WO2000054786A1 - Methodes et compositions destines a rendre des hotes tolerants a la survie a long terme de greffons de tissus - Google Patents

Methodes et compositions destines a rendre des hotes tolerants a la survie a long terme de greffons de tissus Download PDF

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Publication number
WO2000054786A1
WO2000054786A1 PCT/US2000/006737 US0006737W WO0054786A1 WO 2000054786 A1 WO2000054786 A1 WO 2000054786A1 US 0006737 W US0006737 W US 0006737W WO 0054786 A1 WO0054786 A1 WO 0054786A1
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cells
encapsulated
mepo
weeks
rats
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PCT/US2000/006737
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Patrick Aebischer
Giovanni Peduto
Christopher Rinsch
Bernard-Laurent Schneider
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Modex Therapeutiques, S.A.
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Priority to AU37459/00A priority Critical patent/AU3745900A/en
Publication of WO2000054786A1 publication Critical patent/WO2000054786A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates generally to tissue transplantation. More particularly, the invention relates to methods and compositions for tolerizing a host mammal for long term survival of xenogeneic or allogeneic tissue subsequently transplanted into the host.
  • the transplantation of encapsulated cells that have been genetically modified to secrete therapeutic proteins has been used for the treatment of several human diseases including anemia, diabetes, and multiple neurodegenerative disorders.
  • the long-term delivery of therapeutic molecules either locally or systemically via the transplantation of encapsulated cells circumvents many of the problems encountered with conventional techniques of delivering pharmaceuticals.
  • Cell encapsulation allows delivery of engineered cells to isolated locations, such as the brain parenchyma, while eliminating the need for the repeated injections normally required to maintain drug concentration within its therapeutic window. Isolating cells by enclosing them within a semipermeable polymer membrane prevents the cell contact- mediated death of donor cells following transplantation into a recipient. This physical barrier does not, however, protect encapsulated cells from destruction by other immune defense mechanisms that do not require cell contact.
  • encapsulated cells are implanted into the recipient host.
  • the encapsulated cell implant can be retrieved when necessary or desired.
  • the cells can be either xenogeneic or allogeneic to the host.
  • the host is concurrently immunosuppressed transiently (for at least 1 week, preferably at least 2 weeks or 3 weeks, and most preferably for at least 4 weeks).
  • the encapsulated cell devices is then removed, and a second transplant from the same cell or tissue source as the first encapsulated cell device is implanted into the host, according to known techniques.
  • the second implant is preferably an encapsulated tissue or cell implant (in one or more encapsulation devices).
  • the invention demonstrates that host tolerance can be developed in the subcutaneous tissue to encapsulated xenogeneic cells following a transient immunosuppression. This has direct clinical relevance in the field of cell therapy, because capsules can be replaced without additional immunosuppression, thus facilitating long-term cell-based therapies. Long-term treatments are facilitated, because devices can be replaced without supplemental immunosuppression following encapsulated cell senescence. The safety aspect of encapsulation is also preserved, because a recipient host tolerized to encapsulated xenografts maintains the ability of rejecting unencapsulated xenogeneic cells in the rare event that cells escape from the device. BRIEF DESCRIPTION OF THE FIGURES FIG.
  • FIG. 1 is a graphical representation of the hematocrit levels of C3H mice, DBA/2J mice and Fischer rats that were implanted with mEpo-secreting capsules.
  • FIG. 2 is a comparison of the hematocrit in control Fischer rats, inmunosuppressed
  • FIG. 3 is a schematic diagram of the immunosuppression protocol of Fischer rats with FK506.
  • the schematic diagram shows FK506 dosing regimes used for the induction of long- term acceptance of encapsulated C1C12 mEpo cells in xenogeneic rat recipients (a) 1 week, (b) 2 weeks, and 4 weeks immunosuppression with FK506 (hatched bar: administration of
  • FK506 (1 mg/kg/day); * acquired tolerance was evaluated by implanting a second capsule in a separate set of experiments).
  • FIG. 4 is a set of graphical representations of the long-term acceptance of encapsulated C 2 C 12 mEpo cells in Fischer rats immunosuppressed for lweek (FIG. 4A), 2 weeks (FIG. 4B), and 4 weeks (FIG. 4C).
  • FIG. 5 is a graphical representation of the long-term acceptance of encapsulated C 2 C l2 mEpo cells in Fischer rats that were immunosuppressed for 2 weeks.
  • FIG. 6 is a graph showing that C3H mice, DB A/2J mice and Fischer rats were implanted with 280 kDa cut-off capsules containing C 2 C 12 mEpo cells. The hematocrit was monitored for 35 days, at which point all animals were explanted. Data is presented as mean ⁇
  • FIG. 9 is a graph showing that Fischer rats were transiently immunosuppressed for either 1, 2, or 4 weeks with FK506 (1 mg/kg/day) following implantation with mEpo secreting capsules (280 kDa cut-off). Animals in each group increased their hematocrits and maintained these elevated levels for a period of 13 weeks. Data is presented as mean ⁇ SEM.
  • FIG. 10 is a graph showing that Fischer rats were injected intramuscularly with mEpo secreting C 2 C 12 myoblasts and immunosuppressed for 4 weeks with FK506 (1 mg/kg/day).
  • FIG. 13 is a graph showing that long-term tolerance to encapsulated xenogeneic myoblasts.
  • Four Fischer rats were tolerized to capsules containing C 2 C 12 mEpo myoblasts by treating 4 weeks with FK506. On week 13, all devices were explanted, and the hematocrit eventually returned to pre-implant levels. Seven months later (week 41 ), these animals were reimplanted with encapsulated C 2 C 12 mEpo myoblasts leading to an increase in hematocrit.
  • FIG. 14 is a set of graphs showing that antigens implicated in rejection of encapsulated xenogeneic mEpo secreting myoblasts appear to be molecules inherent to myoblasts.
  • This invention provides methods and compositions for tolerizing of hosts for long term survival of allogeneic or xenogeneic tissue or cells. Methods of tolerization according to the invention demonstrate that immunomodulation can be used to prolong the survival of immunoisolated xenografts.
  • Immunoisolation as used herein is a technique in which engineered cells are enclosed within implantable polymeric capsules formed by permiselective membranes. Immunoisolation prevents the cell-to-cell contact between host and implanted tissues, eliminating direct xenorecognition, as the membranes used have a pore size that permits the diffusion of nutrients and bioactive molecules, while reducing passage of antibodies and complement molecules, as described below.
