WO1999045958A1 - Therapie de blocage du cd154 pour la modulation des reponses immunes aux implants - Google Patents

Therapie de blocage du cd154 pour la modulation des reponses immunes aux implants Download PDF

Info

Publication number
WO1999045958A1
WO1999045958A1 PCT/US1999/005016 US9905016W WO9945958A1 WO 1999045958 A1 WO1999045958 A1 WO 1999045958A1 US 9905016 W US9905016 W US 9905016W WO 9945958 A1 WO9945958 A1 WO 9945958A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
tissue
recipient
binding
immunoisolation
Prior art date
Application number
PCT/US1999/005016
Other languages
English (en)
Inventor
Linda Burkly
Camillo Ricordi
Norma Kenyon
Luca Inverardi
Original Assignee
Biogen, Inc.
University Of Miami
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1998/012892 external-priority patent/WO1998058669A2/fr
Application filed by Biogen, Inc., University Of Miami filed Critical Biogen, Inc.
Priority to AU28999/99A priority Critical patent/AU2899999A/en
Publication of WO1999045958A1 publication Critical patent/WO1999045958A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates generally to the suppression of unwanted immune responses, particularly of counter-adaptive T-lymphocyte mediated immune responses.
  • the invention relates in particular to the prevention, treatment, suppression or reversal of immune system driven rejection of an implant or implanted device, such as an immunoisolation device containing foreign cells or tissue, in the body of a recipient.
  • immunoisolation refers to the protection of transplanted living cells or tissue from the immune system of a recipient by enclosure in a semipermeable membrane or barrier.
  • naturally occurring and synthetic polymers have been used to protect transplanted cells and tissues from the recipient's cellular and humoral immune system responses.
  • Several types of immunoisolation systems or devices have been studied, including devices which are anastomosed to the vascular system such as arteriovenous shunts, as well as diffusion chambers, and micro- or macro- encapsulation devices including microcapsules.
  • such encapsulation systems can serve to block the access of antibodies and complement to the tissue within the device, or to block contact with mononuclear cells; however, there remains the possibility that antigens released from the capsule (such as secreted cellular products, shed cell surface components, or cellular debris) could stimulate a recipient immune response. Such an immune response in turn can lead to an allergic response, or immune complex disease.
  • Cell-mediated immunity also can be induced by released antigens, leading to production of proinflam atory cytokines. which are low molecular weight molecules, and therefore able to pass through the semi-permeable membrane.
  • the device itself may also elicit an inflammatory response, such as a foreign body response, including the production of antibodies, proinflammatory mediators such as cytokines and nitric oxide, which ultimately can lead to the deposition of a relatively impermeable fibrotic shell around the device.
  • an inflammatory response such as a foreign body response, including the production of antibodies, proinflammatory mediators such as cytokines and nitric oxide, which ultimately can lead to the deposition of a relatively impermeable fibrotic shell around the device.
  • encapsulation technologies suffer from the risk of breakage or weakening of membrane areas, which impair the effectiveness of the capsule as an immunobarrier.
  • any interaction between implanted devices, such as capsules, and the recipient's immune system depends in part upon the device's composition, contents, and integrity.
  • the recipient's response may depend upon the nature of cellular products which escape the device, such as by crossing a capsule membrane. These products may be antigenic and stimulate a recipient immune reaction.
  • Immunoisolation has potential in the treatment of many chronic conditions.
  • some cells and tissues which have already been used in the study of immunoisolation include: adrenal chromaffin cells for neurodegenerative disease and chronic pain; genetically engineered cells designed to produce clotting factor EX for hemophilia, growth hormone for the treatment of dwarfism, erythropoeitin for anemia, and nerve growth factors or neurotrophic factors for treatment of neurodegenerative conditions; islet cells for producing insulin for diabetics; parathyroid cells for hypocalcemia; and, hepatocytes for the correction of metabolic disorders.
  • a particular object is to provide a CD40:CD154 binding interruptor, such as a CD 154 blocking agent, for use in therapy, particularly for use in therapy to mitigate, delay or reverse immunological rejection of an implanted device.
  • a more general object of the invention is to improve the availability of immunoisolation technology, by providing immunomodulatory compositions that allow functional integration of immunoisolation devices into a
  • a further general object is to prevent, mitigate, attenuate or treat a condition in which the recipient is in need of a product secreted by cells or tissue in an immunoisolation device (e.g., dopamine producing cells needed by an individual with parkinsonism or another neurodegenerative disorder), or in which the recipient is in need of an enzymatic process catalyzed by cells or tissue in the immunoisolation device (e.g., detoxification processes catalyzed by hepatocytes, which are needed by individuals with liver failure).
  • an immunoisolation device e.g., dopamine producing cells needed by an individual with parkinsonism or another neurodegenerative disorder
  • an enzymatic process catalyzed by cells or tissue in the immunoisolation device e.g., detoxification processes catalyzed by hepatocytes, which are needed by individuals with liver failure.
  • the present invention rests on the discovery that use of a CD40:CD154 binding interruptor, such as a CD 154 blocking agent, whether used alone or in combination with another therapeutic agent, such as an immunomodulatory agent, attenuates, suppresses, prevents, delays or reverses counter-adaptive immune system rejection of an implanted device in a recipient, without the need for pan-suppression of the recipient's immune system.
  • a CD40:CD154 binding interruptor such as a CD 154 blocking agent
  • another therapeutic agent such as an immunomodulatory agent
  • the invention accordingly provides methods and compositions for immunomodulatory therapy for recipients of implanted devices, such as immunoisolation devices, drug delivery devices, and prosthetic devices.
  • a first method inhibits rejection of an implanted device by a recipient thereof.
  • a second method prolongs survival of cells or tissue contained within an implanted device.
  • a third method reverses rejection of the implanted device.
  • a fourth method preserves function of cells or tissue contained within the implanted device.
  • a fifth method restores function of an impaired device, particularly of impaired cells or tissue within the device.
  • CD40:CD154 binding interruptor any agent that interrupts the binding of CD40 Ligand (i.e., CD40L, also known as CD154 or the 5c8 antigen, and sometimes referred to in the art as gp39) to its counter or cognate receptor (e.g., CD40).
  • the binding interruptor is a CD 154 (CD40L) blocking agent, by which is meant any agent that binds to CD 154 and prevents or interferes with its binding to counter receptors (e.g., CD40).
  • CD40L CD40L
  • An exemplary CD 154 blocking agent is a monoclonal antibody (MAb), particularly one having the antigen-specific binding characteristics of the 5c8 MAb disclosed in U.S. Patent 5,474,771, the teachings of which are incorporated herein by reference.
  • the invention is particularly suitable for use with immunoisolation devices which contain viable cells or tissue.
