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WO1997011607A9 - Methode d'inhibition de la destruction par le systeme immunitaire de cellules vivantes greffees - Google Patents

Methode d'inhibition de la destruction par le systeme immunitaire de cellules vivantes greffees

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Publication number
WO1997011607A9
WO1997011607A9 PCT/US1996/015577 US9615577W WO9711607A9 WO 1997011607 A9 WO1997011607 A9 WO 1997011607A9 US 9615577 W US9615577 W US 9615577W WO 9711607 A9 WO9711607 A9 WO 9711607A9
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WIPO (PCT)
Prior art keywords
cells
subject
viable
insulin
tissue
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PCT/US1996/015577
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English (en)
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WO1997011607A1 (fr
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Publication date
Application filed filed Critical
Priority to EP96936037A priority Critical patent/EP0877555B1/fr
Priority to JP9513711A priority patent/JP2000500121A/ja
Priority to AU73782/96A priority patent/AU721737B2/en
Priority to DE69637216T priority patent/DE69637216D1/de
Publication of WO1997011607A1 publication Critical patent/WO1997011607A1/fr
Publication of WO1997011607A9 publication Critical patent/WO1997011607A9/fr
Priority to US09/049,865 priority patent/US7041634B2/en

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  • microcapsulated, xenogeneic islets to provide a durable, physiological source of insulin to diabetic patients. It has previously been shown that microcapsules are biocompatible and that xenogeneic islet grafts contained in microcapsules functioned indefinitely in the peritoneal cavity of mice with streptozotocin-induced (SZN) diabetes. Thus, microcapsules may be intact and stable in vivo and factors that may be required for long-term survival and function of the xenogeneic islets are accessible.
  • SZN streptozotocin-induced
  • the microcapsules serve as a mechanical barrier that prevents cell-to-cell contact between recipient lymphocytes and donor islets.
  • the mechanical barrier primarily prevents host sensitization rather than protecting the graft from immune destruction, because encapsulated islets are very rapidly destroyed by recipients that are presensitized to the islet donor cell antigens.
  • encapsulated xenogeneic islets were rejected (in two weeks) by NOD mice, which is possibly due to presensitization of NODs to islet antigens.
  • Xenografts undergoing rejection in NOD mice were surrounded by large numbers of activated macrophages and immunoglobulins, with IL-l ⁇ , TNF ⁇ , both documented by immunocytochemistry, and IL-4 messenger RNA detected by RT- PCR.
  • NOD rejection is initiated by donor antigens that are secreted by or shed from the encapsulated islets and which are processed via the MHC (major histocompatibility complex) class II pathway by host APC (antigen presenting cells) .
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • Islet transplantation is an attractive therapy for patients with IDDM, since problems related to the exocrine pancreas may be avoided.
  • allografts of donor human islets have not been successful long-term (3) ; and availability and yield of human islets are limited.
  • Therapeutic islet transplants for large number of patients almost certainly will require donor islets harvested from animals
  • Islets have been isolated from subhuman primates and xenografted into immunosuppressed, diabetic rodents, with short-term reversal of diabetes (6) .
  • Other promising sources are porcine, bovine, canine, and rabbit islets, which function remarkably well, (i.e., maintaining normoglycemia) in diabetic rodents until transplant rejection occurs (7-11)
  • Long-term human, bovine and porcine islet xenograft survival has been documented in nude mice and rats, suggesting that sufficient islet-specific growth factors are present in xenogeneic recipients (2,12-17) .
  • canine islets are not clinically appropriate. Porcine islets are both difficult to isolate (intact) and to maintain in vi tro; nevertheless, they are extremely promising for eventual clinical application (18-21) . Isolation of bovine islets is technically easier (than porcine islets) , and calf islets are glucose-responsive (22) . Recently, large scale rabbit islets isolation has been developed (23) (see Preliminary Studies) . Rabbit pancreas is an attractive source of islets. Rabbit, like porcine insulin, differs from human insulin at only one amino acid, and rabbit islets are glucose responsive (22,24) . In addition, most humans do not possess natural anti-rabbit antibodies, which might improve the possibility of preventing xenograft rejection
  • acrylic-copolymer hollow fibers placed subcutaneously maintained viability of human islet allografts for two weeks (50 islets per 1.5 cm fiber) (65,000 M.W. permeability) (32) .
  • Nonobese diabetic mice develop diabetes spontaneously, beginning at approximately twelve weeks of age .
  • NOD mice are the most appropriate model for studying the feasibility of islet xenotransplants because their disease resembles human IDDM in several ways. Macrophage, dendritic cell and lymphocytic infiltration of islets can be detected as early as four weeks of age and precedes overt hyperglycemia (43-46) .
  • NOD diabetes is T lymphocyte- dependent (43-45) ; and it is associated with (MHC) Class II genes (47-50) . Cytotoxic T cells and antibodies specific for beta cells or for insulin have been identified, characterized and cloned from NOD mice (44,45,51-55) .
  • the disease can be induced in non-diabetic, syngeneic mice by transfer of both CD8 + and CD4 + T cells or T-cell clones from diabetic NODs (44,52,55,58) ; and inhibition of NOD macrophages or CD4 + T lymphocytes or treatment with anti-Class II monoclonal antibodies prevents or delays diabetes onset in NOD mice (59,50) .
  • Defects in NOD macrophages, C5 complement and NK cell function have been reported (61) . It has been suggested that helper T-cells function to activate CD8 + cells, which damage beta cells by direct cytotoxic attack. However, some recent studies have suggested that beta cell killing may be indirect, from a nonspecific inflammatory response which initially involves
  • CD4* cells but also includes infiltrating macrophages, which release cytokines and oxygen free-radicals
  • NOD mice are the best model in which to study islet xenografts.
