WO2000051630A2 - Procede d'amelioration d'acceptation de greffe chez un receveur par l'administration d'un agent modificateur du profile de la cytokine - Google Patents

Procede d'amelioration d'acceptation de greffe chez un receveur par l'administration d'un agent modificateur du profile de la cytokine Download PDF

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WO2000051630A2
WO2000051630A2 PCT/US2000/005647 US0005647W WO0051630A2 WO 2000051630 A2 WO2000051630 A2 WO 2000051630A2 US 0005647 W US0005647 W US 0005647W WO 0051630 A2 WO0051630 A2 WO 0051630A2
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cell
cells
subject
xenogeneic
cytokine
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WO2000051630A3 (fr
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Albert Edge
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Diacrin, Inc.
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Priority to CA002364279A priority Critical patent/CA2364279A1/fr
Priority to EP00921362A priority patent/EP1158999A2/fr
Priority to AU41698/00A priority patent/AU4169800A/en
Publication of WO2000051630A2 publication Critical patent/WO2000051630A2/fr
Publication of WO2000051630A3 publication Critical patent/WO2000051630A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2026—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/246—IL-2
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • a CD4 + or CD8 + T cell Once a CD4 + or CD8 + T cell has reached the periphery, it can be inactivated by several mechanisms including deletion by apoptosis and inactivation by anergy.
  • the ability of the immune system to develop tolerance to self peripheral antigens provides a possible mechanism by which to promote acceptance of transplanted tissues (Dallman, et al. 1993. Immunol Rev. 133:18). For instance, cells that express Fas ligand have been found to be protected against activated T cells by inducing apoptosis and cell death (Griffith, et al, 1995. Science. 270:1189; Griffith, et al. , Immunity, 1996. 5:7; French, et al., 1996. J Cell Biol.
  • Fas ligand-expressing cells have been demonstrated to have increased survival upon transplantation. (Fandrich et al. 1998. Transplant. Proc. 30:2360) Another way peripheral tolerance has been induced has been by blocking either of the two crucial interactions between a T cell and an antigen presenting cell. Two types of interactions between a T cell and APC are required for a naive T cell to become activated and develop effector function.
  • the T cell receptor must first recognize and bind (signal one) to the antigen peptide-MHC molecule complex on the APC (Schwartz,. 1992. Cell, 71 :1065; Lenschow, D.j. 1993. Curr. Opin. Immunology, 5:747).
  • the second interaction (signal two) is required between the CD28 molecule on T cells and the newly expressed costimulatory B7.1 or B7.2 molecules on the APC (Schwartz, R.H.
  • T cell receptor signaling e.g., using anti-CD3 antibody
  • costimulatory signals e.g., using a soluble form of a costimulatory molecule such as CTL A4Ig
  • Xenograft rejection is thought to involve primarily humoral immunity.
  • the humoral immune response to a xenograft can develop in several ways, both of which ultimately lead to rejection of the transplant.
  • the recipient may have preexisting anti-donor antibodies that fix complement and lead to hyperacute rejection.
  • the xenograft recipient generates anti-graft antibodies over several days post transplantation that lead to rejection. (Bach. 1997.
  • the cellular response to xenografts can develop in several ways. Recognition of an antigen from a foreign species can involve direct recognition by the T cell of the foreign MHC-peptide complexes on the graft or indirect recognition of the foreign antigens as processed peptides in association with recipient antigen presenting cells (APC) (Auchincloss, H., Transplant. Reviews, 1990. 4:1 ; Auchincloss, H., et al, Proc. Natl. Acad. Sci., 1993. 90: p. 3373).
  • APC recipient antigen presenting cells
  • cytokines are not produced constitutively by T cells, but are induced following receptor-mediated T cell activation.
  • methods for reducing the immune response of a transplanted subject which leads to rejection of donor tissue would be of great benefit. Promoting acceptance of an allograft or a xenograft without affecting immune responses to other, unrelated antigens would be highly desirable and would represent a major advance over the use of general immunosuppressants.
  • the instant invention is based, at least in part, on the discovery that biasing the cytokine response of a graft recipient to one dominated by Th2 cytokines promotes graft acceptance.
  • Applicants demonstrate that biasing the cytokine secretion profile of human responder T cells towards a Th2 phenotype leads to an inhibition of the human anti- porcine immune response, thus providing a method for achieving acceptance of xenografts.
  • This enhanced Th2 response leads to long-term graft acceptance even though previous work has demonstrated that the presence of Th2 cytokines correlates with rejection of xenografts (Morris et al. 1995. J Immunol. 154:2470; Wren et al. 1993. Transplantation. 56:905; and Medbury. 1997. Transplantation. 64:1307).
  • This discovery provides methods for promoting the acceptance of grafts.
  • the invention provides methods for promoting xenograft acceptance in a subject by administering an isolated xenogeneic cell and a cytokine profile altering agent to the subject in order to promote acceptance of the xenogeneic cell in the subject.
  • the cytokine profile altering agent is a cytokine selected from the group consisting of IL-4 and IL-10.
  • the cytokine profile altering agent is a cytokine fusion protein selected from the group consisting of IL-4Ig and IL-lOIg.
  • the cytokine profile altering agent is an antibody.
  • the antibody is selected from the group consisting of: an antibody which binds to IL-2, IFN-gamma, and IL-12.
  • the cell is a xenogeneic cell, e.g., a porcine cell.
  • the cell is selected from the group consisting of: a fetal cell, a stem cell, and a progenitor cell.
  • the xenogeneic cell is obtained from a pig which is predetermined to be free from at least one organism selected from the group consisting of: zoonotic, cross-placental and organotropic organisms.
  • the cell is selected from the group consisting of a pancreatic islet cell, a kidney cell, a cardiac cell, a muscle cell, a liver cell, a lung cell, an endothelial cell, a central nervous system cell, a peripheral nervous system cell, an epithelial cell, an eye cell, a skin cell, an ear cell, and a hair follicle cell.
  • the invention pertains to a method for promoting xenograft acceptance by i) modifying a xenogeneic cell to express an exogenous polypeptide comprising a cytokine profile altering agent (e.g., by introducing a heterologous gene encoding a cytokine such as 11-4 or IL-10) to produce a modified xenogeneic cell; ii) administering the modified xenogeneic cell to a subject in order to improve xenograft acceptance in the subject.
  • a cytokine profile altering agent e.g., by introducing a heterologous gene encoding a cytokine such as 11-4 or IL-10
  • the method further includes administering xenogeneic lymphoid cells to the subject.
  • the xenogeneic lymphoid cells are administered intravenously.
  • a lymphoid cell is administered into the portal vasculature of the subject.
  • a xenogeneic cell is administered intravenously.
  • the method further comprises administering an immunosuppressive agent to the subject.
  • an immunosuppressive agent is selected from the group consisting of methylprednisolone, cyclosporin A, and FK506.
  • the method further comprises introducing a soluble form of a costimulatory molecule to the subject.
  • the invention in another aspect, pertains to a method for promoting acceptance of a xenogeneic cell or tissue in a human subject comprising: i) administering an isolated xenogeneic cell to the subject wherein the cell or tissue bears a surface antigen capable of causing an immune response against the cell or tissue in the subject, said antigen being modified, masked, or eliminated to decrease the immune response; and ii) administering a cytokine profile altering agent to the subject such that acceptance of the xenogeneic cell is promoted in the subject.
  • a surface antigen on the transplanted cell is an MHC class I antigen or an MHC class II antigen.