  • Practice of the present invention advantageously attenuates a foreign body response, a humoral immune response, or a cellular immune response, to the implanted device.
  • the invention accordingly is useful wherever the recipient is in need of a product is secreted by cells or tissue that are suitable for containment in an immunoisolation device.
  • the invention is useful wherever the recipient is in need of an enzymatic process that is carried out by cells or tissue suitable for containment in an immunoisolation device.
  • the cells or tissue can be allogeneic, xenogeneic, autologous, postmitotic, mitotically competent, differentiated, partially differentiated, derived from an adult donor, of fetal origin, cultured, immortalized, or genetically modified.
  • the device can be any type of immunoisolation device, including without limitation an arteriovenous shunt, a diffusion chamber, or a micro- or macro-capsule made of any conventional material or combination of materials, that is sufficient to provide a physical barrier to cell/cell contact between tissues of the recipient's body and the cells or tissue contained within the device.
  • the barrier together with any other structural members of the device to which the barrier may be joined, defines an isolation chamber in which the cells or tissue are disposed.
  • the barrier is a semipermeable matrix or membrane which selectively excludes recipient cells and unwanted macromolecules (e.g., complement factor Clq, immunoglobulins) from the interior of the device (i.e., the isolation chamber), yet allows low molecular weight nutrients, metabolites, and dissolved gasses such as oxygen, to pass through freely.
  • the device is implanted at any site within the recipient's body that is appropriate to delivery of the needed product, or enzymatic process, to the recipient.
  • the device may be implanted subcutaneously, intramuscularly, intravascularly, intraperitoneally, intrahepatically, retroperitoneally, within a bone marrow cavity, within a joint capsule, intraocularly, intracranially, intraspinally, or the like.
  • the recipient can be any mammal, but preferably is a primate, most preferably a human.
  • the invention provides a method of implanting cells or tissue into a recipient.
  • the method involves providing an
  • a semipermeable barrier defines an isolation chamber in which the cells or tissue are disposed.
  • the device is implanted into the recipient by any conventional technique, such as by surgery, and a CD40:CD154 binding interruptor is administered to the recipient either concurrently, prior to the implantation, or subsequent to the implantation, in an ammount sufficient to attenuate an immune response (whether a foreign body response, humoral response, or cellular response) to the implanted device.
  • the invention furthermore provides a composition for the implantation of cells or tissue into a recipient.
  • the present composition comprises an immunoisolation device in which a semipermeable barrier defines an isolation chamber (either by completely surrounding the chamber, or by sealing an aperture within or between one or more structural members of the device) and separates the chamber contents physically from tissue surrounding the device once implanted into the body of a recipient.
  • the present composition further provides cells or tissue disposed within the isolation chamber.
  • the composition still further comprises a CD40:CD154 binding interruptor, which can be (without limitation) dispersed in a physiologically acceptable carrier in which the immunoisolation device is immersed; or, adsorbed on the surface of the device; or, produced by cells or tissue within the isolation chamber.
  • the composition is for the delivery of a drug or other therapeutic agent to the recipient, and the composition comprises a reservoir or matrix having the drug/therapeutic agent formulated therein in the isolation chamber in lieu of the above- mentioned cells or tissue.
  • the device is a prosthetic device (including, without limitation, a suture, staple, stent, catheter, pin, bolt, screw, lead line, artificial tooth, bone or joint replacement device, or artificial valve) having adsorbed thereon or formulated therein a CD40:CD154 binding interruptor as defined herein. Any of the foregoing compositions are suitable for use in therapy, including without limitation, for the specific medical uses disclosed herein.
  • T cell activation requires both T cell receptor (TCR) mediated signals and simultaneously delivered costimulatory signals.
  • An important costimulatory signal is delivered by the ligation of CD40 on an antigen-presenting cell, such as a B cell, by CD40L (CD 154) on a T cell.
  • CD40L CD 154 on a T cell.
  • Human CD40 is a 50 kD cell surface protein expressed on mature B cells, as well as on macrophages and activated endothelial cells.
  • CD40 belongs to a class of receptors involved in programmed cell death, including Fas/CD95 and the tumor necrosis factor (TNF) alpha receptor.
  • CD40L Human CD154
  • CD40:CD154 binding has been shown to be required for all T cell-dependent antibody responses.
  • CD40:CD154 binding provides anti- apoptotic and/or lymphokine stimulatory signals.
  • X-linked hyper-IgM syndrome (X-HIGM) in humans is the phenotype resulting from genetic lack of functional CD 154.
  • Affected individuals have normal or high IgM levels, but fail to produce IgG, IgA or IgE antibodies, and suffer from recurrent, sometimes severe, bacterial and parasitic infections, as well as an increased incidence of lymphomas and abdominal cancers.
  • a similar phenotype is observed in non-human animals rendered nullizygous for the gene encoding CD 154 (knockout animals).
  • B cells of CD 154 nullizygotes can produce IgM in the absence of CD40L:CD154 binding, but are unable to undergo isotype switching, or to survive normally after affinity maturation. Histologically, lymph node germinal centers fail to develop properly, and memory B cells are absent or poorly developed. Functionally, these defects contribute to a severe reduction or absence of a secondary (mature) antibody response. Defects in cellular immunity are also observed, manifested by an increased incidence of bacterial and parasitic infections. Many of these cell-mediated defects are reversible by administration of IL-12 or IFN -gamma. These observations substantiate the view that normal CD40:CD 154 binding promotes the development of Type I T- helper cell immunological responses.
  • CD40:CD154 blockade technology thus may provide potentially powerful therapeutic avenues for prevention of immunological complications, including rejection and failure, which currently impede the further development and wider use of implanted devices.
  • immunological studies in rodent model systems have have not correlated consistently with the outcome of testing or therapy of large animals, including humans.
  • immunological studies in induced-disease model systems have not correlated consistently with the outcome of testing in individuals having chronic or ongoing disease states.
  • a preferred CD 154 blocking agent a humanized MAb having the antigen-specific binding properties of MAb 5c8 (Lederman et al.(1992), J. Exp. Med. 175: 1091-1101), in a variety of preclinical models of implant treatment of selected disease states or impaired conditions. Studies using these model systems are expected to yield information of predictive value in developing implant therapies for the corresponding disease states or impairments (such as organ failures) in humans.
  • an "immunoisolation device” is a container or vessel fabricated of a biocompatible material or combination of materials, in which a semipermeable barrier, such as a membrane, defines an isolation chamber in the interior of the device, in which viable cells or tissue can be disposed. The contents of the isolation chamber are separated, and therefore protected, from the environment external to the device by the semipermeable barrier.