  • the methods of the invention include methods for improving survival of a transplant in a recipient host.
  • the methods of the invention comprise implanting one or more encapsulated cell devices into the recipient host.
  • An immunosuppressive agent is administered to the recipient host in an amount effective to permit survival of the encapsulated tissue or cell transplant for a period of at least one week.
  • the transplant has improved survival in the recipient host compared to a recipient host that did not receive an administration of an immunosuppressive agent in an amount effective to permit survival of the encapsulated tissue or cell transplant for a period of at least one week.
  • the transplant has improved survival compared to a recipient host that did not receive an implantation of one or more encapsulated cell devices containing tissue or cells that are allogeneic or xenogeneic to the recipient host, followed by an administration of an immunosuppressive agent in an amount effective to permit survival of the encapsulated tissue or cell transplant for a period of at least one week.
  • the encapsulated cell devices are implanted according to known techniques, and contain cells or tissue that is either allogeneic or xenogeneic to the recipient host.
  • the recipient host is administered an immunosuppressive agent in an amount effective to permit survival of the encapsulated tissue or cell transplant for a period of at least 1 week.
  • the encapsulated cell devices are then removed, and a second transplant from the same cell or tissue source as the first encapsulated cell devices is implanted into the host, according to known techniques (see, or example, EXAMPLES 2, 6, and 7).
  • the second implant is encapsulated.
  • encapsulated xenogeneic cells stimulate an initial-immune response immediately following subcutaneous transplantation, as shown below. Transient immune blockade abrogates this indirect immune attack and allows encapsulated xenogeneic cells to survive indefinitely, or at least until senescence. Therefore, a graft of encapsulated xenogeneic or allogeneic cells, as described below, is a useful alternative to syngeneic graft transplantation.
  • Tolerization encompass the ability to endure or be less responsive to a stimulus, such as an immune response to a transplant or graft, especially over a period of continued exposure.
  • tolerance refers to the inhibition of a graft recipient host's immune response that would otherwise occur, e.g., in response to the introduction of a non-self MHC antigen into the recipient host.
  • Tolerance can involve humoral, cellular, or both humoral and cellular responses.
  • the immunomodulatory or immunosuppressive agent used in the methods of the invention can be any suitable immunosuppressive agent known in the art. Two preferred agents are, e.g., cyclosporin A and FK506.
  • the dosage for effective immunosuppression will vary according to the mammalian host, but typically the effective dosage for, e.g., human patients, are those that maintain the viability of the encapsulated cells, and are well known or could be routinely ascertained by one of ordinary skill in the art.
  • the immunosuppressive agent may be administered according to any suitable regimen known in the art (see, for example, EXAMPLES 3, 6, and 7).
  • the mature adult immune system can be rendered tolerant to specific alloantigens using a number of different strategies.
  • lymphocyte function-associated, antigen (LFA-1) (Isobe et al, 96 Circulation 2247 (1997)).
  • LFA-1 lymphocyte function-associated, antigen
  • the immunosuppressors cyclosporine (Nagao et al, 33 Transplantation 31 (1982), Mottram et al, 50 Transplantation 1033 (1990)) and FK506 (Ochiai et al, 44 Transplantation 734 (1987), Inamura et al, 45 Transplantation 206 (1988)) have been used to establish a state of host unresponsiveness to allografts. Antigen specific tolerance using many of these techniques relies-on a newly generated population of
  • CD4+ T cells that suppress rejection by T cells and eventually recruit them to become tolerant as well (Qin et al, 259 Science 974 (1993)).
  • the invention provides a composition in which cells are encapsulated in an immunoisolatory capsule.
  • An "immunoisolatory capsule” means that the capsule upon implantation into a host minimizes the deleterious effects of the host's immune system on the cells within its core.
  • Encapsulated cell therapy is based on the concept of isolating cells from a host's immune system by surrounding the cells with a semipermeable biocompatible material before implantation within the host.
  • Cells are immunoisolated from the host by enclosing them within implantable polymeric capsules formed by a microporous membrane. This approach prevents the cell-to cell contact between host and implanted tissues, eliminating antigen recognition through direct presentation.
  • the membranes used can also be tailored to control the diffusion of molecules, such as antibody and complement, based on their molecular weight (Lysaght et al, 56 J. Cell Biochem. 196 (1996), Colton, 14 Trends Biotechnol. 158 (1996)).
  • Useful biocompatible polymer capsules usually contain a core which contains a cell or cells, either suspended in a liquid medium or immobilized within an immobilizing matrix, and a surrounding or peripheral region of permselective matrix or membrane ("jacket") which does not contain isolated cells, which is biocompatible, and which is sufficient to protect isolated cells if present in the core from detrimental immunological attack. Encapsulation hinders elements of the immune system from entering the capsule, thereby protecting the encapsulated cells from immune destruction. The semipermeable nature of the capsule membrane also permits the biologically active molecule of interest to easily diffuse from the capsule into the surrounding host tissue ( ee, EXAMPLES 1,6, and 7).
  • the capsule is made from a biocompatible material.
  • a "biocompatible material” is a material that, after implantation in a host, does not elicit a detrimental host response sufficient to result in the rejection of the capsule or to render it inoperable, for example through degradation.
  • the biocompatible material is relatively impermeable to large molecules, such as components of the host's immune system, but is permeable to small molecules, such as insulin, growth factors, and nutrients, while metabolic waste to be removed.
  • a variety of biocompatible materials are suitable for delivery of growth factors by the composition of the invention. Numerous biocompatible materials are known, having various outer surface morphologies and other mechanical and structural characteristics.
  • the capsule of this invention will be similar to those described by PCT International patent applications WO 92/19195 or WO 95/05452, incorporated by reference; or United States patents 5,639,275; 5,653,975; 4,892,538; 5,156,844; 5,283,187; or 5,550,050, incorporated by reference.
  • Such capsules will allow for the passage of metabolites, nutrients and therapeutic substances while minimizing the detrimental effects of the host immune system.
  • Components of the biocompatible material may include a surrounding semipermeable membrane and the internal cell-supporting scaffolding, preferably, the transformed cells are seeded onto the scaffolding, which is encapsulated by the permselective membrane.