  • the masking agent is a non- lytic anti-MHC class I antibody fragment or an anti-MHC class II antibody or fragment thereof.
  • the anti-MHC class I antibody fragment is an anti-MHC class I F(ab')2 fragment.
  • the invention pertains to a method for promoting Th2 cytokine production in a subject comprising: i) administering a xenogeneic cell or tissue to the subject wherein the cell or tissue bears a surface antigen capable of causing an immune response against the cell or tissue in the subject, said antigen being modified, masked, or eliminated to decrease the immune response, such that upon introduction of the composition into the subject lysis of the cell or tissue is prevented; and ii) determining that Th2 cytokine production is promoted in the subject.
  • Figure 1 A shows that PT85 inhibits primary human anti-porcine responses.
  • Human peripheral blood mononuclear cells PBMC
  • MHC-class I + /II porcine smooth muscle cells
  • EBC embryonic brain cells
  • the anti-human MHC class I reactive W6/32 mAb is not reactive with pig MHC class I.
  • F(ab')2 fragment of normal mouse serum (mlg) was used as control.
  • the graph represents the mean and standard deviation (SD) of triplicate wells.
  • Figure IB shows the inhibition of secondary human anti-porcine response. Cells from day 7 primary stimulation were re-stimulated with porcine SMC or EBC for 3 days, and cells were pulsed with ⁇ H-thymidine for the last 20 hours of incubation. No antibody was present in the secondary stimulation.
  • FIG. 2A shows that PT-85 inhibits primary human anti-porcine responses.
  • Tissue cultures were setup as described in Figure legend 1 A. Thymidine incorporation was converted to percentage of the no antibody control values. Responder cells alone gave less than 10% of the full response. Stimulatory cells alone was less than 1% of the full response.
  • the graph represents a summary of 12 experiments with PBMC from 8 individuals.
  • Figure 2B shows inhibition of secondary human anti-porcine responses.
  • Tissue cultures were setup as described in figure legend IB.
  • Thymidine incorporation was converted to percentage of a control (the no Ab group for SMC or mlgG group for EBC).
  • Data represent a summary of 8 experiments with PBMC from 6 individuals for SMC as stimulators, and 2 experiments with PBMC from 2 individuals for EBC as stimulators.
  • Figure 3 shows inhibition of CD8+ T cells in primary human anti-porcine response.
  • Human PBMC, CD8+ T cells or CD4+ T cells were incubated with porcine SMC for 7 days in the presence of absence of antibody (Ab), and cells were pulsed with ⁇ H-thymidine for the last 8 hours of incubation.
  • 9-3 and 74-1 1-10 Ab are reactive with porcine MHC class I (see Table 2).
  • 10.14 is reactive with porcine CD44.
  • Figure 4 shows that PT-85 inhibits IL-2 in the primary PBMC response against porcine SMC.
  • Tissue cultures were set up as Figure 1 A. Supernatants were harvested on the days indicated and used for ELISA. The results show IL-2 levels in the culture supernatants using PBMC from 4 individuals.
  • Figure 5 shows that PT-85 inhibits IFN-gamma in primary PBMC responses against porcine SMC. Tissue cultures were set up as described for Figure 1 A. Supernatants were harvested on days as indicted and used for ELISA. The results show IFN-gamma levels in the culture supernatants using PBMC from 4 individuals.
  • Figure 6 shows sustained IL-10 production in primary PBMC responses against porcine SMC.
  • Tissue cultures were set up as Figure 1A.
  • Supernatants were harvested on days as indicted and used for ELISA.
  • the results show IL-10 levels in the culture supernatants using PBMC from 4 individuals.
  • Figure 7 shows inhibition of IFN-gamma during secondary response against porcine SMC.
  • Tissue cultures were set up as Figure IB.
  • Supernatants were harvested at 48 hours and used for ELISA.
  • the results show IFN-gamma levels in the culture supernatants using PBMC from 4 individuals.
  • Figure 8 shows enhanced IL-10 production during secondary anti -porcine SMC response.
  • Tissue cultures were set up as described in Figure IB.
  • Supernatants were harvested at 48 hours and used for ELISA.
  • the results show IFN-gamma levels in the culture supernatants using PBMC from 4 individuals.
  • Figure 9 shows enhanced IL-10 production during secondary stimulation with anti-CD3 or anti-CD3/CD28 mABs.
  • Cells from day 7 primary stimulation were re- stimulated with immobilized anti-CD3 in the presence or absence of anti-CD28.
  • Culture supernatants were harvested at 48 hours and used for ELISA. The results show IL-10 levels in culture supernatants using PBMC from 4 individuals.
  • Figure 10 shows enhanced IL-4 production during secondary stimulation with anti-CD28 mABs.
  • Cells from day 7 primary stimulation were re-stimulated with immobilized anti-CD3 in the presence or absence of anti-CD28.
  • Culture supernatants were harvested at 48 hours and used for ELISA. The results show IL-4 levels in culture supernatants using PBMC from 4 individuals.
  • the instant invention is based, at least in part, on the discovery that biasing the cytokine response of a subject to a transplanted cell to one dominated by Th2 cytokines inhibits the immune response to the cell and improves graft acceptance.
  • the following terms and phrases shall be defined as follows:
  • cell includes an allogeneic cell or a xenogeneic cell.
  • xenogeneic cell includes any cell which is derived from a different species from the recipient of the cell.
  • isolated refers to a cell which has been separated from its natural environment, e.g., which has been removed from the donor and which is no longer part of an intact organ.
  • Isolated cells of the invention are cells which have been separated from the vascular tissue of the organ from which they originated.
  • the isolated cell can be in the form of a piece of tissue, e.g., an intact sheet of cells, e.g., a monolayer of cells.
  • intact sheet refers to a layer of cells which remain adherent to one another after the cells are harvested.
  • the cell is dissociated from the neighboring cells with which it was in contact in the donor.
  • the isolated cells of the invention can be administered in the form of a cell suspension.
  • More than one of the isolated cells of the invention constitute a population of cells.
  • the term population also includes cells which result from the proliferation of the isolated cells of the invention.
  • a population of cells can be obtained from the same or different source(s), e.g., can originate from different types of donor tissue and can, therefore, comprise different cell types.
  • the isolated cells can also be derived from different donor animals, e.g., the isolated cells can represent a pooled population of cells derived from a number of different animals.
  • T helper cells (Th cells) as used herein includes the meaning that this term is ordinarily given in the art.
  • Prototypical Th cells bear the CD4 cell surface marker and recognize antigen in the context of MHC class II molecules.
  • cytotoxic T cells (TC cells) as used herein includes the meaning that this term is ordinarily given in the art.
  • Prototypical TC cells bear the CD8 cell surface marker and recognize antigen in the context of MHC class I molecules.
  • Thl refers to T helper cells of the type 1 phenotype which primarily secrete Thl cytokines.
  • Thl cytokine refers to a cytokine preferentially produced by a prototypical T helper 1 type cell.
  • Thl cytokines are cytokines which primarily promote cellular immune responses, e.g., delayed type hypersensitivity and cellular cytotoxicity. Thl cytokines include, for example IL-2, IFN-gamma and/or lymphotoxin.
  • Thl cytokines as used herein includes those cytokines which may not be made by Thl cells, but which enhance the development of Thl type cells and, thus, amplify Thl cytokine secretion.
  • Thl -type cytokines need not be exclusively produced by Thl cells.
  • IL-12 is made primarily by monocytes and macrophages and has been found to drive T cell responses to the Thl phenotype (e.g., U.S. patent 5,853,697).