  • NOD background resulting in immuno-deficient NOD-Scid mice (66-69) .
  • These mice homologous for the Scid mutation, which results in an inability to rearrange T-cell receptor and immunoglobulin genes (66,67) .
  • the consequence is an absence of T and B-lymphocytes .
  • These mice do not develop diabetes spontaneously; but they may be rendered diabetic with multiple low-dose streptozotocin (MLD-SZN) regimens, making them an optimal model for adoptive transfer experiments (67-69) .
  • NOD-Scids express NOD MHC genes and other genes that are relevant for development of the disease. They mount robust macrophage and limited NK-cell responses, but are functionally T- and B-lymphocyte deficient (69) .
  • NOD mice Unlike mice with SZN-induced diabetes, diabetic NOD mice rapidly reject unencapsulated islet xenografts, allografts and isografts (7,8,10,19,33,56,70,71) .
  • Conventional immunosuppressive regimens have little effect on this reaction (10,71-73) .
  • Treatment of NOD recipients with monoclonal antibodies directed against CD4 + helper T lymphocytes or FK506 prolongs islet graft function (from 5 to 25 days) (7,8,10,73) ; but long-term islet graft survival in NODs has not been reported.
  • microcapsules like other bioartificial membrane devices promote survival of xenogeneic and allogeneic islets by: (A) preventing or minimizing release of donor antigen (s) , thereby reducing host sensitization, and/or (B) preventing or reducing host effector mechanisms (i.e. T-cell contact, anti-graft antibody binding, cytokine release) .
  • Cytokines known to be products of macrophages including IL-1 and TNF (62,77,85,86) , may be involved destruction of encapsulated islets. Both IL-1 and TNF have been reported to reduce insulin secretion and cause progressive damage of islet cells in vi tro (58,62-64,85- 87) . Cytokine- ediated injury might occur directly or indirectly, by activation of an intraperitoneal inflammatory response (30,77) .
  • Th2 cytokines IL-4, IL-5, IL-10
  • IL-2 messenger RNA increases in IL-2 (11,90)
  • Increased Th2 activity relative to Thl (93-95) activity is distinct from the known NOD 'Thl' anti-islet immune response (56,57,96) .
  • the Th2 response is characteristic of evoked antibody responses to foreign antigens and suggests that humoral reactions to encapsulated xenografts may be of critical importance. Furthermore, strategies designed to abrogate Th2' responses may significantly prolong encapsulated islet xenograft survival.
  • the *Th2' helper T- cell cytokine mRNA profile is characteristic of antibody responses to foreign antigens.
  • APCs In mice, one such costimulatory pathway involves the interaction of the T-cell surface antigen, CD28 with either one of two ligand, B7-1 and B7-2, on the APCs (95,97-102) . Once this full interaction of T-cells and APCs occurs, however, subsequent re-exposure of T-cells to peptide, mitogen, etc. will result in proliferation in the absence of costimulation. (95) .
  • CTLA4 is a cell surface protein that is closely related to CD28; however, unlike CD28, CTLA4 is expressed only on activated T-cells. B7-1 has a high affinity for CLTA4 than CD28; and it has been suggested that CTLA4 may modulate functions of CD28 (97,103,104) .
  • CTLA4Ig is a recombinant soluble fusion protein, combining the extracellular binding domain of the CTLA4 molecule with constant region of the ⁇ fG- gene. Both human and murine CTLA4Ig have been shown to inhibit T-lymphocyte responses in mice (141,142) . Administration of CTLA4Ig to mice has been shown to induce antigen-specific unresponsiveness (in a murine lupus model) (97,99,105) and long-term acceptance of murine cardiac allografts (106,107) . In addition, Lenschow, et al . , found that it induced tolerance to human islets in SZN-diabetic mice (12) .
  • CTLA4Ig has also been reported to reduce the incidence of diabetes in NODs (108) . There are no reports of effects of CTLA4Ig on islet graft survival in spontaneously-diabetic recipients, such as NOD mice. However, our studies show that CTLA4Ig significantly prolongs survival of encapsulated rabbit islets in NOD recipients.
  • helper T-cell-APC interactions with recognition of the importance of binding of the APC-CD40 antigen to its ligand, GP39, on helper T- cells (109,110) .
  • a monoclonal hamster anti-murine GP39 antibody (MRl) blocks helper T-cell interactions with APCs, macrophages, effector T-cells and B-lymphocytes (109,110) .
  • MRl monoclonal hamster anti-murine GP39 antibody
  • Dr. A. Rossini has reported recently (IPITA conf . 6/95) that MRl plus B7 negative donor spleen cells day 7 allows long-term survival of both allo- and xeno-geneic islets in SZN-diabetic mice.
  • Isograft biopsies showed viable islets, intact capsules and no pericapsular immune reaction (36) , while biopsies of failed allografts revealed pericapsular cellular responses and nonviable islets. This is the only report in the literature with encapsulated islet isograft controls. Although the Lewis rat model is not one with autoimmune diabetes, the results are significant, and suggest that donor antigen(s) are the stimulus for subsequent host responses.
  • This invention provides a method of inhibiting viable cells transplanted into a subject from being destroyed by the subject's immune system which comprises: a) containing the viable cells, or tissue comprising the viable cells, prior to transplantation within a device comprising a semipermeable membrane; and b) treating the subject with a substance which inhibits an immune-system costimulation event in an amount effective to inhibit the subject's immune system from responding to said contained cells or tissue.