  • the semipermeable barrier completely surrounds or encapsulates the isolation chamber; however, in other embodiments, the semipermeable barrier seals an aperture within or between one or more structural members of the device. In each case, the barrier separates the chamber contents physically from the external environment, in particular from body tissue(s) of the recipient which surround the device after its implantation into the body of a recipient.
  • the barrier is a semipermeable matrix or membrane which selectively excludes recipient immune effector cells (e.g., leukocytes) and unwanted macromolecules (e.g., complement factor Clq, immunoglobulins) from the interior of the device (i.e., the isolation chamber), yet allows low molecular weight nutrients, metabolites, and dissolved gasses such as oxygen, to pass through freely.
  • recipient immune effector cells e.g., leukocytes
  • macromolecules e.g., complement factor Clq, immunoglobulins
  • preferred barriers whether membranes or matrices
  • a pore size also referred to as
  • some embodiments of the present invention include devices in which the pore size of the semipermeable barrier is larger than 50,000 to 100,000 kD.
  • the invention encompasses devices in which the pore size range is 100,000 to 150,000 kD (large enough to allow diffusion of some immunoglobulins, such as IgG, but small enough to exclude Clq).
  • the present invention can be practiced with any size or shape of immunoisolation device, including without limitation an arteriovenous shunt, a diffusion chamber, or a micro- or macro-capsule, made of any conventional material or combination of materials.
  • the invention can be practiced with devices sufficient to enclose an entire organ, a section of an organ, a sample or biopsy of tissue, a cluster or colony of cells, or individually isolated cells.
  • the configuration and size of the immunoisolation device will vary depending on the site of implantation in the recipient's body, the recipient's body weight, age, sex and health status, and in particular on the number or amount of cells/tissue required to fulfill the recipient's need for a secreted product or an enzymatic process supplied by the cells/tissue within the immunoisolation device.
  • an effective amount of cells or tissue is implanted, by which is meant an amount sufficient to attenuate (detectably mitigate or diminish) the recipient's need for the secreted product or the enzymatic process.
  • the amount is sufficient to restore the recipient to substantially normal
  • Specific immunoisolation devices useful in the present invention include any porous membrane, envelope, or capsule suitable for encapsulating living tissue.
  • Numerous types of microcapsules are known to those of skill in the art; thus, the choice of a particular encapsulation material can be made in light of the desired application.
  • Alginate polylysine microcapsules are well known in the art, but are known to weaken over time.
  • Other microcapsules comprise alginate chemically cross-linked with barium; however, barium is toxic to most cells.
  • Preferred microcapsules comprise biocompatible polymers, free of immunogenic substances, formed into a structurally sound device or capsule which can be tolerated by the recipient.
  • Exemplary alginate microcapsules can be either single walled or double walled, as described in U.S. Patent No. 5,227,298 (July 13, 1993), or uncoated, as described in U.S. Patent No. 5,651,980 (July 29, 1997) the specifications of both of which are hereby incorporated by reference.
  • Additional devices which can be used in the context of the present invention include, without limitation, the biological artificial liver described in U.S. Patent No. 5,270,192 (December 14, 1993); the bioartificial endocrine device of U.S. Patent No. 5,614,205 (March 25, 1997); the vascularizing bioartificial pancreas of U.S.
  • Patent No. 5,674,289 (October 7, 1997); the close vascularizing implant of U.S. Patent No. 5,800,529 (September 1 , 1998) or, the permselective bioartificial organ of U.S. Patent No. 5, 837,234 (November 17, 1998).
  • the present immunoisolation device is implanted at any site within the recipient's body that is appropriate to delivery of the needed product, or enzymatic process, to the recipient.
  • the device can be implanted at a site sufficient to deliver the product or process systemically, for example to the recipient's bloodstream, or locally, for example to a body compartment or a wound site.
  • Suitable sites for implantation for systemic delivery include, without limitation: subcutaneous,
  • Suitable sites for implantation for local delivery include: subcutaneous, intramuscular, subgingival, retroperitoneal, intraosteal (within a bone marrow cavity), subcapsular (within a joint capsule), intraocularly, intracranially, or intraspinally.
  • the implantable device is for the delivery of a drug or other therapeutic agent not of cellular origin to the recipient.
  • the device includes a reservoir or matrix having a drug or other therapeutic agent disposed or formulated therein in lieu of the isolation chamber containing cells or tissue.
  • the device is a prosthetic device, such as a suture, staple, stent, catheter, pin, bolt, screw, lead line, artificial tooth, bone or joint replacement device, or artificial valve.
  • any of the foregoing immunoisolation or other devices can, as desired, be immersed prior to implantation in a physiologically acceptable carrier (such as a preservative solution, a cell culture medium, or a balanced salts solution) in which a CD40:CD154 binding interruptor is dispersed or otherwise formulated.
  • a physiologically acceptable carrier such as a preservative solution, a cell culture medium, or a balanced salts solution
  • the CD40:CD154 binding interruptor can be adsorbed on the surface of the device, or otherwise incorporated or fabricated into the device material.
  • the CD40:CD154 binding interruptor can be a product that is secreted by cells or tissue that are either within the isolation chamber or otherwise associated with the device (for example, adhered to the device surface).
  • the device may deliver products made by two or more distinct cell or tissue types, that is, the device may include cells which produce the CD40:CD154 binding interruptor (facilitating acceptance and engraftment), and in addition also include the isolated cells or tissue which produce a product, or carry out an enzymatic process, which is needed by the recipient.
  • the device may include cells which produce the CD40:CD154 binding interruptor (facilitating acceptance and engraftment), and in addition also include the isolated cells or tissue which produce a product, or carry out an enzymatic process, which is needed by the recipient.
  • the invention can be used for treatment or prophylaxis of any mammalian recipient in need of cells or tissue suitable for implantation in an immunoisolation device.
  • Recipients accordingly are afflicted with, or at risk of, any defect or deficiency which can be overcome by products or processes which can be supplied by living cells or tissue.
  • the recipient can be afflicted with diabetes, which can be
  • the recipient can be afflicted with chronic pain or a neurodegenerative disease, such as epilepsy, amyotrophic lateral sclerosis, parkinsonism, Huntington's disease, Alzheimer's disease, motor neuron disease, bipolar disease, or muscular dystrophy, which can be treated with dopamine, epinephrine, acetylcholine, tryptophan, serotonin, or another neurotransmitter supplied by neural or neuroendocrine cells, such as adrenal chromaffin cells.