  • Th2 refers to T helper cells of the type 2 phenotype which primarily secrete Th2 cytokines.
  • Th2 cytokine refers to a cytokine which is preferentially produced by a prototypical T helper 2 type cell. Th2-type cytokines may not be exclusively produced by Th2 cells.
  • Th2 cytokines are cytokines which primarily promote B cell differentiation and/or activation and humoral immune responses. Th2 cytokines include, for example IL-4, IL-5, IL-6, IL-10, and/or IL-13.
  • Thl and Th2-type cytokines may be produced by cell types other than T helper cells, e.g., by monocytes, macrophages, nonB-nonT cells, cytotoxic T cells or by natural killer cells.
  • Tc cells have been shown to be divisible into two groups based on their cytokine production just as Th cells are.
  • the Thl/Th2 dichotomy may not be absolute, e.g., some T cells may produce both Thl and Th2 cytokines.
  • cytokine profile altering agent includes both Th2 cytokine promoting agents and Thl cytokine inhibitory agents.
  • a cytokine profile altering agent biases T cell cytokine production to a Th2 phenotype.
  • a "Th2 phenotype” can be demonstrated by augmenting levels of Th2 cytokines in the presence of the agent when compared with levels produced in the absence of the agent.
  • a Th2 phenotype can also be demonstrated by the production of qualitatively more types of Th2 cytokines (e.g., IL-4, IL-5, IL-10, or IL-13), i.e., a greater number of Th2 cytokines being produced in the presence of the agent than are produced in the absence of the agent.
  • Cytokine profile altering agent includes agents that lead to the production of decreased levels of Thl cytokines and/or which lead to the production of fewer different Thl cytokines.
  • a cytokine profile altering agent promotes Th2 cytokine production.
  • a cytokine profile altering agent inhibits Thl cytokine production.
  • a cytokine profile altering agent both promotes Th2 cytokine production and inhibits Thl cytokine production.
  • a cytokine profile altering agent promotes an increase in the ratio of Th2/Thl cytokines.
  • the term "fetal" includes cells which are derived from a donor during the fetal development of the donor.
  • fetal cells can be terminally differentiated or can be capable of differentiation into more than one cell type, e.g., can be undifferentiated "precursor cells".
  • stem cell includes an undifferentiated cell which is capable of proliferation and results in additional stem cells having the ability to differentiate into progenitor cells under appropriate conditions.
  • progenitor cell refers to undifferentiated cells derived from stem cells and which, under appropriate conditions, differentiate into terminally differentiated cells.
  • precursor cell also includes totipotent cells (e.g., cells form early stage embryos which are unrestricted in their developmental capabilities). Such precursor cells can be used as sources of the cells, i.e., the cells for use in the invention can be derived from such precursor cells, e.g., in vitro or in vivo.
  • heterologous nucleic acid molecule includes a nucleic acid molecule, preferably DNA, that does not occur naturally as part of the genome in which it is present or which is found in a location or locations in the genome that differs from that in which it occurs in nature, thus, heterologous DNA is not naturally occurring in that position or is not endogenous to the cell into which it is introduced, but ratherhas been obtained from another cell.
  • DNA encodes proteins that are not normally produced by the cell in which it is expressed.
  • Heterologous DNA can be from the same species or, from a different species. In particularly preferred embodiments, it is mammalian, e.g., human.
  • Heterologous DNA may also be referred to as foreign DNA. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is expressed is herein encompassed by the term heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes proteins which provide a benefit to the recipient, e.g., which the recipient is incapable of synthesizing.
  • heterologous protein refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid.
  • soluble form of a costimulatory molecule includes forms of costimulatory molecules which are not expressed on the surface of a cell and which inhibit a costimulatory signal in a T cell.
  • costimulatory molecule collectively refers to costimulatory molecules on APCs (e.g., the B7 molecules B7-1 and B7-2) and their cognate Iigands on T cells (e.g., CD28 and CTLA4).
  • the cell for transplantation is an isolated cell.
  • the cells are xenogeneic cells.
  • Xenogeneic cells for use in the instant methods can be obtained from any donor which is of a different species than the recipient subject.
  • the recipient subject is a human subject and the donor cells are porcine cells.
  • a xenogeneic cell is obtained from a species to which the recipient subject does not have preexisting, natural antibodies. In such a situation, the species combination is said to be concordant.
  • a xenogeneic cell is obtained from a species to which the recipient subject has preexisting, natural antibodies. In such a situation, the species combination is said to be discordant.
  • a xenogeneic cell of the invention is capable of direct presentation of antigen to the T cells of a recipient subject.
  • the xenogeneic cell has class I MHC molecules which are similar enough to those of the recipient that the recipient T cells can directly recognize the MHC antigen on the surface of the xenogeneic cell.
  • a xenogeneic cell of the invention has MHC class I molecules which are different from the class I molecules of the recipient such that recipient T cells cannot directly recognize antigen on the surface of the xenogeneic cell.
  • the recipient T cells recognize xenoantigen indirectly after processing and presentation by recipient antigen presenting cells.
  • a xenogeneic cell of the invention has costimulatory molecules that are not recognized by the costimulatory molecule counter receptors (e.g., CD28 and/or CTLA4) on recipient T cells.
  • costimulatory molecule counter receptors e.g., CD28 and/or CTLA4
  • the combination of donor cell and recipient subject is one in which antigen presentation is primarily direct, but in which indirect presentation may also occur.
  • graft acceptance is improved in a setting where the host response to the graft in the absence of the cytokine profile altering agent is predominantly a CD8+ cellular immune response.
  • graft acceptance is improved in a setting where the host response to the graft in the absence of the cytokine profile altering agent is predominantly a CD4+ cellular immune response.
  • graft acceptance is improved in a setting where the host response to the graft in the absence of the cytokine profile altering agent is predominantly a humoral cellular immune response.
  • the cells for use in the instant methods can be obtained from the donor organism at any point in the donor's life e.g., the cell can be a fetal cell, a juvenile cell, or an adult cell.
  • the cells of the invention can also be obtained at any stage of differentiation, for example, the cells can be stem cells or progenitor cells or can be differentiated.
  • Undifferentiated cells of the invention can either be transplanted as undifferentiated cells and be allowed to differentiate in vivo or can be induced to differentiate in vitro prior to transplantation using methods that are known in the art to cause differentiation into the cell type of interest.
  • the cells described herein can also be grown as a cell culture prior to transplantation, i.e., as a population of cells which grow in vitro, in a medium suitable to support the growth and/or differentiation of the cells.
  • Media which can be used to support the growth of specific cell types is known in the art as are appropriate differentiation factors and/or conditions.
  • Cells can also be derived from any part of a donor, e.g., the cells can be derived from blood or bone marrow, or can be isolated from discrete differentiated organs.
  • a cell is selected from the group consisting of: a pancreatic islet cell, a kidney cell, a cardiac cell, a muscle cell, a liver cell, a lung cell, an endothelial cell, a central nervous system cell, a peripheral nervous system cell, an epithelial cell, an eye cell, a skin cell, an ear cell, and a hair follicle cell.
  • a cell for use in the claimed methods is a brain cell.
  • a population of cells for transplantation is substantially free of endothelial cells, e.g., is separated from any vascular tissue that was associated with the organ from which the cells were derived in the host.
  • the cells of the invention are transplanted as isolated cells, not as whole organs.