  • the filamentous cell-supporting scaffold may be made from any biocompatible material selected from the group consisting of acrylic, polyester, polyethylene, polypropylene polyacetonitrile, polyethylene teraphthalate, nylon, polyamides, polyurethanes, polybutester, silk, cotton, chitin, carbon, or biocompatible metals.
  • bonded fiber structures can be used for cell implantation (United States patent 5,512,600, incorporated by reference).
  • biodegradable polymers can be use as scaffolds for hepatocytes and pancreatic cells, as reviewed by Cima et al, 38 Biotech. Bioeng. 145-58
  • Biodegradable polymers include those comprised of poly(lactic acid) PLA, poly(lactic- coglycolic acid) PLGA, and poly(glycolic acid) PGA and their equivalents.
  • Foam scaffolds have been used to provide surfaces onto which transplanted cells may adhere.
  • PCT International patent application 98/05304 incorporated by reference.
  • Woven mesh tubes have been used as vascular grafts.
  • PCT International patent application WO 99/52573 incorporated by reference.
  • the core can be composed of an immobilizing matrix formed from a hydrogel, which stabilizes the position of the cells.
  • a hydrogel is a 3-dimensional network of cross-linked hydrophilic polymers in the form of a gel, substantially composed of water.
  • the surrounding semipermeable membrane can be used to manufacture the surrounding semipermeable membrane, including polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof.
  • the surrounding semipermeable membrane is a biocompatible semipermeable hollow fiber membrane.
  • the surrounding semipermeable membrane is formed from a polyether sulfone hollow fiber, such as those described by United States-patent 4,976,859 or
  • An alternate surrounding semipermeable membrane material is poly(acrylonitrile/covinyl chloride).
  • the capsule can be any configuration appropriate for maintaining biological activity and providing access for delivery of the product or function, including for example, cylindrical, rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule can be coiled or wrapped into a mesh-like or nested structure. If the capsule is to be retrieved after it is implanted, configurations which tend to lead to migration of the capsules from the site of implantation, such as spherical capsules small enough to travel in the recipient host's blood vessels, are not preferred. Certain shapes, such as rectangles, patches, disks, cylinders, and flat sheets offer greater structural integrity and are preferable where retrieval is desired.
  • the scaffolding may be coated with extracellular matrix (ECM) molecules.
  • ECM molecules include, for example, collagen, laminin, and fibronectin.
  • the surface of the scaffolding may also be modified by treating with plasma irradiation to impart charge to enhance adhesion of cells.
  • Any suitable method of sealing the capsules may be used, including the use of polymer adhesives or crimping, knotting and heat sealing.
  • any suitable "dry” sealing method can also be used, as described, e.g., in United States patent 5,653,687.
  • the encapsulated cell devices are implanted according to known techniques. Many implantation sites are contemplated for the devices and methods of this invention. These implantation sites include, but are not limited to, the central nervous system, including the brain, spinal cord, and aqueous and vitreous humors of the eye.
  • Cell-based therapies have been developed that use cell encapsulation technology to implant primary tissue and established cell lines.
  • Cell lines are most appropriate for use with encapsulated cell technology.
  • Cell lines offer the advantage of unlimited supply, permitting scale-up and establishment of cell banks for potential clinical applications.
  • Using xenogeneic cells provides the additional safety that cell rejection will occur by the host immune system in the case of capsule rupture. It is important to note that cell lines can be screened prior to use for the presence of pathogens which could pose a threat to human recipients.
  • the cells of the invention can be native or recombinant cells.
  • a "recombinant” cell is a cell or progeny of a cell into which has been introduced, by means of recombinant genetic techniques, any desired polynucleotide.
  • tissue e.g., tissue, cell, and “cells” also encompasses any types of transplantable or implantable tissue or cells from a donor other than the recipient host that contains antigen presenting cells (APC's).
  • APC's antigen presenting cells
  • the donor tissue being used in the invention can be any one of a wide variety of tissues, for example, soft tissue such as the amniotic membrane of a newborn, bone marrow, hematopoietic precursor cells, collagen, and bone protein to stimulate cartilage growth; organs such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs (e.g., intestine, blood vessels, or esophagus); parts of organs, such as heart valves; and isolated cells or clusters of cells, such as islet cells of the pancreas or liver cells.
  • the donor tissue or cells can be taken from any source, whether from cadavers or living donors.
  • Suitable donors include live animals such as laboratory animals, for example, dogs, cats, mice, rats, gerbils, guinea pigs, cows, primates, or human beings.
  • Donors are preferably mammalian, including human beings. When both the donor of the graft and the host are human, they are preferably matched for HLA Class II antigens to as to improve histocompatibility.
  • Human donors are preferably of the same or compatible major ABO blood group.
  • Encapsulated xenogeneic cells In the central nervous system, encapsulated xenogeneic cells have displayed extended viability, surviving at least 6 months (Aebischer et al, 1 Hum Gene Ther 851 (1996)). Encapsulated xenogeneic primary islets have displayed long-term survival in the absence of immunosuppression when transplanted intraperitoneally across a variety of species combinations. Encapsulated xenogeneic primary islets implanted intraperitoneally have successfully provided long-term correction of glucose levels in various animal recipients rendered diabetic (Lacy et al, 254 Science 1782 (1991), Lanza et al, 88 Proc. Natl. Acad. Sci. USA 1100 (1991), Sun et al, 98 J. Clin. Invest. 1417 (1996)).
  • xenotransplantation of cells that are highly antigenic is useful, because xenotransplantation now becomes a better therapeutic option.
  • Xenotransplantation offers a method for overcoming the limitations imposed by an insufficient supply of human tissues and organs for transplantation.
  • the terms "transplant” and variations thereof refers to the insertion of a graft into a recipient host, whether the transplantation is syngeneic (where the donor and recipient host are genetically identical), allogeneic (where the donor and recipient host are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient host are from different species).
  • graft refers to biological material derived from a donor for transplantation into a recipient host.
  • recipient host refers to any compatible transplant host.
  • compatible is meant a host that will accept the donated graft according to the present invention.
  • potentially useful recipient hosts includes animals, preferably mammals such as farm animals, for example, horses, cows or sheep; household pets, for example, dogs or cats; laboratory animals, such as mice, rats, gerbils or guinea pigs; or primates, for example, apes or human beings.
