PBS) for 5 minutes at 37° C. PBS is then removed and 2 ml of Trypsin-EDTA 0.5% (Life Technologies No25300054) is added and the cells are incubated for four minutes at 37° C. The trypsin/EDTA allows the detachment of the adherent tumor cells.
  • PBS autoclaved Phosphate buffered Saline
  • the cells are then harvested with a 2 ml pipet and diluted into 5 ml of Hank's balanced salt solution (HBSS Life Technologies No24020091).
  • HBSS Life Technologies No24020091 Hank's balanced salt solution
  • the cells are washed three time with HBSS using a refrigerated centrifuge (Sorvall) 4° C., 5 minutes, 700 rpm) and resuspended in HBSS. Cells are then counted using Trypan blue (Fluka) solution and a neubauer chamber.
  • hGM-CSF The quantity of hGM-CSF produced and secreted by the cells is evaluated Elisa (R&D system and Pharmingen kits) on filtered cell's supernatant. This analysis allows the selection of the best cytokine producing cell-line.
  • the cytokine producing cells are loaded into macrocapsules or embedded in microcapsules.
  • the capsules can be made of various polymers with various sizes and pores, such as PES and TF10/10 capsules with and without PVA (polyvinyl alcohol) matrix.
  • the capsule is loaded with the cell suspension at a rate of 10.5 ul/min. Sealing of the capsule is obtained by polymer glue, but can also be done by heating or surgical clips. Analysis from supernatant of encapsulated cells containing GM-CSF secreting cells showed that a stable, continuous release of GM-CSF is achieved for at least fifteen days after loading, with cytokine levels that are around 70 ng/10 5 cells/24 hrs.
  • the capsule containing the cytokine producing cells is placed in the sub-cutaneous tissue using a small skin incision under local anaesthesia.
  • the skin is closed with surgical tape.
  • Vaccination is repeated every two weeks four times (and more if enough autologous tumour cells are available). The site of vaccination is different at each immunization (abdominal wall, upper arms, thighs, thorax, etc).
  • Onco-Maxi-Vax Autologous Irradiated Tumour Cells +Encapsulated GM-Producers Cells. In Vivo Results in Mouse Model Showing Survival at 50 Days after B16WT Challenge
  • This example concerns in vivo data in mice, which are protected from death induced by the melanoma tumor cell line B16.
  • mice C57B1/6 Strain at Least 8 Weeks of Age
  • Cells Culture in DMEM media with 10% inactivated calf serum +penicillin and streptomycin. Harvest from cell culture plates is performed one to two hours before injection. B16 WT and B16 GM adherent cells are washed with PBS ⁇ 1, detached with Trypsin EDTA (Life tech) then washed ⁇ 3 in HBSS. Cells are then counted and resuspended in HBSS at 2 ⁇ 10 6 cells/ml for vaccination and 4 ⁇ 10 5 cells/ml for tumor challenge.
  • Renca GM GM-CSF secreting Renca cells
  • the capsules were PES capsules with PVA (polyvinyl alcohol) matrix. These cells are cultured in the same DMEM media as above.
  • mice were immunized with either irradiated capsule only (group 1), capsule +irradiated B16 wt (group 2) or irradiated B16-GM-CSF (group 3) on day—7.
  • Each capsule containing 10 5 GM-CSF secreting Renca cells was irradiated (3500 rad) prior to implantation.
  • Mice from groups 1 and 2 were implanted with 2 capsules each, put sub-cutaneously in a V-shape on the abdomen. After 3 hours, the groups 2 and 3 were injected with irradiated B16 cells, 10 6 B16 wt cells for group 2, 10 6 B16-GM-CSF cells for group 3, sub-cutaneously between the two capsules.
  • mice were challenged with live B16 wt 2 ⁇ 10 5 cells on the upper back.
  • mice with growing tumor superior to 1 cm or showing tumor ulceration were sacrificed. All these were tumor-related. Non-sacrificed mice remained tumor-free. Survival represents percentage of tumor-free mice in each group.
  • This animal experiment shows a very good efficacy of encapsulated GM-CSF secreting cells on survival at 50 days (see FIG. 4 ).
  • This experiment was repeated and the results are shown in FIG. 5 with a different graphical representation.
  • This second experiment also illustrates the efficiency of vaccination with encapsulated GM-CSF secreting cells on survival over more than two months.

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EP02013249A EP1374893A1 (fr) 2002-06-17 2002-06-17 Vaccination au moyen de cellules immuno-isolées produisant un immunomodulateur
EP02013249.4 2002-06-17
US46456503A 2003-06-17 2003-06-17
US11/058,883 US20060024318A1 (en) 2002-06-17 2005-02-16 Vaccination with immuno-isolated cells producing an immunomodulator

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WO2018044888A1 (fr) * 2016-08-29 2018-03-08 Chen James C Systèmes, dispositifs et procédés de vaccination de tumeur

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US8551749B2 (en) * 2009-04-23 2013-10-08 The Invention Science Fund I, Llc Device including bone cage and method for treatment of disease in a subject
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WO2018044888A1 (fr) * 2016-08-29 2018-03-08 Chen James C Systèmes, dispositifs et procédés de vaccination de tumeur

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EP2147682A2 (fr) 2010-01-27
US20080107686A1 (en) 2008-05-08
WO2003105895A1 (fr) 2003-12-24
AU2003246369A1 (en) 2003-12-31
ATE447968T1 (de) 2009-11-15
ES2560105T3 (es) 2016-02-17
EP1513551A1 (fr) 2005-03-16
EP2147682A3 (fr) 2012-07-25
EP2147682B1 (fr) 2015-11-11
EP1374893A1 (fr) 2004-01-02
ES2335490T3 (es) 2010-03-29
US20140341982A1 (en) 2014-11-20
DK1513551T3 (da) 2010-03-08
DE60330004D1 (de) 2009-12-24
EP1513551B1 (fr) 2009-11-11
DK2147682T3 (en) 2016-02-01
US9814682B2 (en) 2017-11-14

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