  • Cells can be separated from other cells and tissues which surrounds them using a variety of methods which are known in the art. For example, cells can be subjected to mild protease treatment, gentle trituration, or more vigorous mechanical means of separation.
  • the isolated cells of the invention can be transplanted as a population of cells.
  • populations of cells for administration comprise substantially pure populations of cells.
  • populations comprise cells of one type.
  • Cells for transplantation can be purified using methods known in the art, e.g., using antibodies (e.g., by panning or by cell sorting) or by limiting dilution cloning.
  • a population of cells is at least about 80% pure.
  • a population of cells is at least about 85% pure.
  • a population of cells is at least about 90% pure. More preferably a population of cells is at least about 90% to 95% or about 98% to 99% pure.
  • the purity of a population of cells can be determined by methods known in the art. For example, markers which specifically detect a particular cell type, can be used to detect the presence of the cell type and the percentage of the population of cells which is comprised of that cell type e.g., by staining for that marker.
  • xenogeneic cells are obtained from donor animals which are inbred to minimize genetic variability.
  • donor animals which are inbred to minimize genetic variability.
  • inbred miniature swine are preferred donors for cells.
  • the cells of the invention are cells determined to be free from at least one organism which originates in the donor from which the cells are obtained, which is capable of infecting the donor cells, and which transmits infection or disease to a recipient subject.
  • the cell is determined to be free from at least two organisms.
  • Xenogeneic cells with these characteristics can be obtained by screening the donor to determine if it is essentially free from organisms or substances which are capable of transmitting infection or disease to a xenogeneic recipient, e.g., a human recipient, of the cells prior to isolating the cells.
  • the cells are porcine cells which are obtained from a swine which predetermined to be essentially free from pathogens which detrimentally affect humans.
  • the pathogens from which the swine are free can include, but are not limited to, one or more of pathogens from the following categories of pathogens: zoonotic, cross-placental, and organotropic organisms.
  • zoonotic refers to organisms which can be transferred from pigs to man under natural conditions
  • cross-placental refers to organisms capable of crossing the placenta from mother to fetus
  • organotropic refers to organisms which selectively infect cells of a particular organ.
  • the organism can be a parasite, bacterium, mycoplasma, and/or virus.
  • donor animals e.g., swine
  • donor animals can be free from parasites such as zoonotic parasites (e.g., toxoplasma), cross-placental parasites (e.g., eperythozoon suis or toxoplasma), neurotropic parasites (e.g., toxoplasma), ocular-infecting parasites, and/or mycoplasma, such as M. hypopneumonia.
  • zoonotic parasites e.g., toxoplasma
  • cross-placental parasites e.g., eperythozoon suis or toxoplasma
  • neurotropic parasites e.g., toxoplasma
  • mycoplasma such as M. hypopneumonia.
  • the swine can be free from bacteria such as zoonotic bacteria (e.g., brucella, listeria, mycobacterium TB, leptospirillum), cross- placental bacteria (e.g., brucella, listeria, leptospirillum), neurotropic bacteria (e.g., listeria), and/or ocular-infecting bacteria.
  • bacteria such as zoonotic bacteria (e.g., brucella, listeria, mycobacterium TB, leptospirillum), cross- placental bacteria (e.g., brucella, listeria, leptospirillum), neurotropic bacteria (e.g., listeria), and/or ocular-infecting bacteria.
  • bacteria from which the swine can be free include brucella, clostridium, hemophilus suis, listeria, mycobacterium TB, leptospirillum, salmonella and hemophilus suis.
  • the swine can be free from viruses such as zoonotic viruses, viruses that can cross the placenta in pregnant sows, neurotropic viruses, and ocular-infecting viruses.
  • Zoonotic viruses include, for example, a virus in the rabies virus group, a herpes-like virus which causes pseudorabies, encephalomyocarditis virus, swine influenza Type A, transmissible gastroenteritus virus, parainfluenza virus 3 and vesicular stomatitis virus.
  • Cross- placental viruses include, for example, viruses that cause porcine respiratory reproductive syndrome, a virus in the rabies virus group, a herpes-like virus which causes pseudorabies, parvo virus, a virus that causes swine vesicular disease, teschen (porcine polio virus), hemmaglutinating encephalomyocarditis, cytomegalovirus, suipoxvirus, and swine influenza type A.
  • Organotropic viruses include, e.g., neurotropic viruses, such as viruses in the rabies virus group, a herpes-like virus which causes pseudorabies, parvovirus, encephalomyocarditis virus, a virus which causes swine vesicular disease, porcine poliovirus (teschen), a virus which causes hemmaglutinating encephalomyocarditis, adenovirus, parainfluenza 3 virus.
  • neurotropic viruses such as viruses in the rabies virus group, a herpes-like virus which causes pseudorabies, parvovirus, encephalomyocarditis virus, a virus which causes swine vesicular disease, porcine poliovirus (teschen), a virus which causes hemmaglutinating encephalomyocarditis, adenovirus, parainfluenza 3 virus.
  • viruses from which the swine are free include: a virus which causes (or results in) porcine respiratory reproductive syndrome, a virus in the rabies virus group, a herpes-like virus which causes pseudorabies, parvovirus, encephalomyocarditis virus, a virus which causes swine vesicular disease, porcine poliovirus (teschen), a virus which causes hemmaglutinating encephalomyocarditis, cytomegalovirus, suipoxvirus, swine influenza type A, adenovirus, transmissible gastroenteritus virus, a virus which causes bovine viral diarrhea, parainfluenza virus 3, and vesicular stomatitis virus.
  • the pigs from which the xenogeneic cells are isolated are 5 essentially free from at least one of the organisms selected from the group consisting of: Toxoplasma, eperythrozoon, brucella, listeria, mycobacterium TB, leptospirillum, hemophilus suis, M.
  • hypopneumonia a virus which causes porcine respiratory reproductive syndrome, a virus which causes rabies, a virus which causes pseudorabies, parvovirus, encephalomyocarditis virus, a virus which causes swine vesicular disease, o porcine polio virus (teschen), a virus which causes hemagglutinating encephalomyocarditis, suipoxvirus, swine influenza type A, adenovirus, transmissible gastroenteritis virus, a virus which causes bovine viral diarrhea, and vesicular stomatitis virus.
  • essentially free or free from organisms or substances which are capable of transmitting infection or disease to a xenogeneic recipient when referring to a donor from which cells are isolated or to the cells themselves means that donor does not contain organisms or substances in an amount which transmits infection or disease to a xenogeneic recipient, e.g. a human.
  • a donor animal e.g., a pig harbors infectious agents which are capable of infecting and transmitting disease to a recipient
  • any art recognized method of detecting infectious agents can be used. (See e.g., Fishman. Xenotransplantation. 1 :47. 1994 for exemplary screening methods and methods of maintaining donor animals free of infectious agents).
  • nucleic acid molecules can be extracted from donor tissue using standard procedures.
  • oligonucleotide primers capable of specifically amplifying nucleic acid molecules of an infectious agent can be used to amplify pathogen nucleic acid molecules, e.g., by polymerase chain reaction.
  • the resulting samples can be screened to determine whether or not such sequences were present and amplified in the sample.
  • oligonucleitides capable of specifically hybridizing to infectious agent nucleic acid molecules can be used as probes to detect the presence of infectious agents in a sample taken from the donor.
  • samples from the donor can be concentrated and examined, e.g., visually examined for signs of pathogens, e.g., protozoan cysts of trophozoites, helminth eggs and larvae. Protozoan cyst identification can be confirmed, when required, by trichrome staining.