  • a neurodegenerative disease such as epilepsy, amyotrophic lateral sclerosis, parkinsonism, Huntington's disease, Alzheimer's disease, motor neuron disease, bipolar disease, or muscular dystrophy, which can be treated with dopamine, epinephrine, acetylcholine, tryptophan, serotonin, or another neurotransmitter supplied by neural or neuroendocrine cells, such as adrenal chromaffin cells.
  • the recipient can be afflicted with an endocrine disorder, such as hypocalcemia, which can be treated with a hormone supplied by endocrine cells, such as parathyroid hormone produced by parathyroid gland cells.
  • the recipient alternatively can be afflicted with infertility, which can be treated with reproductive hormone(s) supplied by reproductive endocrine tissue, such as uterine tissue.
  • Other recipients can be afflicted with a metabolic disorder or failure, such as liver failure, which can be treated using immunoisolated hepatocytes to supply enzymatic processes, such as detoxification reactions, to the recipient.
  • the recipient can be in need of any factor which can be supplied by genetically engineered cells, particularly cells which supply a factor genetically missing or defective in the recipient.
  • Exemplary factors include, without limitation, clotting factors, such as factor DC, for treatment of hemophiliac recipients; and, growth hormone for treatment of recipients with dwarfism.
  • the recipient can be in need of a genetically engineered factor to treat an acquired condition (e.g., erythropoeitin for a recipient with anemia), or to assist in healing a wound (e.g., nerve growth factor or neurotrophic factor for treatment of a recipient with nerve tissue trauma, such as spinal cord injury).
  • the recipient is a primate, more preferably a higher primate, most preferably a human.
  • the recipient may be another type of mammal, particularly a mammal of commercial importance, or a companion animal or other animal of value, such as a member of an endangered species.
  • recipient hosts also include, but are not limited to, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice.
  • the invention can be practiced for immunoisolation of any type of cells or tissue which secrete a product needed by the recipient, or supply an enzymatic process needed by the recipient.
  • the invention is particularly suitable for use in any context wherein the cells or tissue are not histocompatible (MHC -compatible) with the recipient, or would not be acceptable to the recipient's immune system, or otherwise would not be compatible with the recipient's body.
  • MHC -compatible histocompatible
  • the invention can be used with allogeneic or even xenogeneic cells or tissue.
  • Differentiated cells or tissue can be derived, by conventional means, from a volunteer or other living donor, or from a cadaveric donor.
  • the cells or tissue have been exposed to cold ischemic conditions for no more than about eight hours, more preferably no more than about two hours.
  • the donor is as histocompatible as practicable with the recipient.
  • the cells or tissue should preferably be autologous or allogeneic to the recipient.
  • the cells or tissue can be sourced from a heterologous species, such as a non-human primate (e.g., a chimpanzee or a baboon), or another relatively compatible mammal (e.g., a pig).
  • the cells or tissue for use in the present invention can be sourced from an adult donor, a neonatal or juvenile donor, or can be of fetal origin, depending on the desired cellular properties, the availability of particular tissue sources, and other parameters.
  • fibroblasts, vascular lining cells, hepatocytes, islet cells, epithelial cells, or other differentiated cell types can be obtained from an adult.
  • the cells or tissue can be obtained from a neonatal or fetal donor (examples would be neural cells, myoblasts, hepatoblasts, and the like).
  • Cells for immunoisolation can be postmitotic or mitotically competent cells; in the latter case, the cells are preferably conditionally mitotic. For example, the cells divide until a specific stimulus is applied, such as contact dependent growth inhibition, whereupon they enter a postmitotic state.
  • Myoblasts are a preferred example of conditionally mitotic cells for immunoisolation.
  • cells for immunoisolation can be derived from biopsied or resected, extracted or excised tissue sourced directly from a donor.
  • Many techniques are known for deriving a desired cell or tissue type from a donor organ, for example for preparing donor islets or islet cell suspensions from whole pancreata (see, e.g., Ricordi
  • the cells for immunoisolation can be cells grown in culture.
  • Such cultured cells can be a population of primary cells, or can be immortalized cells (e.g., a cell line).
  • cultured cells useful in the invention can be tumorigenic cells. The invention makes the use of tumorigenic cells possible because the cells are constrained by the immunoisolation device.
  • the invention still further encompasses the use of genetically modified cells, such as genetically engineered cells which have been (or are derived from ancestor cells which have been) transformed or transfected by conventional means with nucleic acid which encodes a desired secreted product, or encodes one or more enzymes for carrying out a desired enzymatic process.
  • genetically modified cells such as genetically engineered cells which have been (or are derived from ancestor cells which have been) transformed or transfected by conventional means with nucleic acid which encodes a desired secreted product, or encodes one or more enzymes for carrying out a desired enzymatic process.
  • Such cells can be engineered to express the encoded product or enzyme either constitutively or inducibly (e.g., under control of a stimulus-responsive promoter or enhancer).
  • Virtually any of the above-discussed types of cells can be genetically modified for use in this invention.
  • peripheral blood leukocytes, bone marrow, fibroblasts, epithelial cells, hepatocytes or muscle satellite cells can be obtained from the recipient, transfected ex vivo with nucleic acid encoding the desired product, and re-implanted in an immunoisolation device according to the present invention.
  • fetal cells, or cultured neonatal cells such as human umbilical cord vascular endothelial cells (HUVEC) cells, foreskin fibroblasts or myoblasts can be transfected ex vivo and immunoisolated.
  • HUVEC human umbilical cord vascular endothelial cells
  • dedifferentiated or immortalized cells from a cell line can be transfected ex vivo and immunoisolated.
  • myoblast cell lines are transfected with a vector encoding the secreted product of interest, along with a selectable marker such as the herpes simplex thymidine kinase gene, which allows for selective removal of dividing cells (see, e.g., Deglon et al. (1996), 7 Hum. Gene Ther. 2135-2146).
  • cells for immunoisolation can be derived from a transgenic mammal that has been engineered to include foreign nucleic acid encoding the desired product or enzyme in some or all of its body tissues.
  • the cells or tissue for isolation can be insulin-producing tissue, such as islets, islet beta cells, or engineered cells which express recombinant insulin.
  • the cells or tissue can be any naturally-sourced cell or tissue which produces any endocrine hormone, neuroendocrine hormone, neurotransmitter, neurotrophic factor, wound healing factor, angiogenic factor, morphogen, cytokine, lymphokine, chemokine, differentiation promoting factor, survival factor, immunomodulator, histocompatibility factor, blood clotting factor, antithrombotic factor, or other biological signalling molecule or structural protein.