  • Useful recipient hosts can also include aquatic animals, who often live under conditions of lower oxygen tensions than terrestrial animals.
  • the recipient host is a human being.
  • murine C,C ]2 myoblasts were used for encapsulation and subcutaneous implantation.
  • C 2 C I2 myoblasts engineered to secrete murine erythropoietin (Epo; the primary regulator of erythrocyte homeostasis) were used to enable in vivo monitoring of xenograft survival via fluctuations in the hematocrit.
  • Epo murine erythropoietin
  • C 2 C 12 mouse Epo cells were encapsulated and implanted subcutaneously to compare the cell viability in syngeneic. allogeneic and xenogeneic models.
  • Fischer rats were administered FK506 for periods of 1, 2 and 4 weeks after which their hematocrits were monitored until 3 months post-implantation. Animals increased their hematocrits over 70% and sustained these levels for the 3 months, independent of the duration of treatment with FK506.
  • xenografts consisting of free tissues or isolated cells do not possess their own functional vasculature, their outcome depends on a second type of response, which involves cellular rejection.
  • Two separate mechanisms based on direct versus indirect T cell recognition have been considered.
  • Direct xenoantigen recognition requires physical contact between helper T cells and xenogeneic antigen-presenting cells (APC), while indirect xenoantigen recognition occurs when helper T cells respond to xenogeneic peptides presented on host APC.
  • APC xenogeneic antigen-presenting cells
  • CTL cytotoxic T lymphocytes
  • graft rejection can be mediated by a non-classical CTL- independent pathway involving either natural killer cells or macrophages.
  • Encapsulated xenografts elicit an immune response by an indirect presentation of shed or secreted antigens to host T cells.
  • a developed tolerance to xenoantigens must therefore occur through the indirect pathway.
  • Tolerance to minor antigens can be established through indirect presentation alone (Davies et al, 157 J. Immunol. 529 (1996)), suggesting the same mechanism may apply to other types of antigens, including xenoantigens.
  • Antigens can either shed or secreted by the encapsulated xenografts diffuse through the immunoisolating membrane, leading to activation of the host immune system (Loudovaris et al, 24 Transplant Proc.
  • the xenoantigens released by encapsulated xenografts may be proteins naturally produced by these cells, but which show a sufficient difference with their corresponding homologue in the host to initiate an immune response. Once they diffuse outside the capsule membrane, xenoantigens are taken up by antigen presenting cells which in turn stimulate CD4+ T cells to mount an immune attack (Loudovaris et al, 24 Transplant Proc 2291 (1992)).
  • lymphocytes, macrophages, granulocytes and multinucleate giant cells develops around the device, leading to the destruction of the encapsulated xenografts (Loudovaris et al, 24 Transplant Proc. 2291 (1992), Brauker et al, 61 Transplantation 1671 (1996), Weber et al, 49 Transplantation 396 (1990)).
  • the death of the enclosed xenogeneic cells is likely due to the combined effect of locally released immune effectors as well as metabolic stress.
  • the activation of immune response is clinically important, because even a local inflammation caused by an immune response to implanted encapsulated cells can be serious for the host.
  • the second capsule provokes the rejection of cells in the first implant (see, EXAMPLE 7).
  • the immune reaction induced appears to be rather specific against the xenogeneic cells. If the immune reaction was a general inflammatory response following transplantation, the survival of encapsulated cells in the first implant should not be affected. This shows that the induced tolerance threshold to xenoantigens is dependent on the persistence of xenoantigens in an immunosuppressed background.
  • T cells may remain unresponsive to indirect xenoantigen presentation, other immune pathways which function through direct contact, including natural killer cells, macrophages and complement can efficiently act to eliminate unencapsulated xenografts. This is important from a safety point of view, for in the unlikely event of capsule rupture, xenogeneic cells lines would be quickly rejected by the hosts immune system (see, EXAMPLE 6).
  • the murine C 2 C 12 myoblast cell line was used to examine the criteria for the survival of encapsulated xenogeneic cells in the subcutaneous site of a rat recipient host.
  • Mouse C 2 C ]2 myoblasts obtained from the American Type Culture Collection (ATCC; CRL 1772, Rockville, MD), were transfected with the pPI-mEpo-ND plasmid (Regulier et al., 5 Gene Ther. 1014 (1998)) using calcium phosphate precipitation (mammalian transfection kit,
  • the murine C 2 C 12 myoblast cell line is able to secrete high levels of recombinant proteins over prolonged periods (Regulier et al., 5 Gene Ther. 1014 (1998)) and can be induced to differentiate into a post-mitotic state when exposed to low- serum containing medium (Yaffe & Saxel, 270 Nature 725 (1977)).
  • Stably transfected C 2 C 12 cells are capable of secreting therapeutic levels of recombinant proteins, such as erythropoietin
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • C 2 C 12 cells were harvested using a 0.125% trypsin-EDTA solution prepared in modified Puck's saline and were diluted with DMEM in order to achieve a suspension of 1 x 10 5 cells/ ⁇ l.
  • PES microporous polyethersulfone
  • acrylate based glue (460 nm) of acrylate based glue (Luxtrak LCM 23, Ablestik, Electronic Materials & Adhesives, Collinso Dominguez, CA, USA) while the other end was heat-sealed after cell injection.
  • One cm long capsules loaded with approximately 6 x 10 5 C 2 C 12 mEpo cells were implanted subcutaneously on the dorsal flank of mice.
  • Capsules implanted into rats contained 1 x 10 6 cells and measured 2 cm. Their structure was reinforced with an internal titanium coil to prevent kinking.
  • Encapsulated cells were differentiated for 3 days in DMEM containing 10%) Prolifix (Bio Media, Boussens, FRANCE) and subsequently returned to DMEM containing 10% FCS for 4 days in an incubator at 37°C and 5% CO 2 .
  • FCS FCS.
  • the average secretion of the resulting C 2 C 12 mEpo cell line was 51 IU Epo/10 6 cells/day, as determined using a human Epo ELISA test which cross-reacted with mEpo. This relative secretion value was corrected according to bioactivity tests performed on the murine DaE7 cell line (Sakaguchi et al, 15(10) Exp Hematol. 1028-34 (1987)). The normalized value of mEpo secretion was calculated to be 390 IU Epo/10 6 cells/day. EXAMPLE 2 CAPSULE IMPLANTATION
  • Rnu rats (Charles River, Sulzfeld, GERMANY) were used as xenogeneic transplant recipient hosts.