  • the substance which inhibits an immune- system costimulation event is CTLA4.
  • this invention further provides a method of inhibiting viable cells transplanted into a subject from being destroyed by the subject's immune system which comprises: a) containing the viable cells, or tissue comprising the viable cells, prior to transplantation within a device comprising a semipermeable membrane; and b) treating the subject with CTLA4 in an amount effective to inhibit the subject's immune system from responding to said contained cells or tissue .
  • This invention also provides a method of treating diabetes in a subject which comprises: a) containing viable insulin- producing cells, or tissue comprising viable insulin- producing cells, within a device comprising a semipermeable membrane so as to obtain contained viable insulin-producing cells; b) transplanting contained viable insulin-producing cells obtained in step (a) into the subject in an amount effective to treat diabetes in the subject; and c) treating the subject with a substance which inhibits an immune- system costimulation event in an amount effective to inhibit the subject's immune system from responding to an amount of contained viable insulin-producing cells according to step (b) .
  • Figure 1 Encapsulated Lewis rat islet, day #150 after xenografting to unmodified diabetic NOD H&E. (x250) .
  • the microcapsule is a "double-wall" microcapsule.
  • FIG. 3 Comparison of survival of rabbit islets encapsulated in microcapsules with a permeability of up to 70,000 Kd to survival of rabbit islets in microcapsules having a permeability of 100,000 Kd.
  • Figure 4 Effect of Lewis rat splenocyte priming on Lewis rat-to-NOD microencapsulated islet transplantation.
  • Figure 5 Effect of Lewis rat islet priming on Lewis rat-to-NOD encapsulated islet transplantation .
  • FIG. 6 Microencapsulated dog islet, day #80, from peritoneum of NOD mouse treated with Gkl .5. H&E (x250) .
  • Figure 7 Functioning, encapsulated rabbit islets, biopsied day #86, from peritoneum of NOD mouse, treated with CTLA4Ig. Note absence of NOD cell response and the presence of viable islets within capsule. H&E (x400) .
  • Figure 9 Survival of microencapsulated mouse INS- CTLA4 islets transplanted into NODs. These islets express CTLA4.
  • Figure 10 Effects of transplanting rat islets into streptozotocin SZN - diabetic NOD-Scid mice.
  • Figure 12 Effects of transplanting microencapsulated rabbit islets into streptozotocin (SZN) - diabetic NOD-Scid mice.
  • Figure 13 Functioning, encapsulated rabbit islets, biopsied day #86, from peritoneum of NOD mouse, treated with CTLA4Ig. Note absence of NOD cell response and viable islets within capsule. H&E. (x400) . Arrows point to outside of capsule wall .
  • Figure 14 Yield of Islets from Neonatal Porcine Pancreas (Total Islet #) .
  • Nonencapsulated (N) and Encapsulated (E) Neonatal Porcine Islets (uU/1000 islets/24hr. )
  • Figure 16 Dispersed neonatal porcine "islets", in tissue culture, day #5. Anti-insulin - l ⁇ immunocytochemistry demonstrates 5-10% beta cells. Approx. 400X.
  • Neonatal islet in microcapsule biopsied day # 103 from SZN-diabetic NOD-Scid mouse, anti-insulin immunohistochemistry, showing intensely insulin-positive beta cells, occupying approximately 80% of islet. Approx. 40OX. Arrow points to outer surface of microcapsule membrane .
  • Figure 18 Non-encapsulated intrasplenic/portal neonatal procine islet xenograft in streptozotocin diabetic NOD-Scid mouse.
  • Biopsies revealed viable porcine islets in both liver and splenic parenchyma.
  • Figure 19 Intraperitoneal microencapsulated neonatal porcine islet xenograft into streptozotocin-diabetic NOD-Scid mouse.
  • FIG. 20 Neonatal porcine islet in mirocapsule, biopsied day #103 after xenotransplantation to SZN-diabetic NOD-Scid mouse. H & E, X 400. Arrow points to inner surface of microcapsule membrane.
  • Figure 21 Encapsuled Neonatal Porcine Islet
  • CTLA4Ig 200 ⁇ g i.p. Q.O.D., x 20 days.
  • NOD 880 was biopsied at day #101 (see Fig.
  • FIG. 22 Microencapsulated neonatal porcine islet, biopsied 101 days after xenotransplantation i.p. to spontaneously diabetic NOD mouse.
  • CTLA4Ig 200 ⁇ g i.p. Q.O.D., days # 0-21.
  • Figure 24 Intraperitoneal microencapsulated neonatal porcine islet xenografts in NOD mice treated with CTLA4Ig * , which does not fix complement .
  • Spleen cells were cultured at 2xl0 6 cells/ml in 96-well plates with no antigen, 10 empty capsules, 10 capsules containing neonatal pig islets, 4 x 10 3 neonatal pig islets that were unirradiated or irradiated with 2000R.
  • Spleen cells were obtained from normal NOD mice (panel A) ; diabetic NOD mice (panel B) ,- diabetice NOD mice that were transplanted with encapsulated, neonatal pig islets and injected with CTLA4Ig * (panel D) as described in Fig.24. After 48 hrs incubation, 3 H-TdR was added and the cells harvested 18 hrs later. Results represent the average ⁇ SD of triplicate cultures.
  • FIG. 26 Lymphokine production in cultures of spleen cells from the mice described in Fig. 24 were determined by ELISA. Spleen cells from normal or diabetic NOD mice were cultured with unirradiated neonatal, pig islets as described in Fig. 24. Supernatent fluids were harvested after 24 hrs of incubation and assayed for IL-4, IL- 10 and IFN ⁇ using a sandwich ELISA and the appropriate recombinant cytokines as standards.