  • tibodies include, without limitation: insulin, growth hormone, estrogen, progesterone, testosterone, parathyroid hormone, dopamine, epinephrine, acetylcholine, tryptophan, serotonin, substance P, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), transforming growth factor beta (TGF-beta) (or any TGF-beta superfamily member), any colony stimulating factor (CSF), erythropoeitin, any interleukin, tumor necrosis factor (TNF) (or any TNF superfamily member).
  • NGF nerve growth factor
  • CNTF ciliary neurotrophic factor
  • FGF fibroblast growth factor
  • TGF-beta transforming growth factor beta
  • CSF colony stimulating factor
  • erythropoeitin any interleukin
  • tumor necrosis factor (TNF) or any TNF superfamily member
  • tissue and cell types include, without limitation, those sourced from solid organs, such as brain, spinal cord, liver, heart, kidney, lung, uterus, pituitary, thyroid gland, parathyroid gland, adrenal gland, etc.; those sourced from tissue samples, organ sections, or biopsies, such as pancreatic islets, adrenal chromaffin cells, sertoli cells, blood vessels, nerve fibers or ganglia, skin plugs or sections (full- or partial-thickness), fat, bone, or cartilage; or those sourced from cell suspensions, isolates, or aspirates, such as peripheral blood leukocytes, or bone marrow cells (including sorted cells, such as stem cells or colony forming cells).
  • engineered cells useful in the present invention include cells which secrete recombinant versions of the above-mentioned naturally occurring products, or engineered versions thereof
  • cells or tissue useful in the present invention include those which naturally carry out needed enzymatic processes needed by the recipient, or which have been engineered to do so.
  • Hepatic tissue or hepatocytes are nonlimiting examples of such cells.
  • Therapeutic compounds useful for practice of the invention include any compound that blocks the interaction of cell surface CD40 (e.g., on B cells) with
  • CD40:CD154 binding interruptor compounds such as CD 154 blocking agents, that are specifically contemplated include polyclonal antibodies and monoclonal antibodies (MAbs), as well as antibody de ⁇ vatives such as chime ⁇ c molecules, humanized molecules, molecules with reduced effector functions, bispecific molecules, and conjugates of antibodies.
  • the antibody has the antigen-specific binding characteristics of MAb 5c8, as desc ⁇ bed in U.S. Patent 5,474,771, the disclosure of which is hereby incorporated by reference.
  • the antibody is a humanized 5c8.
  • antibodies against CD 154 include antibodies ImxM90, ImxM91 and ImxM92 (obtained from Immunex), an ant ⁇ -CD40L mAb commercially available from Ancell (clone 24-31, catalog # 353-020, Bayport, MN), and an anti- CD40L mAb commercially available from Genzyme (Cambridge, MA, catalog # 80- 3703-01). Also commercially available is an ant ⁇ -CD40L mAb from PharMmgen (San Diego, catalog #33580D) Numerous additional ant ⁇ -CD40L antibodies have been produced and characterized (see, e.g., WO 96/23071 of Bristol-Myers Squibb, the specification of which is hereby incorporated by reference).
  • the invention also includes use of CD 154 blocking agents of other types, such as complete Fab fragments, F(ab 2 compounds, V H regions, Fv regions, single chain antibodies (see, e.g., WO 96/23071), polypeptides. fusion constructs of polypeptides, fusions of CD40 (such as CD40Ig, as in HoUenbaugh et al., J. Immunol. Meth. 188: 1-7, 1995, which is hereby incorporated by reference), and small molecule compounds such as small semi-peptidic compounds or non-peptide compounds, all capable of blocking or interrupting CD40.CD154 binding. Procedures for designing, screening and optimizing small molecules are provided in PCT US96/ 10664, filed June 21, 1996, the specification of which is hereby incorporated by reference.
  • chimeric antibodies may be constructed, in which the antigen binding domain from an animal antibody is linked to a human constant domain (an antibody de ⁇ ved initially from a nonhuman mammal in which recombinant DNA technology has been used to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human
  • Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e.g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of the human antibody genes in which the antigen binding regions have been deleted.
  • Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly.
  • Another embodiment of the invention includes the use of human antibodies, which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, as described in U.S. 5,569,825.
  • Antibody fragments and univalent antibodies also can be used in practice of this invention.
  • Univalent antibodies comprise a heavy chain/light chain dimer bound to the Fc (or stem) region of a second heavy chain.
  • Fab region refers to those portions of the chains which are roughly equivalent, or analogous, to the sequences which comprise the Y branch portions of the heavy chain and to the light chain in its entirety, and which collectively (in aggregates) have been shown to exhibit antibody activity.
  • a Fab protein includes aggregates of one heavy and one light chain (commonly known as Fab'), as well as tetramers which correspond to the two branch segments of the antibody Y, (commonly known as F(ab) ), whether any of the above are covalently or
  • standard recombinant DNA techniques can be used to alter the binding affinities of recombinant antibodies with their antigens by altering amino acid residues in the vicinity of the antigen binding sites.
  • the antigen binding affinity of a humanized antibody may be increased by mutagenesis based on molecular modeling (Queen et al., Proc. Natl. Acad. Sci. 86:10029-33, 1989; PCT patent application WO 94/04679). It may be desirable to increase or to decrease the affinity of the antibodies for CD40L, depending on the targeted tissue type or the particular treatment schedule envisioned. This may be done utilizing phage display technology (see, e.g., Winter et al., Ann. Rev. Immunol.
  • the CD40:CD154 binding interruptors, including CD 154 blocking agents, used in the invention can be administered in any manner which is medically acceptable. Depending on the specific circumstances, local or systemic administration may be desirable.
  • the agent is administered via a parenteral route such as by an intravenous, intraarterial, subcutaneous, intramuscular, intraorbital, intraventricular, intraperitoneal, subcapsular, intracranial, intraspinal. or intranasal injection, infusion or inhalation.
  • the agent also can be administered by implantation of an infusion pump, a biocompatible or bioerodable sustained release implant or other drug delivery device (e.g., capsules, liposomes), into the recipient, either before or after implantation of the immunoisolation device.
  • the implant for supplying the interruptor is disposed at or near the site of implantation of the immunoisolation device, so as to deliver the interruptor to the local environment of the immunoisolation device.
  • certain compounds of the invention, or formulations thereof may be appropriate for oral or enteral administration. Still other compounds of the invention will be suitable for topical administration.
  • the CD40:CD154 binding interruptor is provided indirectly to the recipient, by administration of a vector or other expressible genetic material encoding the interruptor.
  • the genetic material is internalized and expressed in cells or tissue of the recipient, thereby producing the interruptor in situ.
  • a suitable nucleic acid construct would comprise sequence encoding one or more of the MAb 5c8 immunoglobulin (Ig) chains as disclosed in U.S. Pat. 5,474,771.
  • Other suitable constructs would comprise sequences encoding chimeric or humanized versions of the MAb 5c8 Ig chains or antigen-binding fragments thereof.
  • Still other suitable constructs would comprise sequences encoding part or all of other CD 154- specific MAbs.