  • animals were anesthetized by inhalation of isoflurane (Forene, Abbott Laboratories, Cham, SWITZERLAND).
  • Capsules were implanted subcutaneously in the dorsal flank of the animals by means of a trocar (Abbocath-T 16 G, Abbott Laboratories, Cham, SWITZERLAND). The entry site in the skin was closed using a nonresorbable suture
  • Immunocompetent Fischer rats consistently rejected encapsulated C 2 C 12 mEpo secreting cells implanted subcutaneously.
  • Nude rats subcutaneously implanted with encapsulated C 2 C ]2 mEpo cells maintained elevated hematocrits and capsules explanted after 1 month continued to secrete high levels of erythropoietin.
  • C 2 C ]2 mEpo cells were harvested from confluent cultures by trypsinization. Subsequently, they were washed 3x in
  • HBSS Hank's balanced salt solution
  • HBSS Hank's balanced salt solution
  • encapsulated C 2 C, 2 cells secreting mEpo were transplanted subcutaneously in C3H mice, DBA/2J mice and Fischer rats. C3H and DBA/2J mice were each implanted with a single 1 cm long capsule. Prior to implantation, encapsulated cells secreted 18.1 ⁇ 9.62 IU/24hrs of mEpo (C3H mice) and 35.3 ⁇ . 8.42 IU/day (DBA/2J mice) (TABLE 1). Both mice strains experienced a significant increase in their hematocrit and maintained these elevated levels for the 5 week trial period.
  • C3H and DBA/2J mice had attained hematocrits of 79.7 ⁇ 4.18%o and 89.5 ⁇ 3.7%>, respectively (FIG. 1).
  • mEpo secretion had decreased to 24%o for C3H mice and 32% for DBA/2J mice, relative to pre-implantation levels.
  • Histological analysis revealed living myoblasts in most explanted devices, with the occasional presence of multinucleated myotubes. In a few instances, necrosis was observed at the capsule core. The biocompatibility of the capsules appeared to be excellent in the subcutaneous site, as they were surrounded by an extensive neovascular network with only a thin layer of fibroblasts adhering to membrane.
  • Fischer rats were implanted with reinforced capsules 2 cm long containing C 2 C 12 mEpo cells.
  • the mean secretion pre-implantation was 23.3 ⁇ 9.07 IU mEpo/day. (TABLE 1).
  • the delivered Epo induced a significant, but only a transient increase in the hematocrit.
  • the hematocrits rose to a high of 65.9 ⁇ 2.38% > and then progressively decreased to pre-implantation levels (FIG. 1).
  • histological examination of the devices revealed that none of the capsules contained viable myoblasts.
  • the pericapsular tissue was composed of a thick fibroblast layer infiltrated principally by lymphocytes and neutrophil granulocytes. This suggested that the decline in the hematocrit levels was due to a gradual, immune mediated destruction of the encapsulated cells following implantation.
  • nude rats possess a rnu autosomal recessive locus which provokes hairlessness and thymic aplasia, rendering them severely immune deficient (Hougen HS).
  • Selected rats were pretreated for 3 days with FK506 (lmg/kg BW) (Prograf, Fujisawa GmbH, M ⁇ nchen, GERMANY). After transplantation, these animals were treated daily (5 days out of 7) for 1, 2 or 4 weeks at a dose of lmg/kg body weight.
  • FK506 doses were injected i.m. into the quadriceps muscle, alternating daily between the left and right leg. Blood was drawn weekly from the tail vein into heparinized capillary tubes. The hematocrit was then measured by a standard microhematocrit method. At the end of the test period, the capsules were carefully explanted, fixed in Lang's fixative for 3 hours and dehydrated using alcohol in preparation for glycol-me hacrylate embedding (Leica Instruments GmbH, Nussloch, GERMANY). After retrieval of the titanium reinforcement, the capsules were cut at 5 ⁇ m thickness and stained with cresyl violet and hematoxylin eosin.
  • the immunosuppressor FK506 was administered to Fischer rats to evaluate if it could similarly protect the encapsulated C 2 C 12 cells against immune mediated destruction. Seven Fischer rats were each implanted following the protocol previously described. The mean capsule secretion before implantation was 34.4 ⁇ 7.99 UI mEpo/day (TABLE 3). Beginning 3 days prior to transplantation, 5 of Fischer rats were immunosuppressed with FK506 for the test period of 4 weeks. The other 2 non-immunosuppressed rats served as internal controls.
  • both the immunosuppressed and the control rats showed an upward trend in their hematocrits, as previously observed.
  • immunosuppressed rats continued to increase their hematocrits, while the levels of the control rats began to decline.
  • rats treated with FK506 continued to maintain hematocrits above 64%, while untreated animals had decreased to pre-implantation levels (47.5 ⁇ 2.12%) (FIG. 2).
  • capsules were explanted from the animals and assessed for their secretion of mEpo.
  • the 4 week treatment regime with FK506 proved to be the most effective in assuring the long-term survival of encapsulated C 2 C 12 mEpo cells following xenotransplantation.
  • implanted rats administered FK506 for 4 weeks sustained elevated hematocrits exceeding 70% throughout the study period (FIG. 4C)
  • capsules retrieved from rats implanted with only one device continued to secrete 37% of their original, pre-implantation levels (TABLE 4). Histology of devices at explant revealed a clear improvement of cell survival in animals receiving FK506 for 4 weeks versus 1 week.
  • Rats which received a second device in the absence of immunosuppression showed less of a tendency to reject the second implant as compared to the previous groups treated with FK506 for 1 and 2 weeks.
  • the initial capsules continued to secrete high levels of mEpo at explant, with 1 capsule showing no change in its secretion from implant to explant (TABLE 4).
  • the second implants made in the absence of immuosuppression showed significant mEpo secretion following 4 weeks in vivo. An average cell survival of 27% was observed in these 2 cases as compared the 4.6% and 2.3% viability observed in animals immunosuppressed for 1 and 2, respectively.
  • FK506 a long-term tolerance can be developed. Xenografted capsules implanted for 13 weeks (37%) for rats treated with FK506 for 4 weeks) had a superior survival to allografted capsules implanted for only 5 weeks (32% for DBA/2J mice) (TABLE 1 and TABLE 4).