  • FIG. 27 Model of immune response to micro encapsulated, xenogeneic islets by autoimmune, NOD mice. Secreted insulin clearly crosses the membrane of double walled microcapsules and regulated glucose levels in engrafted mice.
  • 1) Potentially, other donor proteins or protein fragments of less than 100,000mw (AgX) that are shed or secreted by islets diffuse out of microcapsules and are endocytosed by dendritic cells.
  • 2) Dendritic cells process proteins via the MHC class II pathway and present peptide X complexed with class II and co-stimulatory molecules to CD4* T cells. In the presence of the appropriate cytokines, CD4 * T cells are activated and develop into Th2 cells that express CD40L (GP39) .
  • This invention provides a method of inhibiting viable cells transplanted into a subject from being destroyed by the subject's immune system which comprises: a) containing the viable cells, or tissue comprising the viable cells, prior to transplantation within a device comprising a semipermeable membrane; and b) treating the subject with a substance which inhibits an immune-system costimulation event in an amount effective to inhibit the subject's immune system from responding to said contained cells or tissue.
  • an "immune-system costimulation event” is an interaction between an APC and a T-cell required in conjunction with the binding of an MHC-bound antigen on the surface of the APC to the T cell receptor.
  • Immune-system costimulation events include any specific binding of an APC cell-surface molecule (other than an MHC-bound antigen) to a specific ligand on a T cell.
  • specific bindings include, but are not limited to, binding of a B7 molecule (present on the surface of an APC) to a CTLA4 receptor or a CD28 receptor on the surface of a T cell, and binding of a CD40 molecule (present on the surface of an APC) to GP39 (on the surface of a T cell) .
  • Substances which inhibit immune-system costimulation events are known in the art and include, but are not limited to,
  • T cell or APC cell-surface-molecule analogs such as MRl
  • CTLA4 (which blocks the binding of a B7 molecule to a CD28 receptor or a CTLA4 receptor) .
  • CTLA4 the substance which inhibits an immune-system costimulation event is CTLA4.
  • CTLA for purposes of this invention, is meant to indicate any proteinaceous construct which comprises an amino acid sequence which is the same as or sufficiently the same as the amino acid sequence of the CTLA4 receptor such that the proteinaceous construct is capable of binding to a B7 molecule, thereby blocking the B7 molecule from binding to a CTLA4 receptor on a T cell.
  • Proteinaceous constructs are well known in the art and indicate any molecule which comprises amino acid moieties linked to one another by peptide bonds; including peptides, polypeptides, and molecules comprising peptide and/or peptide subunits.
  • CTLA4 includes, but is not limited to, molecules expressed by the gene encoding the B7-binding site of the CTLA4 receptor in genetically engineered cells, molecules expressed by mutants of the gene encoding the B7-binding site of the CTLA4 receptor which molecules are capable of binding to a B7 molecule, and synthetic amino acid chains having an amino acid sequence which is the same as or sufficiently the same as the amino acid sequence of the CTLA4 receptor such that they are able to bind to B7.
  • CTLA4 also includes soluble CTLA4 comprising the extracellular binding domain of the CTLA4 receptor, such as CTLA4Ig.
  • CTLA4Ig i.e. a recombinant soluble fusion protein which combines the extracellular binding domain of the CTLA4 receptor with the constant region of IgG j .
  • the substance which inhibits an immune-system costimulation event also alters the cytokine profile of the subject so as to protect the contained cells or tissue from the subject's immune system.
  • cytokine profile means the type and quantity of each type of cytokine produced in a subject at a given time.
  • Cytokines are proteins which have an immune effect and which are released by white blood cells. Examples of cytokines include, but are not limited to interferon (such as gamma-interferon) , tumor necrosis factor, interleukin (ID 1, IL-2, IL-4, IL-6, and IL-10.
  • the substance may be a substance which increases the production of gamma-interferon in the subject.
  • An example of a substance which alters the cytokine profile of a subject so as to protect contained cells or tissue grafted into the subject is CTLA4Ig.
  • the substance which inhibits an immune-system costimulation event binds complement.
  • Substances which bind complement favor prolonged survival of contained cells or tissue grafted into the subject.
  • An example of a substance which binds complement is CTLA4Ig.
  • This invention also provides a method of inhibiting viable cells transplanted into a subject from being destroyed by the subject's immune system which comprises: a) containing the viable cells, or tissue comprising the viable cells, prior to transplantation within a device comprising a semipermeable membrane; and b) treating the subject with CTLA4 in an amount effective to inhibit the subject's immune system from responding to said contained cells or tissue .
  • Devices comprising a semipermeable membrane useful for transplantation of viable cells or tissue are well-known to those of ordinary skill in the art, and any such device may be used in the subject invention.
  • Devices useful for the subject invention may be comprised of various materials and may be formed into various shapes, such materials and shapes being well known in the art. Any particular device for an application of this invention is selectable based on factors including, but not limited to, the biocompatibility of the material with the subject, the site of transplantation, whether the transplantation is intravascular or extravascular, the method of transplantation, availability, and economy.
  • suitable shapes for devices include, but are not limited to, hollow fibers, discs, and spheres.
  • Suitable materials include, but are not limited to, agarose hydrogel, plastics, polymers, and polyamino acids.
  • a device may be comprised of more than one material.
  • the device is a microcapsule.
  • microcapsule means any polyamino acid spherical capsule. Microcapsules as defined herein and their methods of manufacture are well known in the art and include, but are not limited, single layered, double layered, or multilayered polyamino acid spheres, as well as polyamino acid spheres comprising a layer or more than one layer of alginate.
  • the viable cells or the tissue comprising the viable cells in the aforementioned method of this invention may be derived from any source for viable cells.
  • the viable cells or the tissue are derived from a xenogeneic donor, i.e. a subject which is a different species from the subject into which the viable cells or tissue are transplanted.
  • the viable cells or the tissue comprising the viable cells are derived from an allogeneic donor, i.e. a subject which is of the same species as the subject into which the viable cells or tissue are transplanted.
  • the viable cells or the tissue comprising the viable cells are derived from the subject into which they are transplanted, i.e. they are, inter alia, obtained from the subject, contained within the device, and transplanted back into the subject. Viable cells obtained from the subject may, for example, be genetically engineered after they are obtained and before they are transplanted back into the subject.
  • the viable cells or tissue comprising viable cells may be obtained from any donor.
  • the donor is a mammal.
  • a mammalian donor may, for example, be a calve, a pig, a rabbit, a rat, a mouse, or a human.
  • the viable cells or tissue comprising viable cells may be obtained from a mammalian neonate, such as a neonatal pig.
  • the subject of the invented method described herein may be any subject into which transplantation of viable cells is desired.
  • the subject is a human. If the subject is a human, the viable cells, or tissue containing them, are in one embodiment derived from a mammal, for example a human.
  • the subject is a domesticated animal.
  • a domesticated animal is any animal subjected to human intervention.
  • domesticated animals include, for example, farm animals which are raised by humans and which are used as a resource for products for human consumption. Such products include, but are not limited to, meat, milk, and leather.
  • Examples of domesticated animals include, but are not limited to, cows, pigs, sheep, horses, and chickens.
  • domesticated animals useful in applications of the subject invention may be adults, infants, or domesticated animals at any other developmental stage.
  • the viable cells comprise cells which secrete a hormone which promotes growth in the domesticated animal.
  • hormones are well known to those of ordinary skill, including hormones such as growth hormone and insulin.
  • the viable cells secreting such a hormone are in one embodiment genetically engineered to secrete the hormone. That is they have been genetically engineered to contain the gene encoding the hormone and are capable of expressing the gene.
  • the viable cells in one embodiment comprise cells which secrete a biologically active substance.
  • biologically active substance as used herein means any substance which is capable of eliciting a physiological response in a subject.
  • the biologically active substance may illicit a response in the subject into which the cells producing it are transplanted.
  • Cells which secrete biologically active substances are well known in the art, and any such cells may be used in the subject invention.
  • the cells which secrete a biologically active substance are endocrine cells.
  • Endocrine cells are well known to those of ordinary skill in the art and include, but are not limited to, insulin-producing cells, hepatocytes, parathyroid cells, and pituitary cells.
  • the cells which secrete a biologically active substance are neuroectodermal cells.
  • Neuroectodermal cells are also well known in the art, and include, but are not limited to, adrenal cells and lymphocytes.
  • the cells are genetically engineered to secrete a biologically active substance.
  • the cells may be genetically engineered to secrete a biologically active substance useful for treating the subject into which they are transplanted.
  • the subject method provides a novel, useful, and advantageous drug delivery system for treatment of subjects afflicted with conditions including, but not limited to, cancer and HIV infection.
  • the transplanted viable cells may, for example, be genetically engineered to secrete Interleukin-2, a cytokine, or a lymphokine.
  • the transplanted viable cells may, for example, be genetically engineered to secrete a substance which stimulates lymphocyte production in the subject, such as a T cell growth factor or the HIV T cell receptor.
  • the permeability of the semipermeable membrane of the device is determined based on factors well known in the art, for example, the size of the cells or tissue being contained, the size of any substances needed to permeate the membrane in order to sustain the cells or tissue, and the size of any biologically active substances secreted by the cells which are desired to permeate from the device.
  • the semipermeable membrane is impermeable to lymphocytes.
  • the semipermeable membrane is impermeable to lymphocytes and immunoglobulins .
  • Using a semipermeable membrane which is impermeable to immunoglobulins and/or lymphocytes prevents contact between the immunoglobulins and/or lymphocytes of the subject and the contained viable cells, and thereby prevents destruction of the contained cells which would result from such contact .
  • any suitable method of treatment may be used in the subject invention to treat the subject with the substance which inhibits an immune-system costimulation event, and such methods are well-known in the art.
  • the substance may be administered by injection to the subject in the form of a pharmaceutically acceptable composition.
  • CTLA4Ig may be directly administered to the subject, or in another embodiment, cells genetically engineered to secrete CTLA4 , that is cells which have been genetically engineered to contain a gene encoding a molecule capable of binding to a B7 molecule and to express that molecule, may be transplanted into the subject.
  • treatment of the subject with the substance comprises transplanting into the subject cells genetically engineered to secrete the substance.
  • cells genetically engineered to secrete the substance are transplanted into the subject, such cells may themselves be contained within a device comprising a semipermeable membrane prior to transplantation.
  • the semipermeable membrane of the device containing the cells secreting the substance is impermeable to immunoglobulins and/or lymphocytes, thereby preventing destruction of these cells which would otherwise result from such contact .
  • treatment with the substance may occur before, after, of contemporaneously with transplantation of the viable cells or tissue.
  • treating the subject with the substance comprises containing cells genetically engineered to secrete the substance within the device containing the viable cells or tissue prior to transplantation.
  • treating the subject with the substance comprises genetically engineering the viable cells transplanted into the subject to secrete the substance prior to transplantation.
  • the amount of the substance effective to inhibit the subject's immune system from responding to said contained cells or tissue is determined by factors well-known to those of skill in the art, including, but not limited to, the amount of viable cells or tissue transplanted into the subject, and the size and weight of the subject.
  • Inhibiting the subject's immune system from responding to the contained viable cells or tissue by the method of the subject invention involves an inhibition of immunoglobulin production in the subject and an inhibition of macrophage activation in the subject.
  • Such immunoglobulins and activated macrophages would otherwise be capable of reacting with, and destroying, the contained viable cells or tissue.
  • This invention also provides a method of treating diabetes in a subject which comprises: a) containing viable insulin- producing cells, or tissue comprising viable insulin- producing cells, within a device comprising a semipermeable membrane so as to obtain contained viable insulin-producing cells; b) transplanting contained viable insulin-producing cells obtained in step (a) into the subject in an amount effective to treat diabetes in the subject; and c) treating the subject with a substance which inhibits an immune- system costimulation event in an amount effective to inhibit the subject's immune system from responding to an amount of contained viable insulin-producing cells according to step (b) .
  • Substances which inhibit an immune-system costimulation event are known in the art, and any such substance may be used in the method for treating diabetes described herein. Substances which inhibit an immune-system costimulation event which may be used in the subject method for treating diabetes are described above. In one embodiment, the substance is CTLA4.
  • the viable insulin-producing cells, or tissue comprising viable insulin-producing cells may be obtained from any known source for insulin-producing cells or tissue comprising insulin-producing cells.
  • viable insulin- producing cells are derived from pancreatic islet tissue.
  • the viable insulin-producing cells comprise cells which have been genetically engineered prior to transplantation to secrete insulin.
  • the viable cells or tissue may be derived from a xenogeneic donor, an allogeneic donor, or they may be derived from the subject prior to transplantation. If the cells are derived from the subject, in one embodiment, they are genetically engineered to produce insulin after they have been removed from the subject, prior to being transplanted back into the subject.
  • the viable insulin-producing cells or tissue comprising viable insulin-producing cells may be obtained from any donor.
  • the donor is a mammal.
  • a mammalian donor may, for example, be a calve, a pig, a rabbit, a rat, a mouse, or a human.
  • the viable insulin-producing cells or tissue comprising viable insulin-producing cells, such as pancreatic islet tissue may be obtained from a mammalian neonate, such as a neonatal pig.
  • the viable insulin-producing cells or tissue comprising viable insulin-producing cells used in the subject invention comprises neonatal porcine (pig) pancreatic cells.
  • the subject of the invented method described herein may be any subject into which transplantation of viable cells is desired.
  • the subject is a human. If the subject is a human, the viable cells, or tissue containing them, are in one embodiment derived from a mammal, for example a human.
  • Devices comprising a semipermeable membrane are well-known to those of ordinary skill as described above, and any such device may be used in the subject method of treating diabetes.
  • the device is a hollow fiber, a disk, and a sphere.
  • the device is a microcapsule as described above.
  • the method of treating diabetes described herein may be applied to any subject for whom diabetes treatment is desired.
  • the subject is afflicted with insulin-dependent diabetes mellitus (IDDM) .
  • IDDM insulin-dependent diabetes mellitus
  • the subject is a mammal, for example a human.
  • the amount of contained viable insulin-producing cells transplanted into the subject effective to treat diabetes in the subject depends on factors known to those of ordinary skill, including, but not limited to, factors such as the weight of the subject, and the severity of the diabetes.
  • the permeability of the semipermeable membrane of the device in the subject method of treating diabetes is determined by factors known to those of ordinary skill, including those factors for determining permeability described above.
  • the semipermeable membrane is impermeable to immunoglobulins and/or lymphocytes.
  • Treatment of the subject with the substance which inhibits an immune-system costimulation event in the subject method of treating diabetes includes those methods of treatment described above.
  • treatment may comprise administering CTLA4Ig to the subject, for example by injecting CTLA4Ig into the subject.
  • Treatment with the substance may, as described above, comprise transplanting into the subject cells genetically engineered to secrete the substance.
  • Such genetically engineered cells may themselves be contained within a device comprising a semipermeable membrane prior to transplantation.
  • treatment with the substance comprises transplanting into the subject cells genetically engineered to secrete the substance contained within a device comprising a semipermeable membrane, the device is in different embodiments impermeable to immunoglobulins and/or lymphocytes .
  • treatment may occur before, after, or contemporaneously with transplantation of the contained viable insulin-producing cells into the subject .
  • treating the subject with the substance capable of inhibiting an immune-system costimulation event comprises containing cells genetically engineered to secrete the substance within the device containing the viable insulin-producing cells or tissue prior to transplantation.
  • treating the subject with the substance comprises genetically engineering the viable insulin-producing cells to secrete the substance prior to transplantation.
  • Inhibiting the subject's immune system from responding to contained viable insulin-producing cells or tissue by the subject method of treating diabetes involves an inhibition of immunoglobulin production and of macrophage activation in the subject which would otherwise react with and lead to the destruction of the viable insulin-producing cells or tissue.
  • Double-wall microcapsule ( Figures 1 and 2) .
  • This double- wall microcapsule is more durable than conventional microcapsules, with fewer capsule wall defects, has a measured membrane permeability of approximately 100,000 Kd, and excludes IgG (unlike conventional design capsules, which allowed passage of IgG and 148,000 Kd fluoresceinated dextran) (9,19,20,118) .
  • IgG unlike conventional design capsules, which allowed passage of IgG and 148,000 Kd fluoresceinated dextran
  • PLL Poly-L-Lysine
  • microencapsulated islet xenograft survival would be influenced by microcapsule permeability.
  • microcapsule permeability may be altered by increasing or decreasing the concentration of PLL (poly-1- lysine) in the microcapsule formula.
  • Red blood cells were encapsulated in alginate via an air jet system and then incubated with various polyamino acids including PLL.
  • PLL concentration was the most critical factor in altering capsule diffusion. These observations are supported by the recent findings of other investigators (119) . There was a thirteen fold decrease in hemoglobin efflux occurring in capsules that had a fourfold increase in PLL (see Table 1) . In experiments, encapsulated rabbit islet survival in NODs is prolonged using microcapsules with permeability ⁇ 70,000 Kd vs. 100,000 Kd (see Figure 3) .
  • Microcapsules Prevent or Delay Host Sensitization
  • P. microencapsulated islet graft to peritoneal cavity
  • Splenic Nonencapsulated islets grafted beneath splenic capsule.
  • mRNA Cytokine Messenger RNA
  • mRNA was extracted from recipient NOD peritoneal cells and expression of mRNA for IL-2, IL-4, and IL-10 was studied by RT-PCR, as previously described (121) . Integrity of RNA samples was assessed by inspection of northern transfer and hybridization with the probe for the 3' untranslated region of beta actin (121) . IL-4 was detected in the majority of xenografts undergoing rejection. IL-10 expression was variable (Table 4) . IL-2 was detected during autoimmune destruction of NOD isografts, (and in one allograft) but only rarely in rejecting xenografts (Table 4) .
  • NOD-Scid Mice Accept Rat and Rabbit Islet Xenografts Long- Term
  • MLD-SZN diabetes (30mg/kg daily x5)
  • reversal of NOD-scid diabetes with xenografts of nonencapsulated and encapsulated rat and rabbit islets for greater than 50 days is documented (see Figures 10,11, and 12 and Table 2 . ) .
  • the NOD-scid mice will serve as a good recipient model for the transfer of antibodies and/or T cells for studies of the mechanisms by which encapsulated islets are rejected.
  • Rabbit islets were isolated by duct- injection, collagenase digestion. Rabbit islets (approx. 2000) were encapsulated in double-wall, poly-1-lysine- alginate microcapsules and xenografted intraperitoneally in NODs, as previously reported (7,20) . Controls received approximately 2000 unencapsulated rabbit islets xenografted beneath the splenic or renal capsule, as previously described (7,20) .
  • Murine CLTA4Ig provided by Bristol-Myers-Squibb, Seattle, WA, was administered at 200ug intraperitoneally (i.p.) , day-1 and then Q.O.D. for 14 or 92 days, or until graft rejection.
  • Controls included NODs receiving identically encapsulated rabbit islets (i.p) , and given no additional treatments, cyclosporine 30mg/kg s.c, day-1, and then daily, or monoclonal anti-CD8 antibody #53.6.7.7 (A.T.C.C) , lOO ⁇ g i.p. day-5, +2, and then weekly.
  • Biopsies of long-term functioning peritoneal microcapsules were done periodically, using metafane anesthesia and sterile technique. Removal of 100-200 microcapsules allowed histologic light microscopic studies without altering graft-related normogycemia.
  • splenectomy was performed on one long-term functioning, biopsy-proven, CTLA4Ig-treated NOD.
  • These splenocytes (10 7 ) were passively transferred, intraperitoneally, to two naive diabetic NODs, which subsequently received identically encapsulated fresh rabbit islets (donor-type New Zealand, not inbred) , intraperitoneally, on day 10-14 after splenocyte transfer.
  • Statistical difference between groups were assessed by use Student's "t"-tested and by ANOVA.
  • Biopsies of failed CTLA4Ig-treated, encapsulated rabbit islet xenografts showed primarily disrupted (broken) microcapsules, few viable islets, and minimal pericapsular cellular reaction.
  • Biopsies of intrasplenic rabbit islets at rejection showed nuclear and cytoplasmic damage and nonviable islets.
  • CTLA4Ig 200 ⁇ g day -1, then Q.O.D., i.p.
  • biopsies of these "islets” 100 days following xenotransplantation reveal increased numbers of intensely insulin-positive islet cells (Figure 17) .
  • These neonatal pig islets have an added advantage over adult islets, in that they appear to differentiate and proliferate within microcapsules after transplantation.
  • NOD-Scid mice do not develop diabetes spontaneously; but they may be rendered diabetic with multiple low-dose streptozotocin (MLD-SZN) , (67,68,69) NOD-Scids express NOD MHC genes and other genes that are required for development of diabetes, upon transfer of lymphocytes from diabetic NODs.
  • MLD-SZN multiple low-dose streptozotocin
  • CTLA4Ig significantly prolonged survival of encapsulated rabbit and porcine islets in NOD recipients, whereas CTLA4Ig alone did not protect non- encapsulated islet xenografts in NOD mice (Table 6 and Figure 21) .
  • Biopsies of long-term functioning encapsulated neonatal porcine islet xenografts showed viable porcine islets within intact microcapsules and absence of host NOD pericapsular reactivity was observed in biopsies of long- term normoglycemic NODs ( Figure 22 and 23) .
  • CTLA4Ig which does not fix complement (CTLA4Ig * ) (145) .
  • CTLA4Ig * CTLA4Ig *
  • CTLA4Ig * does not prolong graft survival above that of capsules alone.
  • the data are distinct from findings with murine allografts, which are prolonged significantly by either conventional CTLA4Ig or mutant CTLA4IgP.
  • These results suggest that mechanisms of prolongation of graft survival by CTLA4Ig * may be different for allogeneic and xenogeneic islet grafts.
  • the results suggest that the cytokine profile in a subject can be altered in favor of graft protection.
  • conventional CTLA4Ig altered the cytokine production so as to protect the graft by increasing gamma-interferon production in the host.
  • an increase in IL-10 production induced by CTLA4Ig * treatment favored graft rejection.
  • CD4 * T cells are activated and develop into Th2 cells that express CD40L.
  • Th2 specific peptide X complexed with class II binds B cells and the interaction of CD40 with CD40L causes the activation of B cells.
  • Activated B cells mature into plasma cells under the direction of Th2 lymphokines. Plasma cells secrete specific antibody that forms complexes with AgX.
  • Antibodies are not able to directly damage the encapsulated islets because they are too large to enter the capsules. However, antibodies could be involved in the recruitment and activation of macrophages which are the predominant population in the peritoneal cavity of NODs rejecting encapsulated islet xenografts. Specific antibodies in the peritoneal cavity could form complexes with antigens shed or secreted from the capsules. Such antigen-antibody complexes efficiently bind to FcR expressed on the surface of peritoneal macrophages.
  • Binding of complexes to FcR activates macrophages to secrete a variety of mediators including IL-1, TNF ⁇ and nitric oxide (NO) (122,123) , all of which have toxic effects on islets and all of which are small enough to cross a double walled microcapsule.
  • the effector arm could be further augmented by the activation of complement (c) by antigen complexes.
  • C3b bound to the complexes enhances the activation of macrophages by increasing the binding of the complexes via the C3b receptor (124) and small peptides such as C3b released during complement activation induce local inflammatory responses thereby attracting more macrophages into the peritoneal cavity (125) .
  • Vanenbossche G. Van Oostveldt P., Demeester J. , Remon J.
  • the molecular weight cut-off of microcapsules is determined by the reaction between alginate and polylysine. Biotechnology and Bioengineering 1993 ;42 : 381-386.
  • nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: Increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered N 9 -monomethyl-1-arginine. J. Exp Med 1994;179: 651-660.
  • Xenogeneic proliferation and lymphokine production are dependent on CD4+ helper T cells and self antigen- presenting cells in the mouse. J Exp Med 1990;172: 567 - 575 .
  • Lacy P. Lacy E., Finke E., Yasunami Y. Diabetes 1982;31:109-111.
  • CTLA-4 is a second receptor for the B cell activation antigen B7. J Exp Med 1991; 74: 561-569.

Abstract

L'invention concerne un procédé permettant d'inhiber la destruction de cellules vivantes greffées chez un sujet par le système immunitaire dudit sujet. Ce procédé consiste à a) enfermer, avant la greffe, les cellules vivantes, ou du tissu contenant les cellules vivantes, dans un dispositif comportant une membrane semi-perméable, et b) à traiter le sujet avec une substance qui inhibe un phénomène de costimulation du système immunitaire, administrée en quantité utile pour empêcher le système immunitaire du sujet de répondre auxdites cellules ou audit tissu ainsi enfermés. Selon un mode de réalisation, la substance inhibant un phénomène de costimulation du système immunitaire est CTLA4. Cette invention concerne également un mode de traitement du diabète chez un sujet consistant à a) enfermer des cellules vivantes productrices d'insuline, ou du tissu contenant de telles cellules, dans un dispositif comportant une membrane semi-perméable; b) à greffer chez le sujet une quantité efficace de cellules vivantes productrices d'insuline ainsi enfermées; et c) à traiter le sujet avec une quantité utile d'une substance qui inhibe un phénomène de costimulation du système immunitaire.
PCT/US1996/015577 1995-09-27 1996-09-27 Methode d'inhibition de la destruction par le systeme immunitaire de cellules vivantes greffees WO1997011607A1 (fr)

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EP96936037A EP0877555B1 (fr) 1995-09-27 1996-09-27 Utilisation d'un inhibiteur d'un processus de costimulation du système immunitaire pour la préparation d'un médicament pour la prévention de la destruction par le systeme immunitaire de cellules vivantes non embryonnaires greffées
JP9513711A JP2000500121A (ja) 1995-09-27 1996-09-27 免疫系による生きた被移植細胞の破壊を阻止する方法
AU73782/96A AU721737B2 (en) 1995-09-27 1996-09-27 Method of inhibiting immune system destruction of transplanted viable cells
DE69637216T DE69637216D1 (de) 1995-09-27 1996-09-27 Verwendung eines inhibitors eines kostimulatorischen vorgangs des immunsystems zur herstellung eines medikaments zum schutz transplantierter, nichtembryonaler lebender zellen vor zerstörung durch das immunsystem
US09/049,865 US7041634B2 (en) 1995-09-27 1998-03-27 Method of inhibiting immune system destruction of transplanted viable cells

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US7041634B2 (en) * 1995-09-27 2006-05-09 Emory University Method of inhibiting immune system destruction of transplanted viable cells
US6197294B1 (en) 1998-10-26 2001-03-06 Neurotech S.A. Cell surface molecule-induced macrophage activation
SE523817C2 (sv) 1999-02-05 2004-05-18 Corline Systems Ab Användning av ett koagulationsförebyggande ämne i samband med transplantation av insulinproducerande celler
JP5298134B2 (ja) 2007-11-01 2013-09-25 パーシード セラピューティクス リミテッド ライアビリティ カンパニー 免疫抑制ポリペプチドおよび核酸
US20140112958A1 (en) 2012-10-24 2014-04-24 Mwm Biomodels Gmbh Pancreatic islets of transgenic LEA29Y animals for treating diabetes

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