  • the construct is delivered systemically or locally, e.g., to a site vicinal to the site of device implantation.
  • the vector or other genetic material encoding the interruptor is internalized within a suitable population of isolated cells to produce interuptor- producing host cells. These host cells then are implanted or infused into the recipient, either locally or systemically, to provide in situ production of the CD40:CD154 binding interruptor.
  • Appropriate host cells include cultured cells, such as immortalized cells, as well as cells obtained from the recipient (e.g., autologous peripheral blood or lymph node cells, such as natural killer (NK) cells, or fibroblasts or muscle satellite cells).
  • the host cells are implanted at or near the site of device implantation, and optionally may be attached to a support matrix or enclosed within a macroporous device (which allows the interruptor to diffuse freely).
  • the CD40:CD154 binding interruptor compounds used in practice of the invention are suspended, dissolved or dispersed in a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient comprises normal physiologic saline (0.15M NaCl, pH 7.0 to 7.4).
  • acceptable carriers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences. Gennaro, ed., Mack Publishing Co., 1990. Acceptable carriers can include biocompatible, inert or bioabsorbable salts, buffering
  • agents oligo- or polysaccharides, polymers, viscosity-improving agents, preservatives, and the like.
  • Any CD40:CD154 binding interruptor, such as a CD 154 blocking agent, that is used in practice of the invention is formulated to deliver a pharmaceutically-effective or therapeutically-effective amount or dose, which is an amount sufficient to produce a detectable, preferably medically beneficial effect on the recipient.
  • Medically beneficial effects would include preventing, delaying or attenuating deterioration of, or detectably improving, the recipient's medical condition.
  • renal function and health of a kidney allograft or xenograft can be monitored by routinely measuring the concentrations of blood urea nitrogen or creatinine, or the volume or solute contents of urine, or the rate of clearance of relevant solutes from the blood into the urine.
  • an effective amount of a therapeutic compound of the invention is any amount which detectably decreases the recipient's dependence on exogenous drug or other therapy.
  • An optimal effective amount is one which substantially frees the recipient of dependence on exogenous drug. More specifically, an effective amount is one which induces partial or substantially complete engraftment (acceptance and function) of the implanted immunoisolation device.
  • the amount of and frequency of dosing for any particular compound to be used in practice of the invention is within the skills and clinical judgement of ordinary practitioners of the tissue transplant arts, such as transplant surgeons.
  • the general dosage and administration regime is established by preclinical and clinical trials, which involve extensive but routine studies to determine effective, e.g., optimal, administration parameters for the desired compound. Even after such recommendations are made, the practitioner will often vary these dosages for different recipient hosts based on a variety of considerations, such as the recipient's age, medical status, weight, sex, and concurrent treatment with other pharmaceuticals. Determining
  • an effective dosage and administrauon regime for each CD40:CD154 binding interruptor used to inhibit graft rejection is a routine matter for those of skill in the pharmaceutical and medical arts.
  • the dosage amount and timecourse of should be sufficient to produce a clinically beneficial change in one or more indicia of the recipient's health status. Numerous exemplary timecourse and dosage regimes will be apparent to skilled practitioners.
  • the invention involves administration of a CD40:CD154 binding interruptor (exemplified by a humanized MAb 5c8, hu5c8) in an acceptance-inducing regime, followed if deemed prudent by an acceptance-maintaining regime, as described in prior PCT/US98/12892.
  • an effective dose of antibodies ranges from about 1 mg/kg body weight to about 20 mg/kg body weight, administered daily for a period of about 1 to 5 days, preferably by bolus intravenous administration.
  • the same dosage and dosing schedule may be used in the load phase of a load- maintenance regimen, with the maintenance phase involving intravenous or intramuscular administration of antibodies in a range of about 0.1 mg/kg body weight to about 20 mg/kg body weight, for a treatment period of anywhere from weekly to 3 month intervals.
  • Chronic treatment may also be carried out by a maintenance regimen, in which antibodies are administered by intravenous or intramuscular route, in a range of about 0.1 mg/kg body weight to about 20 mg/kg body weight, with interdose intervals ranging from about 1 week to about 3 months.
  • chronic treatment may be effected by an intermittent bolus intravenous regimen, in which between about 1.0 mg/kg body weight and about 100 mg/kg body weight of antibodies are administered, with the interval between successive treatments being from 1 to 6 months.
  • administration may also be by oral, pulmonary, nasal or subcutaneous routes.
  • the effectiveness of the antibodies can be increased by administration serially or in combination with conventional anti-rejection therapeutic agents or drugs such as, for example, corticosteroids or immunosuppressants.
  • the antibodies may be conjugated to a conventional agent. This advantageously permits the administration of the conventional agent in an amount less than the conventional dosage, for example, less than about 50% of the conventional dosage, when the agent is administered as monotherapy. Accordingly, the occurrence of many side effects associated with that agent should be avoided.
  • Combination therapies according to this invention for treatment of implant rejection include the use of anti-CD40L antibodies together with agents targeted at B cells, such as anti-CD19, anti-CD28 or anti-CD20 antibody (unconjugated or radiolabeled), IL-14 antagonists, LIP394 (LaJolla Pharmaceuticals receptor blocker), IR-1116 (Takeda small molecule) and anti-Ig idiotype monoclonal antibodies.
  • agents targeted at B cells such as anti-CD19, anti-CD28 or anti-CD20 antibody (unconjugated or radiolabeled), IL-14 antagonists, LIP394 (LaJolla Pharmaceuticals receptor blocker), IR-1116 (Takeda small molecule) and anti-Ig idiotype monoclonal antibodies.
  • the combinations may include T cell/B cell targeted agents, such as CTLA4Ig, IL-2 antagonists, IL-4 antagonists, IL-6 antagonists, receptor antagonists, anti-CD80/CD86 monoclonal antibodies, TNF, LFA1/ICAM antagonists, VLA4/VCAM antagonists, brequinar and IL-2 toxin conjugates (e.g., DAB), prednisone, anti-CD3 MAb (OKT3), mycophenolate mofetil (MMF), cyclophosphamide, and other immunosuppressants such as calcineurin signal blockers, including without limitation, tacrolimus (FK506).
  • T cell targeted agents such as CD4 antagonists, CD2 antagonists and IL-12.
  • a maintenance dose of anti-CD40L antibodies is administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced. Where no sign of implant rejection is evident, treatment might cease, with vigilant monitoring for signs of rejection. In other instances, as determined by the ordinarily skilled practitioner, occasional treatment might be administered, for example at intervals of four weeks or more. Recipients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • CD40:CD154 interrupting compound e.g., an anti-CD40L compound or a CD 154 blocking agent, such as a MAb having the specificity of MAb 5c8
  • a CD40:CD154 interrupting compound e.g., an anti-CD40L compound or a CD 154 blocking agent, such as a MAb having the specificity of MAb 5c8