  • a threshold dose of immunosuppression is needed immediately following xenograft implant for its development.
  • the unstimulated host immune system is exposed to xenoantigens continually shed by the encapsulated cells. This prolonged exposure to xenoantigens in an immunosuppressed background leads to the tolerization of the host.
  • Murine C 2 C I2 myoblasts engineered to secrete murine erythropoietin (Epo) were used to enable in vivo monitoring of xenograft survival by fluctuations in the hematocrit.
  • These C 2 C, 2 mEpo cells were encapsulated in a semipermeable membrane and subsequently implanted in the subcutaneous site of xenogeneic rat recipients.
  • the C 2 C 12 myoblasts were then used to evaluate the response of control versus FK506 treated xenogeneic recipients (Fischer rats) to encapsulated myoblasts implanted in the subcutaneous site.
  • Encapsulated C 2 C 12 mEpo cells were rapidly eliminated in immunocompetent Fischer rats.
  • This EXAMPLE shows the importance of combining the technique of cell encapsulation with transient immunosuppression to achieve long-term survival of xenografted myoblasts in a peripheral inimunoreactive site. Encapsulation alone cannot protect xenogeneic myoblasts from immune destruction in the subcutaneous site.
  • C2C12 mEpo myoblasts Transplantation of encapsulated C2C12 mEpo myoblasts in C3H mice, DBA/2 J mice and Fischer rats.
  • C 2 C 12 myoblasts stably transfected with the pPI-mEpo-ND expression vector released 163 IU Epo/1 06 cells/day, as determined using a human Epo ELISA test which cross- reacted with mEpo (Regulier et al, 5 Gene Ther. 1014 (1998)).
  • mouse C 2 C 12 myoblasts obtained from the American Type Culture Collection (ATCC; CRL 1772,
  • Rockville, MD were transfected with the pPI-mEpo-ND plasmid (Regulier et al, 5 Gene Ther. 1014 (1998)) using calcium phosphate precipitation (mammalian transfection kit, Stratagen, Basel, SWITZERLAND).
  • the cells were selected for 2 weeks in 0.8 mg/ml G418. Subsequently, selected cells were incubated with increasing concentrations of methotrexate (1 to 200 M) over 6 weeks to amplify the copy number of the integrated plasmid. Selected pools of high expressing cells were obtained by diluting the total population to a final concentration of 10 cells per well.
  • the C 2 C 12 mEpo myoblasts used in this EXAMPLE were derived from the original pool previously described (Regulier et al, 5 Gene Ther. 1014 (1998)). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FC S), 2 mM L- glutamine, 4.5 g/1 glucose, 100 U/ml penicillin and 100 U/ml streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • FC S fetal calf serum
  • FC S fetal calf serum
  • C 2 C 12 cells were harvested using a 0.125%> trypsin-EDTA solution prepared in modified Puck's saline and were diluted with DMEM in order to achieve a suspension of lxl 0 5 cells/ ⁇ l.
  • Encapsulated cells were differentiated for 3 days in DMEM containing 10% > Prolifix (Bio Media, Boussens, FRANCE), a serum free medium capable of inducing myoblast differentiation. The encapsulated cells were subsequently returned to DMEM containing 10% > FCS for 4 days.
  • Prolifix Bio Media, Boussens, FRANCE
  • C3H mice, DBA/2J mice and Fischer rats were transplanted subcutaneously in C3H mice, DBA/2J mice and Fischer rats in order to compare their survival in syngeneic, allogeneic and xenogeneic recipients.
  • C3H and DBA/2J mice were each implanted with 1 capsule containing C,C I2 mEpo myoblasts.
  • Capsule implantation was performed as follows: C3H and DBA/2J mice (Iffa Credo, Saint-German sur l'Abresle, FRANCE) were chosen for syngeneic and allogeneic transplantation, respectively.
  • Fischer rats Iffa Credo, FRANCE
  • Rnu rats were used as xenogeneic transplant recipients.
  • mice were anesthetized by inhalation of isoflurane (Forene, Abbott Laboratories, Cham, SWITZERLAND). Capsules were implanted subcutaneously in the dorsal flank of the animals using a trocar (Abbocath-T 16 G, Abbott Laboratories, Chain, SWITZERLAND). The entry site in the skin was closed using a nonresorbable suture (Prolene 6-0). Upon recovery, the animals were returned to the animal care facility, where they had access to food and water ad libitum.
  • Capsules implanted into the mice were 1 cm long capsules loaded with approximately 6xl0 5 C 2 C 12 mEpo cells and implanted subcutaneously on the dorsal flank of the mice. Capsules implanted into the rats contained lxl 0 6 cells and measured 2 cm long. Their structure was reinforced with an internal titanium coil to prevent kinking. The membrane used for immunoisolation had a mean molecular weight cut-off of 280 kDa. Immunosuppression was performed as follows: Selected rats were pretreated for 3 days with FK506 (1 mg/kg BW) (Fujisawa GmbH, Munchen, GERMANY).
  • FK506 doses were injected intramuscularly into the quadriceps muscle, alternating daily between the left and right legs. Intramuscular injections of cells was performed as follows: C 2 C 12 mEpo cells were harvested from confluent cultures by trypsinization.
  • HBSS Hank's balanced salt solution
  • mEpo The secretion of mEpo from encapsulated mEpo cells was measured both preimplantation and following explant by incubating the capsules for 1 hr in 1 ml DMEM containing 10%> FCS. Epo levels in the conditioned media were measured using an enzyme linked immunosorbent assay (ELISA) (Quantikine IVD, R&D systems, Minneapolis). Cross- reaction of the kit allowed detection of mEpo in culture supernatants (Regulier et al, 5 Gene
  • mice were each implanted with 1 capsule containing C 2 C 12 mEpo myoblasts. Prior to implantation, capsules containing C 2 C 12 mEpo myoblasts were measured for their secretion of Epo (TABLE 5). Both mice strains experienced a significant increase in their hematocrit and maintained these elevated levels for the 5 week trial period (FIG. 6).
  • hematocrit measurement blood was drawn weekly from the tail vein into heparinized capillary tubes. The hematocrit was then measured by a standard microhematocrit method (Koepke, Microhematocrit method. In Koepke JA, ed., Practical Laboratory Hematology.
  • the capsules were cut at 5 ⁇ m thickness nd stained with hematoxylin and eosin.
  • animals were sacrificed by pentobarbital overdose and perfused transcardially with 4%> paraformaldehyde. Muscles were dissected and embedded in paraffin for histology. Sections were cut at 7 ⁇ m thickness and stained with hematoxylin and eosin.
  • Histological analysis revealed living myoblasts in explanted devices. In a few instances, a necrotic core was observed at the capsules' center. The biocompatibility of the system was characterized by an extensive neovascular network surrounding the capsules, with only a thin fibrotic reaction adhering to the membrane.
  • Fischer rats were each implanted with one 2 cm long capsule releasing 23.3 ⁇ 2.9 IU mEpo/day (TABLE 5).
  • the delivered mEpo induced a significant, but only transient increase in the hematocrit.
  • the hematocrit levels had risen to a high of 65.9 + 24% which then progressively decreased to pre-implantation levels (FIG. 6).
  • histology of the devices revealed that the encapsulated cells had all died.
  • the capsules were surrounded by a pronounced pericapsular tissue reaction characterized by an extensive infiltration of neutrophils and lymphocytes, suggesting an immune-mediated cell destruction.
  • nude rats Eight nude rats were implanted for 28 days following the protocol previously used. Fischer rats were implanted in parallel to serve as controls. Pre-implantation, devices implanted in immunodeficient rats secreted 33.0 ⁇ 1.9 IU mEpo/day (TABLE 5).
  • the hematocrit of nude rats increased in a manner similar to that of control rats, reaching 53.4 ⁇ 3.2% (FIG. 7).
  • a continued increase of the hematocrit was observed in nude rats (78.4 ⁇ 3.7% on day 28) (FIG. 7), whereas the hematocrit in control rats had already substantially declined (54 ⁇ 1.7 on day 28) at that time.
  • Fischer rats were implanted with C 2 C 12 mEpo myoblasts encapsulated in identical membranes. These capsules induced only a transient increase in the hematocrit before returning to baseline levels 5 weeks later (FIG. 8). No difference was observed between the hematocrit profiles of Fischer rats receiving capsules having a 280 kDa versus 32 kDa cutoff (FIG. 8). At explant, devices contained no viable cells and secretion of mEpo was undetectable. Histology of capsules having a 32 kDa membrane was comparable to those with a 280 kDa cut-off, with numerous lymphocytes observed around the capsules.
  • Fischer rats were implanted with encapsulated C 2 C ⁇ 2 mEpo myoblasts and immunosuppressed for either 1 , 2, or 4 weeks.
  • a gradual increase in the hematocrit was observed, with levels reaching over 70% on week 13 (FIG. 9).
  • a long-term unresponsiveness to encapsulated C 2 C 12 mEpo myoblasts developed following only a 1 week administration of FK506. No difference was observed in the progression of hematocrit in the 3 animal groups during the EXAMPLE. All animals maintained an elevated hematocrit exceeding 65% throughout the 3 month trial period (FIG. 9). Devices explanted at 13 weeks continued to secrete measurable quantities of mEpo (TABLE 6). TABLE 6 '
  • Rat #3 36 13 36.1% mean 34.7 7.5 21.6 %
  • Fischer rats were immunosuppressed with FK506 (1 mg/kg) ( m a daily basis, 5 out of 7 days, for either 1 , 2, or 4 weeks. Capsules were retrieved and measured for mEpo secretion following 91 days residence in vivo.
  • the 4 week treatment regimen with FK506 appeared to be slightly more effective than 1 and 2 week regimens in assuring the long-term survival of encapsulated C 2 C 12 mEpo cells (TABLE 6).
  • the capsules retrieved from implanted rats treated 4 weeks secreted slightly higher levels of mEpo than rats administered FK506 for 1 and 2 weeks (TABLE 6).
  • Histology of devices at explant revealed viable cells with some central necrosis in animals from all 1, 2, and 4 week FK506 treatment groups. Outside the capsules, an extensive network of blood vessels was observed close to the membrane, with few lymphocytes in the vicinity.
  • murine C 2 C 12 myoblasts engineered to secrete mEpo were used to examine the criteria for the survival of encapsulated xenogeneic myoblasts in the subcutaneous site of a rat recipient.
  • Immunocompetent Fischer rats consistently rejected encapsulated C 2 C 12 mEpo secreting cells implanted subcutaneously and capsules retrieved following 1 month in vivo were found to contain only cellular debris.
  • the pericapsular tissue was characterized by a massive lymphocytic infiltration, suggesting an immune mediated destruction.
  • This EXAMPLE documents transient immunosuppression as a method of achieving long-term host unresponsiveness to encapsulated xenogeneic myoblasts grafted outside the central nervous system.
  • an initial treatment with FK506 permitted encapsulated C 2 C 12 mEpo myoblasts to survive at least 3 months when transplanted into the subcutaneous site of Fischer rats. No differences were observed in the hematocrit level attained at 13 weeks in animals given longer initial treatments with FK506. However, slightly higher levels of mEpo were produced by devices retrieved from animals immunosuppressed 4 weeks versus only 1 week.
  • capsules secreting as little as 2 IU mEpo/day are able to maintain rats at their threshold hematocrit levels, making device secretion at explant a more quantitative means of evaluating the survival of xenogeneic myoblasts.
  • Fischer rats were rendered unresponsive to encapsulated murine C 2 C, 2 myoblasts secreting mouse erythropoietin by either a 1 or 4 week initial treatment of FK506.
  • animal were challenged with a second implant 9 weeks after the initial implantation. Challenging animals treated only 1 week with FK506 led to rejection of both primary and secondary implants.
  • This EXAMPLE shows that a host tolerance can be established to xenoantigens released by encapsulated xenogeneic cells by using a short-term immunomodulation.
  • Murine erythropoietin (mEpo) was used as a reporter gene to permit monitoring of xenograft viability by fluctuations in animal hematocrit and by comparing mEpo secretion from devices at explant versus implant.
  • This EXAMPLE further examined the nature of the host's acceptance of encapsulated xenografts following FK506 treatment.
  • Fischer rats rendered unresponsive to encapsulated C 2 C 12 mEpo cells by transient immunosuppression were challenged with a secondary implant containing identical cells, in the absence of immunosuppression, to establish if animals had been tolerized.
  • the role of the length of initial immunosuppression on the survival of cells within the 2 implants was tested.