  • pathogens e.g., protozoan cysts of trophozoites, helminth eggs and larvae.
  • Protozoan cyst identification can be confirmed, when required, by trichrome staining.
  • cells from a donor can be cocultured with cells which are capable of being infected by the pathogen which is being tested for and the ability of the donor cells to infect the test cells can be determined.
  • test cells can be observed for viral cytopathic effects: Hemadsorbing viruses and hemaglutination testing can be detected using standard methods.
  • a hemagglutination test is a test that detects viruses with the property to agglutinate erythrocytes, such as swine influenza type A, parainfluenza, and encephalomyocarditus viruses, in the test article. Hsuing, G.D. (1982) Diagnostic Virology (Yale University Press, New Haven, CT);.
  • Fluorescent antibodies can also be used to stain cell suspensions of porcine cells to detect infectious agents, e.g., viruses.
  • samples taken from donors can be cultured to determine whether bacteria are present in the samples. If signs of bacterial growth are observed when the samples are initially screened for bacterial growth, a Gram stain is prepared and viewed microscopically at lOOx oil immersion for the presence of any bacteria or fungi. Positive cultures can then be subcultured onto both chocolate agar plates with Iso Vitlex and onto BMB plates. The chocolate plate is incubated at 35-37°C in 5% CO2 for 24 hours and the BMB anaerobically at 35-37°C for 48 hours. Any yeast or fungi seen by gram stain is subcultured onto a Sabaroud dextrose/Emmons plate.
  • the Vitek automated system can be to identify bacteria and yeast. Fungi can be identified via their macroscopic and microscopic characteristic.
  • the cells of the invention can be modified prior to use in transplantation.
  • the cells to be modified are xenogeneic cells.
  • the cells of the invention are altered prior to administration to the recipient.
  • the cells In an unaltered state, the cells have one or more antigen on the cell surface which stimulates an immune response against the cell when the cell is administered to a subject (also referred to herein as recipient or recipient subject).
  • a subject also referred to herein as recipient or recipient subject.
  • recipient or recipient subject By altering the antigen, the normal immunological recognition of the cell, e.g., by the immune system cells of the recipient is disrupted. An "abnormal" immunological recognition of this altered form of the antigen can further augment graft survival.
  • alteration of an antigen on the cell prior to introducing the cell into a subject provides another method by which the production of Th2 cytokines is promoted. Accordingly such alteration can be used, e.g., in conjunction with a cytokine profile altering agent to further bias the T cell cytokine profile of the recipient towards a Th2 phenotype.
  • the term “altered” encompasses changes that are made to at least one cell antigen(s) which reduces the immunogenicity of the antigen to thereby interfere with immunological recognition of the antigen(s) by the recipient's immune system.
  • Cells can be altered, e.g., by modifying, masking, or eliminating the antigen such that such that upon introduction of the composition into the recipient, lysis of said cell is prevented.
  • Antigens that can be altered according to the current invention include antigens on a xenogeneic cell, e.g., a porcine cell, which can interact with an immune cell in a xenogeneic recipient subject and thereby stimulate a specific immune response against the xenogeneic cell in the recipient.
  • the interaction between the antigen and the immune cell may be an indirect interaction or a direct interaction between the antigen and a molecule present on the surface of the immune cell.
  • the term “altered” encompasses changes that are made to at least one cell antigen(s) which reduces the
  • immune cell is intended to include a cell which plays a role in specific immunity (e.g., is involved in an immune response) or plays a role in natural immunity.
  • immune cells include all distinct classes of lymphocytes (T lymphocytes, such as helper T cells and cytotoxic T cells, B lymphocytes, and natural killer cells), monocytes, macrophages, other antigen presenting cells, dendritic cells, and leukocytes (e.g., neutrophils, eosinophils, and basophils).
  • T lymphocytes such as helper T cells and cytotoxic T cells, B lymphocytes, and natural killer cells
  • monocytes e.g., macrophages, other antigen presenting cells, dendritic cells, and leukocytes (e.g., neutrophils, eosinophils, and basophils).
  • the antigen is one which interacts with a T lymphocyte in the recipient (e.g., the antigen normally binds to a receptor on the surface
  • the antigen is altered by masking the cell, e.g., hiding an epitope of the antigen, for example using an antibody that binds to the antigen.
  • the antigen is altered by modifying the antigen, e.g., by expressing a form of the antigen which comprises an alteration in amino acid sequence such that an epitope of the antigen which would normally be recognized by the recipient's immune system is no longer so recognized.
  • a mutant form of a cell surface molecule can be expressed by the cell using standard techniques, e.g., by using site directed mutagenesis.
  • an antigen is altered eliminating the expression of the antigen from the surface of the cell. This can be accomplished, e.g., using enzymatic treatment, or by making a knock-out donor animal that no longer expresses the antigen to be altered.
  • the antigen to be altered on the cell is an MHC class I antigen.
  • an adhesion molecule on the cell surface such as NCAM-1 or ICAM-1
  • An antigen which stimulates a cellular immune response against the cell such as an MHC class I antigen, can be altered prior to transplantation by contacting the cell with a molecule which binds to the antigen.
  • a preferred molecule for binding to the antigen is an antibody, or fragment thereof (e.g., an anti-MHC class I antibody, or fragment thereof, an anti-ICAM-1 antibody or fragment thereof, an anti- LFA-3 antibody or fragment thereof, or an anti- ⁇ 2 microglobulin antibody or fragment thereof).
  • a preferred antibody fragment is an F(ab')2 fragment.
  • Polyclonal or, more preferably, monoclonal antibodies can be used.
  • Other molecules which can be used to alter an antigen include peptides and small organic molecules which bind to the antigen.
  • two or more different epitomes on the same or different antigens on the cell surface can be altered.
  • a particularly preferred monoclonal antibody for alteration of MHC class I antigens on xenogeneic cells is PT85 (commercially available from Veterinary Medicine Research Development, Pullman WA).
  • PT85 can be used alone to alter MHC class I antigens or, if each antibody is specific for a different epitope, PT85 can be used in combination with another antibody known to bind MHC class I antigens to alter the antigens on the cell surface.
  • Suitable methods for altering a surface antigen on a cell for transplantation are described in greater detail in Faustman and Coe 1991. Science 252:1700-1702 and PCT publication WO 92/04033. Methods for altering multiple epitopes on a surface antigen on a cell for transplantation are described in greater detail in PCT publication WO 95/26741 , published on October 12, 1995, the contents of which are inco ⁇ orated herein by reference.
  • the xenogeneic cells of the present invention can be altered to inhibit natural antibody-mediated hyperacute rejection of the cells.
  • the cells of the invention may, in unaltered form, express an epitope on their surface which stimulates hyperacute rejection of the by natural antibodies in a recipient subject.
  • Such an epitope can be modified, reduced or substantially eliminated. This treatment of the cell inhibits subsequent binding of the epitope by natural antibodies in a recipient, thereby inhibiting hyperacute rejection.
  • the epitope is a carbohydrate, preferably galactosyl ( l,3)galactose (Gal ( ⁇ l,3) Gal).
  • Epitopes on the surface of the xenogeneic cells are removed from the surface of a cell, such as by enzymatic or chemical treatment of the cell.
  • a cell such as by enzymatic or chemical treatment of the cell.
  • Gal ( ⁇ l,3)Gal epitopes can be cleaved from a xenogeneic cell surface by treatment of the cell with an alpha-galactosidase.
  • formation of the epitope on the cell surface is inhibited. This can be accomplished by inhibiting the activity of an enzyme which forms the epitope.