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Transplantation (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des procédés et des compositions destinés à inhiber le rejet d'un implant chez un receveur, notamment d'un dispositif d'isolation immunologique. Les procédés et compositions de l'invention sont d'usage approprié concernant des dispositifs qui comprennent des cellules ou des tissus, tels que des tissus endocriniens ou neuroendocriniens, ou du tissu hépatique, afin d'apporter, au receveur d'implant, une variété de produits sécrétés ou de processus enzymatiques. Le rejet est inhibé par l'usage d'un interrupteur de liaison CD40:CD154, tel qu'un agent bloquant de CD154, de préférence un anticorps monoclonal qui se lie spécifiquement au CD154 et empêche sont interaction avec le CD40.
PCT/US1999/005016 1998-03-09 1999-03-09 Therapie de blocage du cd154 pour la modulation des reponses immunes aux implants WO1999045958A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU28999/99A AU2899999A (en) 1998-03-09 1999-03-09 Cd154 blockade therapy for modulation of immune responses to implanted devices

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7726598P 1998-03-09 1998-03-09
US60/077,265 1998-03-09
USPCT/US98/12892 1998-06-19
PCT/US1998/012892 WO1998058669A2 (fr) 1997-06-20 1998-06-19 Therapie de blocage par cd154 pour transplantation de tissus d'ilots pancreatiques