  • the extent of host acceptance was considered by tolerizing animals to encapsulated naive myoblasts and challenging animals with encapsulated genetically modified myoblasts. Challenging host unresponsiveness to encapsulated xenografts.
  • FCS Fluorescence Activated Cell Sorting
  • 2 mM L-glutamine 2 mM L-glutamine, 4.5 g/1 glucose, 100 U/ml penicillin and 100 U/ml streptomycin.
  • PES polyethersulfone
  • the encapsulated cells were differentiated for 3 days in DMEM containing 10% Prolifix (Bio Media, Boussens, FRANCE), a serum free medium capable of inducing myoblast differentiation (Regulier et al, 5 Gene Ther. 1014 (1998)). The encapsulated cells were subsequently returned to DMEM containing 10% FCS for 4 days.
  • Prolifix Bio Media, Boussens, FRANCE
  • FCS a serum free medium capable of inducing myoblast differentiation
  • Capsules were implanted subcutaneously on the dorsal flank of Fischer rats. Capsule implantation was performed as follows: Fischer rats (Iffa Credo, FRANCE) were used as concordant xenogeneic transplant recipients. Animals were anesthetized using isoflurane (Forene, Abbott Laboratories, SWITZERLAND) and implanted using a trocar (Abbocath-T 16 G, Abbott Laboratories, SWITZERLAND). One or two cm long capsules were subcutaneously implanted in the dorsal flank. Upon recovery, the animals were returned to the animal care facility, where they had access to food and water ad libitum.
  • Fischer rats were pretreated for 3 days with FK506 (1 mg/kg BW) (Fujisawa GmbH, M ⁇ nchen, GERMANY). Following transplantation, these animals were treated daily (5 days out of 7) for either 1 or 4 weeks at a dose of 1 mg/kg BW. FK506 doses were injected intramuscularly into the quadriceps muscle, alternating daily between the left and right legs.
  • FK506 for either 1 or 4 weeks.
  • Fischer rats were immunosuppressed with FK506(1 mg/kg) on a daily basis, 5 out of 7 days, for either 1 or 4 weeks.
  • Fischer rats were immunosuppressed with FK506 (1 mg/kg) on a daily basis, 5 out of 7 days, for 4 weeks.
  • Capsules retrieved from immunosuppressed animals contained viable cells while implants removed from untreated animals were empty and showed an extensive lymphocytic reaction around the capsule membrane. Five weeks later (week 9), these animals were challenged with a second implant, this time containing C 2 C )2 mEpo cells (FIG. 14A, FIG 14B). In response to the new devices, the tolerized animals progressively increased their hematocrit over the 4 weeks following implantation (FIG. 14B). Control rats showed only a transient rise in hematocrit that began to decrease by the second week and returned to base line levels at week 4 (FIG. 14A).
  • Fischer rats were immunosuppressed with FK506 (1 mg/kg) on a daily basis, 5 out of 7 days, for 4 weeks were indicated.
  • the second capsule provokes the rejection of cells in the first implant.
  • both devices are positioned at different locations, with inflammation localized around each capsule exterior, the immune reaction induced appears to be rather specific against the xenogeneic cells. If the immune reaction was a general inflammatory response following transplantation, the survival of encapsulated cells in the first implant should not be affected. This shows that the induced tolerance threshold to xenoantigens is dependent on the persistence of xenoantigens in an immunosuppressed background. Studies in allotransplantation have highlighted the importance of antigen persistence on the efficacy of T cell tolerization (Scully et al, 24 Eur. J. Immunol. 2383 (1994), Ehl et al, 4 Nature Med. 1015 (1998)), suggesting that this may similarly apply to xenotransplantation.
  • animals tolerized to shed xenoantigens maintain this state of unresponsiveness over an extended period in the absence of transplant specific antigens.
  • a lack of continued xenoantigen presence may moderate host tolerance as xenogeneic myoblast survival in this case was reduced in comparison to challenges given at shorter intervals between the last host exposure to xenoantigens.

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Abstract

L'invention concerne des cellules encapsulées et des méthodes permettant de rendre un receveur tolérant à une greffe, les cellules encapsulées permettant d'induire ladite tolérance chez l'hôte. L'administration concurrente d'un agent immunosuppresseur garantit le succès de la transplantation d'un greffon voulu. Une fois la tolérance induite, un greffon peut être transplanté au niveau du site voulu sans utilisation prolongée d'une immunosuppression ou d'une immunothérapie. Ainsi, l'invention concerne une méthode permettant de transplanter des myoblastes xénogènes génétiquement modifiés au niveau d'un site immunoréacteur périphérique et de garantir leur survie à long terme. En combinant ces techniques d'encapsulation et l'utilisation d'immunosuppresseurs déterminés, on peut désormais transplanter divers types de cellules xénogènes différentes.
PCT/US2000/006737 1999-03-15 2000-03-15 Methodes et compositions destines a rendre des hotes tolerants a la survie a long terme de greffons de tissus WO2000054786A1 (fr)

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US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs

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US4352883A (en) * 1979-03-28 1982-10-05 Damon Corporation Encapsulation of biological material
WO1996038178A1 (fr) * 1995-06-01 1996-12-05 The General Hospital Corporation Transplantation allogenique et exogenique
WO1997015243A1 (fr) * 1995-10-26 1997-05-01 Latta Paul P Induction d'une tolerance immunologique
WO1997041863A1 (fr) * 1996-05-09 1997-11-13 The General Hospital Corporation Chimerisme mixte et tolerance

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US4352883A (en) * 1979-03-28 1982-10-05 Damon Corporation Encapsulation of biological material
WO1996038178A1 (fr) * 1995-06-01 1996-12-05 The General Hospital Corporation Transplantation allogenique et exogenique
WO1997015243A1 (fr) * 1995-10-26 1997-05-01 Latta Paul P Induction d'une tolerance immunologique
WO1997041863A1 (fr) * 1996-05-09 1997-11-13 The General Hospital Corporation Chimerisme mixte et tolerance

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MORRIS P J: "Immunoprotection of therapeutic cell transplants by encapsulation", TRENDS IN BIOTECHNOLOGY,GB,ELSEVIER PUBLICATIONS, CAMBRIDGE, vol. 14, no. 5, 1 May 1996 (1996-05-01), pages 163 - 167, XP004035787, ISSN: 0167-7799 *

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US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs

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