  • Gal ( l,3)Gal epitopes on the surface of a xenogeneic cell can be interfered with by inhibiting the activity of an alpha-l,3-galactosyltransferase within the cell, such as by introducing into the cell a nucleic acid which is antisense to a coding or regulatory region of an alpha- 1,3-galactosyltransferase gene or by treating the cell with a chemical inhibitor of the enzyme.
  • epitopes on the surface of a cell are altered by binding a molecule to the epitope, thereby inhibiting its subsequent recognition by natural antibodies in a recipient.
  • lectins, antibodies or antibody fragments can be bound to an epitope to inhibit its subsequent recognition by natural antibodies.
  • Methods for altering epitopes on xenogeneic cell surfaces which stimulate hyperacute rejection of the cells by natural antibodies in a recipient subject are described in greater detail in PCT Publication WO 95/33828, published on December 14, 1995, the contents of which are incorporated herein by reference.
  • a cell of the invention is modified to express a heterologous gene product.
  • the term "modified" to express a heterologous gene product is intended to mean that the cell is treated in a manner that results in the production of a heterologous gene product by the cell.
  • the cell does not express the gene product prior to modification.
  • modification of the cell may result in an increased production of a gene product already expressed by the cell or result in production of a gene product (e.g., an antisense RNA molecule) which decreases production of another, undesirable gene product normally expressed by the cell.
  • a cell is modified to express a gene product by introducing genetic material, such as a nucleic acid molecule (e.g., RNA or, more preferably, DNA) into the cell.
  • the nucleic acid molecule introduced into the cell encodes a gene product to be expressed by the cell.
  • the term "gene product" as used herein is intended to include proteins, peptides and functional RNA molecules.
  • the gene product encoded by the nucleic acid molecule is the desired gene product to be supplied to a subject.
  • the encoded gene product is one which induces the expression of the desired gene product by the cell (e.g., the introduced genetic material encodes a transcription factor which induces the transcription of the gene product to be supplied to the subject).
  • a nucleic acid molecule introduced into a cell is in a form suitable for expression in the cell of the gene product encoded by the nucleic acid.
  • the nucleic acid molecule includes coding and regulatory sequences required for transcription of a gene (or portion thereof) and, when the gene product is a protein or peptide, translation of the gene product encoded by the gene.
  • Regulatory sequences which can be included in the nucleic acid molecule include promoters, enhancers and polyadenylation signals, as well as sequences necessary for transport of an encoded protein or peptide, for example N-terminal signal sequences for transport of proteins or peptides to the surface of the cell or for secretion.
  • Nucleotide sequences which regulate expression of a gene product are selected based upon the type of cell in which the gene product is to be expressed and the desired level of expression of the gene product. For example, a promoter known to confer cell-type specific expression of a gene linked to the promoter can be used. A promoter specific for myoblast gene expression can be linked to a gene of interest to confer muscle-specific expression of that gene product. Muscle-specific regulatory elements which are known in the art include upstream regions from the dystrophin gene (Klamut et al, 1989. Mol. Cell. Biol. 9:2396), the creatine kinase gene (Buskin and Hauschka, 1989. Mol. Cell Biol.
  • Regulatory elements specific for other cell types are known in the art (e.g., the albumin enhancer for liver-specific expression; insulin regulatory elements for pancreatic islet cell-specific expression; various neural cell-specific regulatory elements, including neural dystrophin, neural enolase and A4 amyloid promoters).
  • a regulatory element which can direct constitutive expression of a gene in a variety of different cell types such as a viral regulatory element, can be used.
  • viral promoters commonly used to drive gene expression include those derived from polyoma virus, adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.
  • a regulatory element which provides inducible expression of a gene linked thereto can be used.
  • the use of an inducible regulatory element e.g., an inducible promoter
  • examples of potentially useful inducible regulatory systems for use in eukaryotic cells include hormone- regulated elements (e.g., see Mader, S. and White, J.H. 1993. Proc. Natl. Acad. Sci.
  • the nucleic acid is in the form of a naked nucleic acid molecule.
  • the nucleic acid molecule introduced into a cell to be modified consists only of 5 the nucleic acid encoding the gene product and the necessary regulatory elements.
  • the nucleic acid encoding the gene product (including the necessary regulatory elements) is contained within a plasmid vector. Examples of plasmid expression vectors include CDM8 (Seed, B., 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195).
  • the nucleic acid o molecule to be introduced into a cell is contained within a viral vector.
  • the nucleic acid encoding the gene product is inserted into the viral genome (or a partial viral genome).
  • the regulatory elements directing the expression of the gene product can be included with the nucleic acid inserted into the viral genome (i.e, linked to the gene inserted into the viral genome) or can be provided by the viral genome itself. Examples of methods which can be used to introduce naked nucleic acid into cells and viral - mediated transfer of nucleic acid into cells are described separately in the subsections below.
  • Transfection mediated by CaPO ⁇ Naked DNA can be introduced into cells by forming a precipitate containing the DNA and calcium phosphate.
  • a HEPES-buffered saline solution can be mixed with a solution containing calcium chloride and DNA to form a precipitate and the precipitate is then incubated with cells.
  • a glycerol or dimethyl sulfoxide shock step can be added to increase the amount of DNA taken up by certain cells.
  • CaPO4-mediated transfection can be used to stably (or transiently) transfect cells and is only applicable to in vitro modification of cells. Protocols for CaPO4- mediated transfection can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, 1989., Section 9.1 and in Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, 1989., Sections 16.32-16.40 or other standard laboratory manuals.
  • Electroporation Naked DNA can also be introduced into cells by incubating the cells and the DNA together in an appropriate buffer and subjecting the cells to a high- voltage electric pulse.
  • the efficiency with which DNA is introduced into cells by electroporation is influenced by the strength of the applied field, the length of the electric pulse, the temperature, the conformation and concentration of the DNA and the ionic composition of the media.
  • Electroporation can be used to stably (or transiently) transfect a wide variety of cell types and is only applicable to in vitro modification of cells. Protocols for electroporating cells can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, 1989., Section 9.3 and in Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, 1989., Sections 16.54-16.55 or other standard laboratory manuals.
  • Liposome-mediated transfection Naked DNA can be introduced into cells by mixing the DNA with a liposome suspension containing cationic lipids. The DNA/liposome complex is then incubated with cells. Liposome mediated transfection can be used to stably (or transiently) transfect cells in culture in vitro. Protocols can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, 1989., Section 9.4 and other standard laboratory manuals. Additionally, gene delivery in vivo has been accomplished using liposomes. See for example Nicolau et al. 1987. Meth. Enz. 149:157-176; Wang and Huang 1987. Proc. Natl.
  • DNA can be introduced into cells by directly injecting the DNA into the cells.
  • DNA can be introduced by microinjection. Since each cell is microinjected individually, this approach is very labor intensive when modifying large numbers of cells.
  • microinjection is a method of choice is in the production of transgenic animals (discussed in greater detail below).
  • the DNA is stably introduced into a fertilized oocyte which is then allowed to develop into an animal.
  • the resultant animal contains cells carrying the DNA introduced into the oocyte.
  • Direct injection has also been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. 1991.
  • a delivery apparatus e.g., a "gene gun" for injecting DNA into cells in vivo can be used.
  • a delivery apparatus e.g., a "gene gun”
  • Such an apparatus is commercially available (e.g., from BioRad).