Publications (1)

Publication Number Publication Date
WO1999045958A1 true WO1999045958A1 (fr) 1999-09-16

Family

ID=22137063

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/005016 WO1999045958A1 (fr) 1998-03-09 1999-03-09 Therapie de blocage du cd154 pour la modulation des reponses immunes aux implants

Country Status (1)

Country Link
WO (1) WO1999045958A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6534277B1 (en) 2000-04-14 2003-03-18 Millennium Pharmaceuticals, Inc. Method for identifying a compound to be tested for an ability to reduce immune rejection by determining Stat4 and Stat6 proteins
US8958896B2 (en) 2007-06-28 2015-02-17 Apple Inc. Dynamic routing of audio among multiple audio devices

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058669A2 (fr) * 1997-06-20 1998-12-30 Biogen, Inc. Therapie de blocage par cd154 pour transplantation de tissus d'ilots pancreatiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058669A2 (fr) * 1997-06-20 1998-12-30 Biogen, Inc. Therapie de blocage par cd154 pour transplantation de tissus d'ilots pancreatiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRAUKER J H ET AL: "PROTECTION OF ALLOGRAFT TISSUES FROM REJECTION BY IMPLANTATION WITHIN AN IMMUNOISOLATION DEVICE.", KEYSTONE SYMPOSIUM ON TISSUE ENGINEERING, KEYSTONE, COLORADO, USA, APRIL 3-10, 1992. J CELL BIOCHEM SUPPL. (1992) 0 (16 PART F), 128., XP002109831 *
BUHLMANN, JANET E. ET AL: "Therapeutic potential for blockade of the CD40 ligand, gp39", J. CLIN. IMMUNOL. (1996), 16(2), 83-9, XP002080039 *
LARSEN, CHRISTIAN P. ET AL: "CD40-gp39 interactions play a critical role during allograft rejection: Suppression of allograft rejection by blockade of the CD40-gp39 pathway", TRANSPLANTATION (1996), 61(1), 4-9, XP002109830 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6534277B1 (en) 2000-04-14 2003-03-18 Millennium Pharmaceuticals, Inc. Method for identifying a compound to be tested for an ability to reduce immune rejection by determining Stat4 and Stat6 proteins
US8958896B2 (en) 2007-06-28 2015-02-17 Apple Inc. Dynamic routing of audio among multiple audio devices

Similar Documents

Publication Publication Date Title
US20070292440A1 (en) CD154 blockade therapy for pancreatic islet tissue transplantation
US20070244053A1 (en) Use of a CD40:CD154 binding interruptor to prevent counter-adaptive immune responses, particularly graft rejection
JP2002502824A (ja) 同種移植における補刺激遮断および混合キメラ現象
US20030170239A1 (en) Immunotherapeutic method to prevent islet cell rejection
WO2006052660A2 (fr) Blocage du recepteur il-7 pour supprimer l'immunite
WO1999045958A1 (fr) Therapie de blocage du cd154 pour la modulation des reponses immunes aux implants
EP1754490A2 (fr) Thérapie de blocage par CD154 pour transplantation de tissus d'ilots pancréatiques chez les primates
PL191122B1 (pl) Zastosowania przerywacza wiązania CD40:CD154
CZ461699A3 (cs) Použití činidla rušícího vazbu CD40:CD154 pro přípravu léku k zabránění odhojení štěpu tkáně tvořící inzulín
CN1736483A (zh) 应用cd40：cd154结合阻断物预防反适应性免疫应答,特别是移植物排斥反应
MXPA99011740A (en) Cd154 blockade therapy for pancreatic islet tissue transplantation
CZ403599A3 (cs) Použití činidla rušícího vazbu CD40:CD154 pro výrobu léku k inhibici odhojení tkáňového štěpu a přípravek obsahující činidlo rušící vazbu CD40:CD154
MXPA99010571A (en) Use of a cd40:cd154 binding interruptor to prevent counter adaptive immune responses, particularly graft rejection

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase