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WO2009125332A1 - Procédé nouveau et sans danger pour une immunisation contre des agents de maladies infectieuses - Google Patents

Procédé nouveau et sans danger pour une immunisation contre des agents de maladies infectieuses Download PDF

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WO2009125332A1
WO2009125332A1 PCT/IB2009/051437 IB2009051437W WO2009125332A1 WO 2009125332 A1 WO2009125332 A1 WO 2009125332A1 IB 2009051437 W IB2009051437 W IB 2009051437W WO 2009125332 A1 WO2009125332 A1 WO 2009125332A1
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cells
bao
antigens
cell
viruses
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PCT/IB2009/051437
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Peter Bromley
Jean-Jacques Sunier
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Peter Bromley
Jean-Jacques Sunier
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Publication of WO2009125332A1 publication Critical patent/WO2009125332A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • 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 swine influenza (flu) vaccine was associated with a severe paralytic illness called Guillain-Barre Syndrome (GBS).
  • GBS Guillain-Barre Syndrome
  • Some live virus vaccines such as the chickenpox vaccine, can cause mild versions of the disease they protect against, but this is usually only a serious problem if the patient has a severely compromised immune system.
  • Subunit vaccines raised against purified antigens of infectious agents have had some success, but a problem with this approach is that purifying the infectious agent, isolating the surface and other antigens, purifying these proteins etc, frequently changes the presentation form of these antigens when subsequently injected into patients.
  • the technology of this invention totally avoids these drawbacks; the subunit vaccines are produced by the cells in the Bio- artificial organs (BAO), are secreted in their natural format, with all associated post- translational modifications, ensuring an effective immune response.
  • BAO Bio- artificial organs
  • This invention ensures that infectious organism antigens capable of eliciting neutralizing antibody formation, are correctly formed, and presented to the immune system in an optimal manner. This invention avoids alterations to such sub-unit vaccines that result from purification procedures and storage prior to employment.
  • This new vaccination procedure of this invention can be applied to essentially all vaccination needs today, these include childhood infections, tropical diseases and cancer vaccines, and nosocomial infections.
  • childhood vaccines that can be treated using this invention include, but are not limited to:
  • Measles + Mumps + Rubella MMR II or Priorix
  • Pneumococcal Prevenar
  • the BCG vaccine to prevent tuberculosis is no longer given routinely to school-age children. Instead, the vaccine is now only recommended for infants and children at high risk of the disease.
  • Further applications include vaccination against essentially any organism that presents a health risk, and can be produced using this invention; for instance the antigens of organisms specific to certain parts of the world, such as tropical diseases, and cancer-associated vaccines, such as by the human papilloma viruses.
  • a major and growing health risk is that of hospital-acquired infections, which encompass almost all clinically evident infections that do not originate from a patient's original admitting diagnosis. Within hours after admission, a patient's flora begins to acquire characteristics of the surrounding bacterial pool. Most infections that become clinically evident after 48 hours of hospitalization are considered hospital-acquired. Infections that occur after the patient's discharge from the hospital can be considered to have a nosocomial origin if the organisms were acquired during the hospital stay.
  • Iatrogenic risk factors include pathogens that are present on medical personnel hands, invasive procedures (e.g., intubation, indwelling vascular lines, urine catheterization), and antibiotic use and prophylaxis.
  • Organizational risk factors include contaminated air-conditioning systems, contaminated water systems, and staffing and physical layout of the facility (e.g., nurse-to-patient ratio, open beds close together).
  • Patient risk factors include the severity of illness, underlying immunocompromised state, and length of stay.
  • NIS Surveillance System of the Centers for Disease Control and Prevention performed a survey from October 1986 to April 1998. They ranked hospital wards according to their association with central-line bloodstream infections. The highest rates of infection occurred in the burn, neonatal, and pediatric departments. Nosocomial infections are estimated to more than double the mortality and morbidity risks of any admitted patient, and they probably result in about 90,000 deaths a year in the United States alone.
  • Nosocomial infections are caused by viral, bacterial, and fungal pathogens. These pathogens should be investigated in all febrile patients who are admitted for a nonfebrile illness.
  • Viruses are the leading etiologies of nosocomial infections in pediatric patients (responsible for up to 14% of Hospital Associated Infections (HAI) with identifiable pathogens).
  • HAI Hospital Associated Infections
  • Bacterial and fungal infections are less common. However they are significantly associated with more morbidity and mortality. Most patients who are infected with nosocomial bacterial and fungal pathogens have a predisposition caused by invasive supportive measures such as intubation and the placement of intravascular lines and urinary catheters. Fungal infections more likely to arise from the patient's own flora; occasionally, they are caused by contaminated solutions (e.g., those used in parenteral nutrition).
  • Nosocomial etiologies in bloodstream infections include the following: o Coagulase-negative staphylococci, 40% o Enterococci, 11.2% o Fungi, 9.65% o Staphylococcus aureus, 9.3% o Enterobacter species, 6.2% o Pseudomonads, 4.9% o Acinetobacter baumannii with substantial antimicrobial resistance - Reported with increasing frequency
  • Urinary Tract Infections include the following: o Gram-negative enterics, 50% o Fungi, 25% o Enterococci, 10%
  • Nosocomial etiologies in surgical-site infections include the following:
  • antibiotics Besides increased drug resistance, high-dose and prolonged antimicrobial therapy can eliminate helpful bacterial flora and predispose people to infection.
  • a common adverse effect of antibiotics is diarrhea, which can lead to loss of essential vitamins and minerals, especially vitamin K, magnesium, and zinc.
  • Other adverse effects of antibiotic therapy include vitamin deficiencies, seizures, allergic shock (in people who are allergic to antibiotics), autoimmune disease, decreased platelets, kidney injury, drug/drug interaction.
  • the invention features the use of encapsulated cells in a BAO that have been genetically modified to produce antigens of the most dangerous viral, bacterial and fungal infective agents, and this under the control of a heat-inducible promoter. Such expression control allows the optimization of the production of an immune reaction against these antigens.
  • "Boosting" by repeated production of antigens is simply provided by this invention, and is achieved by repeating the heat induction cycles of protein production at defined times.
  • BAO may be used to supply biologically active molecules for the treatment or prevention of neurodegenerative conditions such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and Acquired Immune Deficiency Syndrome-related dementia. Additionally, lymphokines and cytokines may also be supplied by BAOs to modulate the host immune system. Other biologically active molecules which may be provided by bioartificial organs include, catecholamines, endorphins, enkephalins, and other opioid or non-opioid peptides that are useful for treating pain. Enzymatic deficiencies may also be treated by using BAOs. Alternatively, the biologically active molecule may remove or eliminate deleterious molecules from the host. For example, a BAO may contain cells which produce a biologically active molecule that can be used to "scavenge" cholesterol from a host.
  • BAOs are known. See, e.g., Aebischer (U.S. Pat. No. 5,158,881), Dionne et al. (WO 92/03327), Mandel et al. (WO 91/00119), Aebischer (WO 93/00128). BAOs also include extravascular diffusion chambers, intravascular diffusion chambers, intravascular ultrafiltration chambers, and microcapsules. See, e.g., Lim et al., Science 210:908-910 (1980); Sun, A.M., Methods in Enzymology 137: 575-579 (1988); Dunleavy et al. (WO 93/03901) and Chick et al. (U.S. Pat. No. 5,002,661).
  • differentiated, non-dividing cells may be preferred over dividing cells for use in BAOs because they allow for the optimal production of the desired biologically active molecule.
  • differentiated, non-dividing cells produce a greater quantity of a desired therapeutic protein than dividing cells because the expression of differentiation specific genes and cell division are thought to be antagonistic processes. Wollheim, "Establishment and Culture of Insulin- Secreting beta-cell lines," Methods in Enzymology, 192, p. 223-235 (1990). Cellular replication capacity decreases as cells differentiate. In many cases, proliferation and differentiation are mutually exclusive. Gonos, "Oncogenes in Cellular Immortalisation and Differentiation," 13, Anticancer Research, p. 1117 (1993).
  • differentiated tissue is advantageous because the functional properties of tissue desired for incorporation into a BAO have most often been defined by the properties of differentiated tissue in vivo.
  • Another advantage to the use of differentiated, non-dividing cells is that the cell number within the BAO will remain relatively constant. This, in turn, leads to more predictable results and stable dosage for the recipient host.
  • differentiated cells are better suited for use in BAOs which encapsulate more than one cell type secreting biologically active molecules. In such BAOs, if dividing cells are used, different cell types may grow at different rates, resulting in the overgrowth of one cell type. By using differentiated, non-dividing cells, the relative proportions of two or more synergistic cell types can be more readily controlled.
  • necrotic tissue may also release excess cellular proteins which unnecessarily flood the host with xeno-proteins or other factors which are detrimental to the surviving cells, e.g., factors which elicit a macrophage or other immune response.
  • Controlling the cell number and cell location within the BAO also provides the advantage of facilitating optimization of the BAO membrane and other device parameters to the particular encapsulated cell type. This is because the required device characteristics are more readily determined for a fixed cell population than for a dividing cell population in the BAO. Additionally, long term delivery of biologically active molecules can be achieved.
  • the invention addresses the such problems by providing methods and compositions for controlling the distribution of cells (i.e. cell number or cell location in the BAO, or both) when encapsulated in a BAO.
  • the methods and compositions of this invention include (1) methods and compositions for modification of the cells that are encapsulated within the BAO and (2) methods and compositions for modifying the growth surfaces within the BAO.
  • Methods and compositions for cellular manipulation include genetic alteration of the cells with a gene which encodes a product that influences cell proliferation or differentiation.
  • the treatment may comprise providing a chemical compound or growth factor which inhibits proliferation or induces differentiation.
  • the treatment may comprise removing from the growth medium a chemical compound or growth factor which stimulates proliferation or inhibits differentiation.
  • the treatment may be before or after encapsulation in the BAO, preferably before encapsulation. Additionally, cell proliferation may be controlled by irradiation.
  • Methods and compositions for growth surface modification include coating at least one growth surface within the BAO with one or more extracellular matrix molecules ("ECM").
  • ECMs may be coated directly onto the luminal surface or any inner support within the BAO, or onto microsphere carriers ("microcarriers").
  • Cells or cell-seeded microcarriers may additionally be suspended in a matrix material that physically inhibits cell proliferation. Further, the matrix material may be derivatized with chemical or peptide derivatives.
  • a growth surface of the BAO can be modified by chemical treatment to inhibit cell attachment or to enhance cell attachment to the BAO's luminal surface.
  • the growth surface can be modified by addition of an inert scaffold prior to cell loading. The scaffold physically inhibits cell outgrowth and provides additional sites for cell attachment. It is to be understood that the various methods and compositions for cell modification and for growth surface modification are not mutually exclusive and may be used in combination.
  • This invention relates to a biocompatible, immuno-isolatory, implantable vehicle.
  • the instant vehicle is suitable for isolating biologically active cells or substances from the body's protective mechanisms following implantation into an individual.
  • the instant vehicle is comprised of (a) a core which contains isolated cells, either suspended in a liquid medium or immobilized within a hydrogel matrix, and (b) a surrounding or peripheral region ("jacket") of permselective matrix or membrane which does not contain isolated cells, which is biocompatible, and which is sufficient to protect the isolated cells in the core from immunological attack.
  • This immuno-isolatory vehicle is useful (a) to deliver a wide range of biologically active moieties, including high molecular weight products, to an individual in need of them, and/or (b) to provide needed metabolic functions to an individual, such as the removal of harmful substances.
  • the instant vehicle is contains a multiplicity of cells, such that implantation of a few or a single vehicle is sufficient to provide an effective amount of the needed substance or function to an individual.
  • a further advantage offered by the instant vehicle is practicality of retrieval.
  • An immuno-isolatory vehicle of the present invention is implanted into the individual (referred to as the recipient), using known techniques or methods and selected for the particular immuno- isolatory vehicle and site of implantation. Once implanted, cells isolated within the biocompatible immuno-isolatory vehicle produce the desired moieties or perform the desired function(s). If moieties are released by the isolated cells, they pass through the surrounding or peripheral permselective membrane or hydrogel matrix into the recipient's body.
  • They also relate a method of isolating cells within a biocompatible, immuno-isolatory implantable vehicle, thereby protecting the cells within the vehicle from immunological attack after being implanted into an individual.
  • some low molecular weight mediators of the immune responses e.g. cytokines
  • the isolated cells are protected from attack by the recipient's immune system and from potentially deleterious inflammatory responses from the tissues which surround the implanted vehicle.
  • the isolated cells are maintained in a suitable local environment. In this manner, needed substances or metabolic functions can be delivered to the recipient even for extended periods of time, and without the need to treat the recipient with potentially dangerous immunosuppressive drugs.
  • the vehicle is formed by co-extruding from a nested-bore extrusion nozzle materials which form the core and surrounding or peripheral regions, under conditions sufficient to gel, harden, or cast the matrix or membrane precursor(s) of the surrounding or peripheral region (and of the core region).
  • a particular advantage of this co-extrusion embodiment is that the cells in the core are isolated from the moment of formation of the vehicle, ensuring that the core materials do not become contaminated or adulterated during handling of the vehicle prior to implantation.
  • a further advantage of the co-extrusion process is that it ensures that the surrounding or peripheral region is free of cells and other core materials.
  • the permeability and biocompatibility characteristics of the surrounding or peripheral region are determined by both the matrix or membrane precursor materials used, and the conditions under which the matrix or membrane is formed.
  • the immuno-isolatory vehicle is formed stepwise.
  • the immuno-isolatory vehicle being made includes a hydrogel core containing the isolated cells
  • the core can be formed initially, and the surrounding or peripheral matrix or membrane can be assembled or applied subsequently.
  • the surrounding or peripheral matrix or membrane can be preformed, and then filled with the preformed isolated-cell containing core material or with materials which will form the core (i.e., core precursor materials).
  • the vehicle is sealed in such a manner that the core materials are completely enclosed. If a core precursor material is used, the vehicle is then exposed to conditions which result in formation of the core.
  • This present invention differs from the above mentioned BAO technologies in that the encapsulated cells contain gene constructs in which the antigens, including but not limited to those of the most dangerous bacterial and fungal nosocomial infective agents, have been placed under the expression control of a heat-inducible promoter.
  • cells encapsulated in a BAO are induced to produce and to secrete the selected antigens including but not limited to those of the most dangerous bacterial and fungal nosocomial infective agents. Placing their genes under hsp-70B expression control allows these antigens to be produced by the effect of local heat applied over the site of implantation of the BAO. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • Preferred antigens of this invention include but are not limited to those involved in chidhood infections, tropical diseases, cancer vaccines and nosocomial diseases, and include those of the following organisms:
  • Nosocomial etiologies in bloodstream infections include the following: Coagulase-negative staphylococci, Enterococci, Fungi, Staphylococcus aureus, Enterobacter species, Pseudomonads, Acinetobacter baumannii with substantial antimicrobial resistance - Reported with increasing frequency.
  • Nosocomial etiologies in UTI include Gram-negative enterics, Fungi, Enterococci,
  • Nosocomial etiologies in surgical-site infections include S aureus, Pseudomonads, Coagulase- negative staphylococci, Enterococci, fungi, Enterobacter species, and Escherichia coli.
  • Viruses are the leading etiologies of nosocomial infections in pediatric patients (responsible for up to 14% of hospital acquired diseases with identifiable pathogens). Neutralization-inducing antigens of these viruses are included in preferred embodiments of this invention.
  • This invention can also be employed to vaccinate against tropical diseases; African trypanosomiasis, Dengue fever, Leishmaniasis, Malaria, Schistosomiasis, Tuberculosis, Chagas disease, Leprosy, Lymphatic filariasis, and Onchocerciasis
  • This invention also provides a safer vaccination approach to viral diseases such as influenza, human papilloma viruses and the AIDS viruses.
  • a subunit vaccine has been shown to be effective in immunization
  • this invention provides a safe and convenient approach; in addition, vaccination against multiple infectious agents can be performed with no increase in risk.
  • the immune response to a protective antigen can be influenced by the location of the antigen. Although good immune responses have been observed for antigens retained in the cytoplasm or secreted into the periplasm of Gram-negative vaccines (Roberts, M., Chatfield, S.N., and Dougan, G. 1994. Salmonella as carriers of heterologous antigens. In Novel delivery systems for oral vaccines. D. T. O'Hagan, editor. CRC Press.
  • Hess, J. et al.2000 Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG. FEMS Immunol. Med. Microbiol. 27:283-289) or homologous (Hess, J. et al.2000. Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination.
  • Microbes Infect. 2:1799-1806 secretion mechanisms can further enhance the level and type of immune response induced.
  • type III secretion apparatus of Salmonella and Yersinia antigens with T cell epitopes can be delivered into the cytoplasm of antigen-presenting cells within the immunized eukaryotic host, resulting in a CD 8 -restricted CTL response (R ⁇ ssmann, H. et all 998. Delivery of epitopes by the Salmonella Type III secretion system for vaccine development. Science. 281 :565-568).
  • SCOTS Selective Capture of Transcribed Sequences
  • a rapidly developing approach is the use of DNA vaccines.
  • the gene for a pathogen protein is introduced into human cells and is then expressed to produce the protein inside the body.
  • the DNA vaccination method There are many advantages to the DNA vaccination method. For example, it is much cheaper to produce and distribute large amounts of DNA than it is to produce and distribute large amounts of protein. Also, the same strategy can be used to tackle virtually any pathogen, so multiple vaccinations are possible.
  • Technical hurdles that need to be overcome include finding efficient ways of getting the DNA into human cells, making sure the gene is expressed once it is inside the cell, and making sure the DNA does not integrate into the genome and disrupt our own genes.
  • DNA vaccines in clinical and pre-clinical trials, including vaccines for HIV, herpes, hepatitis and influenza.
  • This invention describes a new approach to vaccination, whereby cells are genetically modified to produce viral, bacterial and fungal antigens, in a heat-inducible manner; these cells are introduced into the patient's body as an encapsulated BAO.
  • the organisms selected in this invention are those associated with nosocomial diseases, although the invention is not in any way limited to such organisms.
  • any organism that presents a health risk can be produced using this invention, for instance the antigens of organisms specific to certain parts of the world, such as tropical diseases, and cancer-associated infections such as by the human papilloma viruses.
  • This invention completely avoids the risks associated with attenuated organism based vaccines, and also avoids the complications encountered with DNA vaccines.
  • a variety of cell types can be employed in this invention, from animal or human origin.
  • a preferred embodiment is to have cells that do not divide extensively once introduced into the BAO.
  • Many transformed cells or cell lines are most advantageously isolated within a vehicle having a liquid core comprising a nutrient medium, optionally containing a liquid source of additional factors to sustain cell viability and function, such as fetal bovine serum.
  • the term "cells" means cells in any form, including but not limited to cells retained in tissue, cell clusters, and individually isolated cells.
  • the vehicle employed in the current invention may be prepared of sufficient size to deliver an immune response from a single or just a few (less than 10) implanted and easily retrievable vehicles.
  • the core may be composed of a matrix formed by a hydrogel which stabilizes the position of the cells in cell clumps.
  • hydrogel herein refers to a three dimensional network of cross-linked hydrophilic polymers. The network is in the form of a gel substantially composed of water, preferably but not limited to gels being greater than 90% water.
  • Cross-linked hydrogels can also be considered solids because they do not flow or deform without appreciable applied shear stress, compositions which form hydrogels fall into three classes for the purposes of this application.
  • the first class carries a net negative charge and is typified by alginate.
  • the second class carries a net positive charge and is typified by extracellular matrix components such as collagen and laminin. Examples of commercially available extracellular matrix components include Matrigel.TM. and Vitrogen.TM..
  • the third class is net neutral in charge.
  • An example of a net neutral hydrogel is highly crosslinked polyethylene oxide, or polyvinyalcohol.
  • One particularly advantageous use of hydrogel cores pertains to the encapsulation of actively dividing cells.
  • Alginate or other hydrogels may be included in suspensions of actively dividing cells to be encapsulated. Following encapsulation and generation of the gel, the encapsulated cells are somewhat immobilized within the gel and new cells produced during cell division stay localized near the parent cell. In this manner clusters of cells are produced within the core.
  • Stable human hepatocyte cell lines include, but are not limited to Hep G2, Hep 3B, C3A, C3 A/Hep G2, Chang cells, Fa2N4, HuH7, Hepa RG, LH 86, NKNT-3, OUMS-29, TTNT- 16-3 as well as cell lines immortalised using the human telemorase reverse transcriptase gene via a retroviral vector.
  • Secretory cells of the immune system include, but are not limited to DAUDI (Burkitt lymphoma cells), JURKAT and Molt 4 (human acute T cell leukaemia cell lines), and K562 human chronic myelogenous leukaemia cells).
  • Cell lines are derived from a reversibly immortalized human hepatocyte line such as TTNT- 16-3. Briefly, human hepatocytes are transduced with a recombinant retroviral vector (SSR#197) (Westerman KA, Leboulch P: Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A93 :8971 -8976,1996) containing cDNAs expressing hTERT, for immortalization, and enhanced green fluorescent protein (EGFP), for a selection marker, flanked by a pair of recombination target loxPs.
  • SSR#197 Westerman KA, Leboulch P: Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A93 :8971 -8976,1996) containing cDNAs expressing hTERT
  • EGFP-positive immortalized clones Two days after three rounds of SSR#197 transduction, cells are sorted using a FACSCalibur system (BD Biosciences Immunocytochemistry Systems, San Jose, CA) for recovering EGFP-positive cell populations.
  • FACSCalibur system BD Biosciences Immunocytochemistry Systems, San Jose, CA
  • One of the EGFP-positive immortalized clones is selected on the basis of its hepatocyte-specif ⁇ c gene expression profile and negative tumorigenesis.
  • These cells are transduced with a plasmid encoding a tamoxifen-inducible Cre recombinase fused with paired mutant estrogen receptor ligand-binding domains (MerCreMer) under the control of the CAG promoter (cytomegalovirus IE enhancer, chicken ⁇ -actin promoter, and ⁇ -globin splicing acceptor) with a puromycin resistance gene.
  • the resultant puromycin-resistant clones grow steadily in tissue culture with serum-free ISE- RPMI medium (Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J: Growth of human hepatoma cell lines with differentiated functions in chemically defined medium.
  • An expression vector is constructed which allows for the selection of stable transfectants by selection for the zeocin antibiotic (Cayla) in both prokaryotic and eukaryotic cells.
  • the Zeocin resistance gene is obtained as a restriction digest fragment from the pZeoSV plasmid (Invitrogen) and is ligated to a fragment containing a bacterial origin of replication obtained by PCR amplification from pUC19 (New England Biolabs). This ligation mixture is then used to transform competent E. coli cells and the presence of the desired recombinant plasmid (pUC-Zeo) is selected for on Zeocin-containing bacterial plates.
  • a synthetic poly(A) sequence obtained as a restriction fragment from a digest of pGL3-Basic (Promega) is ligated into pUC-Zeo upstream of the HSP70B promoter, and the desired recombinant (pUC-ZeoA) will selected for Zeocin resistance.
  • the HSP70B driven expression cassette drives the expression of genes for the antigens of this invention. These expression cassettes can be transfected into for instance JURKAT cells, as an example of secretory cells of the immune system.

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention porte sur un organe artificiel biologique (BAO) contenant des cellules eucaryotes, lesdites cellules eucaryotes a) étant transformées de façon stable par des gènes codant pour des antigènes viraux, bactériens ou fongiques, b) de tels gènes étant placés sous le contrôle d'expression fonctionnel d'un promoteur inductible par la chaleur, c) le BAO étant introduit de façon rétractable sous la peau du patient devant être immunisé, d) les cellules à l'intérieur du BAO étant induites à exprimer et secréter lesdits antigènes à l'aide d'une chaleur locale, e) cette induction étant répétée si nécessaire pour induire une neutralisation efficace de la réponse d'anticorps à l'encontre desdits antigènes.
PCT/IB2009/051437 2008-04-07 2009-04-06 Procédé nouveau et sans danger pour une immunisation contre des agents de maladies infectieuses WO2009125332A1 (fr)

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IBPCT/IB2008/051300 2008-04-07

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