  • Receptor-Mediated DNA Uptake Naked DNA can also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, CH. 1988. J. Biol. Chem. 263:14621 ; Wilson et al. 1992. J Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320). Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis. Receptors to which a DNA-ligand complex have targeted include the transferrin receptor and the asialoglycoprotein receptor.
  • a DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. 1991. Proc. Natl 0 Acad. Sci. USA 88:8850; Cristiano et al. 1993. Proc. Natl Acad. Sci. USA 90:2122- 2126).
  • Receptor-mediated DNA uptake can be used to introduce DNA into cells either in vitro or in vivo and, additionally, has the added feature that DNA can be selectively targeted to a particular cell type by use of a ligand which binds to a receptor selectively expressed on a target cell of interest.
  • An alternative method for generating a cell that is modified to express a gene product involving introducing naked DNA into cells is to create a transgenic animal which contains cells modified to express the gene product of interest.
  • a transgenic animal is an animal having cells that contain a transgene, wherein the transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.
  • a transgene is a DNA molecule which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a transgenic animal expressing a gene product of interest in one or more cell types within the animal can be created, for example, by introducing a nucleic acid molecule encoding the gene product (typically linked to appropriate regulatory elements, such as a tissue-specific enhancer) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Methods for generating transgenic animals, particularly animals such as mice have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 and Hogan, B.
  • transgenic founder animal can be used to breed more animals carrying the transgene.
  • Cells of the transgenic animal which express a gene product of interest can then be used to deliver the gene product to a subject in accordance with the invention.
  • an animal containing a gene which has been modified by homologous recombination can be constructed to express a gene product of interest.
  • an endogenous gene carried in the genome of the animal can be altered by homologous recombination (for instance, all or a portion of a gene could be replaced by the human homologue of the gene to "humanize” the gene product encoded by the gene) or an endogenous gene can be "knocked out” (i.e., inactivated by mutation).
  • an endogenous gene in a cell can be knocked out to prevent production of that gene product and then nucleic acid encoding a different (preferred) gene product is introduced into the cell.
  • a vector which contains the DNA which is to replace or interrupt the endogenous DNA flanked by DNA homologous to the endogenous DNA (see for example Thomas, K.R. and Capecchi, M. R. 1987. Cell 5 503).
  • the vector is introduced into an embryonal stem cell line (e.g., by electroporation) and cells which have homologously recombined the DNA are selected (see for example Li, E. et al. 1992. Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see for example Bradley, A.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Cells of the animal containing the homologously recombined DNA which express a gene product of interest can then be used to deliver the gene product to a subject in accordance with the invention.
  • a preferred approach for introducing nucleic acid encoding a gene product into a 0 cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product.
  • a viral vector containing nucleic acid e.g. a cDNA
  • Infection of cells with a viral vector has the advantage that a large proportion of cells receive the nucleic acid, which can obviate the need for selection of cells which have received the nucleic acid.
  • molecules encoded within the viral vector e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which 5 have taken up viral vector nucleic acid and viral vector systems can be used either in vitro or in vivo.
  • Retroviruses Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. 1990. Blood 6:271).
  • a o recombinant retrovirus can be constructed having a nucleic acid encoding a gene product of interest inserted into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. 1985. Science 230:1395-1398; Danos and Mulligan 1988.
  • Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell. 5
  • Adenoviruses The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. 1988. BioTechmques 6:616; Rosenfeld et al. 1991. Science 252:431-434; and Rosenfeld et al. o 1992. Cell 68: 143-155.
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. 1992. cited supra), endothelial cells (Lemarchand et al. 1992. Proc. Natl. Acad. Sci. USA 89:6482- 6486), hepatocytes (Herz and Gerard 1993. Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. 1992. Proc. Natl. Acad. Sci. USA 89:2581-2584).
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj- Ahmand and Graham 1986. J. Virol. 57:267).
  • Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material.
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • AAV Adeno-associated virus
  • Vectors containing as little as 300 base pairs of AAV 5 can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
  • An AAV vector such as that described in Tratschin et al. 1985. Mol. Cell. Biol. 5:3251- 3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. 1984. Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. 1985. Mol. Cell Biol.
  • DNA introduced into a cell can be detected by a filter hybridization technique (e.g., Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • the gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product, such as an enzymatic assay. If the gene product of interest to be expressed by a cell is not readily assayable, an expression system can first be optimized using a reporter gene linked to the regulatory elements and vector to be used.
  • the reporter gene encodes a gene product which is easily detectable and, thus, can be used to evaluate the efficacy of the system.
  • Standard reporter genes used in the art include genes encoding ⁇ -gammalactosidase, chloramphenicol acetyl transferase, luciferase and human growth hormone .
  • the modified population of cells may be used without further isolation or subcloning of individual cells within the population. That is, there may be sufficient production of the gene product by the population of cells such that no further cell isolation is needed.
  • Such a population of uniform cells can be prepared by isolating a single modified cell by limiting dilution cloning followed by expanding the single cell in culture into a clonal population of cells by standard techniques.
  • a cell can be modified by inducing or increasing the level of expression of the gene product by a cell.
  • a cell may be capable of expressing a particular gene product but fails to do so without additional treatment of the cell.
  • the cell may express insufficient amounts of the gene product for the desired purpose.
  • an agent which stimulates expression of a gene product can be used to induce or increase expression of a gene product by the cell.
  • cells can be contacted with an agent in vitro in a culture medium.
  • the agent which stimulates expression of a gene product may function, for instance, by increasing transcription of the gene encoding the product, by increasing the rate of translation or stability (e.g., a post transcriptional modification such as a poly A tail) of an mRNA encoding the product or by increasing stability, transport or localization of the gene product.
  • agents which can be used to induce expression of a gene product include cytokines and growth factors.
  • a transcription factor which upregulates the expression of a gene encoding a gene product of interest can be provided to a cell, for example, by introducing into the cell a nucleic acid molecule encoding the transcription factor.
  • this approach represents an alternative type of nucleic acid molecule which can be introduced into the cell (for example by one of the previously discussed methods).
  • the introduced nucleic acid does not directly encode the gene product of interest but rather causes production of the gene product by the cell indirectly by inducing expression of the gene product.
  • a cell is modified to express a gene product by coupling the gene product to the cell, preferably to the surface of the cell.
  • a protein can be obtained by purifying the cell from a biological source or expressing the protein recombinantly using standard recombinant DNA technology. The isolated protein can then be coupled to the cell.
  • the terms "coupled” or “coupling” refer to a chemical, enzymatic or other means (e.g., by binding to an antibody on the surface of the cell or genetic engineering of linkages) by which a gene product can be linked to a cell such that the gene product is in a form suitable for delivering the gene product to a subject.
  • a protein can be chemically crosslinked to a cell surface using commercially available crosslinking reagents (Pierce, Rockford IL).
  • crosslinking reagents Pierce, Rockford IL.
  • Other approaches to coupling a gene product to a cell include the use of a bispecific antibody which binds both the gene product and a cell-surface molecule on the cell or modification of the gene product to include a lipophilic tail (e.g., by inositol phosphate linkage) which can insert into a cell membrane.
  • a CD4+ T cell response can be modulated toward a Thl or Th2 profile (Kuchroo et al. 1995. Cell. 80:707).
  • certain cytokines once produced, can alter the profile of cytokines that are subsequently produced.
  • a cytokine profile altering agent alters the profile of cytokine production from any cell type that produces cyokines.
  • such an agent alters the profile of cytokines produced by T cells.
  • T helper subset 1 and T helper subset 2 (Th2) subpopulations of T helper cells regulate either cell-mediated (DTH) or antibody-mediated immune responses is based on the work of Mosmann and colleagues who identified a cytokine production profile for each T cell subset (Mosmann, T.R. 1986. J Immunol.136:2348; Cher, D.J. and T.R. Mosmann, 1987 J Immunol, 138: p. 3688; Stout, R.D. and K.
  • Thl cytokines are primarily involved in promoting cellular responses.
  • the major function of IL-2 is the activation of T cells and NK cells.
  • IL-2 is secreted by helper T lymphocytes 4-12 hours following stimulation by antigen.
  • the subsequent binding of IL-2 to its receptor results in proliferation of the antigen activated T cells, enhanced secretion of Iymphokines, and heightened expression of membrane receptors for the other growth factors.
  • IL-2 principally enhances expansion of the Thl subset of T cells.
  • IL-2 has been demonstrated to influence B cells and monocytes. In particular, activation of monocytes by IL-2 induces IL-1 secretion, enhances monocyte-mediated cytotoxicity, promotes proliferation of macrophage precursors, and increases phagocytosis.
  • IFN-gamma augments the induction of the Thl cells (Swain, S.L. 1991. Immunol Rev. 123: 1 15) and is capable of regulating specific immune effector mechanisms by direct actions on helper T cells, cytotoxic T cells, and B cells. Conversely, IFN-gamma impedes the induction, proliferation, and effector functions of the Th2 cell subset. IFN-gamma up regulates the expression of class I and class II MHC antigens on a variety of cell types. The consequence of this up-regulated class II MHC antigen expression is an augmented and accelerated immune response.
  • IFN-gamma can induce the de novo expression of class II MHC antigens on epithelial, endothelial, and connective tissue cells, allowing these cells to become active in antigen presentation and induction of specific T-cell immunity.
  • IFN-gamma is a potent activator of macrophage and monocytes.
  • IFN-gamma augments cytotoxic immune responses by directly activating NK cells and CD8 + T cells.
  • Many of the in vitro properties of IFN-gamma have been confirmed by in vivo animal model experiments.
  • IL-12 has been identified as an important cytokine in the initiation of cell mediated Thl response. Unlike IFN-gamma, the presence of IL-12 during priming directly augments the Thl cells, but has no effect on Th2 cells.
  • Th2 cytokines are important in the development of humoral immune responses e.g., through their affects on B cells.
  • Th2 cytokines have been found to positively amplify Th2 responses.
  • IL-4 and IL-10 have an important role in initiating the Th2 response (Swain, S.L., et al. 1990. J Immunol, 145:3796).
  • the inclusion of anti-IL-4 antibody during priming of an immune response completely abrogates the generation of Th2 cells, suggesting that the presence of IL-4, even if endogenously derived, is essential for Th2 differentiation.
  • IL-10 has also been reported to promote the development of Th2 cells, its major effect may be in suppressing Thl cells ( Bromberg, J.S. 1995. Curr Opin Immunol. 7:639; de Vries, J.E. 1995. Ann Med. 27:537).
  • CD8 + T cells are Thl -like (TCI ) or Th2-like (TC2) in their cytokine production profiles (Sad, S., R. et. al. 1995. Immunity. 2:271).
  • TCI Thl -like
  • TC2 Th2-like
  • Many of the CD8 + T cells that have been examined produce a pattern of cytokines very similar to that of Thl clones, with the exception that they tend to make little, if any, IL-2 (Fong, T.A. and T.R. Mosmann, 1990. J Immunol, 144:1744.
  • Th2-like CD8 + T cell clones have been established from such patients; these clones can suppress the response of Thl -like cells, in part, through the activities of IL-4 (Salgame, P., et al. 1991. Science. 254:279.
  • the cytokine profile of a recipient of a graft can be biased by the administration of a cytokine profile altering agent such that Th2 cytokine production after exposure to the agent is enhanced when compared with Th2 cytokine production in the absence of 0 the agent and/or Thl cytokine production is diminished upon exposure to cytokine profile altering agent.
  • a cytokine profile altering agent is not a general immunosuppressant, e.g., cyclosporin
  • a cytokine profile altering agent is a protein or polypeptide.
  • a cytokine profile altering agent can lead to an increase the production of Th2 cytokines overall (or an increased ratio of Th2 to Thl cytokines 5 overall), or can specifically lead to an increase in the production of one or more Th2 cytokines (or an increase in the ratio of a specific Th2 cytokine to Thl cytokines.
  • the cytokine profile altering agent leads to decrease in the production of IL-2 and/or IFN-8.
  • the cytokine profile altering agent leads to an increase in the production of IL-4 and/or IL-10. Examples of cytokine profile o altering agents are provided below: Cytokines And Cytokine Fusion Proteins
  • the cytokine profile altering agent is a cytokine which is administered to the transplant recipient.
  • cytokines such as IL-4 and IL-10 have been found to bias T cell responses towards a Th2 phenotype.
  • These cytokine genes have been cloned and their sequences are available in the art (e.g., Viera et al. 1991. Proc. Natl. Acad. Sci 88:1172 (for IL-10) and Yokota et al. 1986. Proc. Natl. Acad. Sci. 83:5894 or Arai et al. 1989. J Immunol. 142:274 (for IL-4).
  • cytokine that biases a T cell response towards a Th2 phenotype can be administered to transplant recipient.
  • the cytokine or cytokines to be administered can be derived from any source, but are preferably human.
  • modified forms of cytokines can also be used, e.g., mutated forms that contain conservative substitutions, as long as they retain the ability to bind to their cognate cytokine receptor and bias a T cell response towards a Th2 phenotype.
  • a cytokine can be modified to reduce immunogenicity in the subject or to promote increased half life. Exemplary methods of modifying amino acid sequences are known in the art.
  • the cytokine that alters a T cell cytokine secretion profile is a fusion protein comprising the active portion of the cytokine (i.e., the portion that is capable of bias's a T cell cytokine secretion profile to a Th2 phenotype) and a second non-cytokine protein.
  • the second non-cytokine protein is a peptide derived from immunoglobulin molecule.
  • a fusion protein e.g., IL-4Ig or IL-lOIg, can be made using methods known in the art (see e.g., Linsley 1994.
  • cytokine analogs that mimic the function of cytokines can be used to bias an immune response towards a Th2 phenotype (e.g., U.S. patent 5,837,293).
  • the cytokine profile altering agent is an antibody that biases a response towards a Th2 phenotype can be administered to a transplant recipient.
  • antibodies to cytokines that reduce Thl cytokine secretion can be administered.
  • antibodies to IL-12, IL-2, and/or IFN-gamma can be administered.
  • Antibodies for use in the claimed methods can be obtained commercially or can be made by immunization with the agent of interest.
  • IL-12 has been cloned and sequenced (e.g., Wolf et al. 1991. J Immunol. 146:3074) as have IL-2 (Taniguchi et al. 1983. Nature 302:305; Maeda et al. 1983. Biochem. Biophys. Res. Commun. 115:1040; Devos et al. 1983. Nucleic Acids Res. 11 :4307) and antibodies can be generated against one or more of these cytokines and tested for their ability to bias an immune response towards a Th2 profile.
  • Antibodies can be from any source.
  • antibodies are preferably of human origin or, if generated in other species, are "humanized” for administration to humans as described in the art. (e.g., Cancer Research. 1990. 50:1495; Brown et al. 1991. Proc. Natl. Acad. Sci. USA 88:2663; Kettleborough et al. 1991. Protein Engineering 4:773; US patent 5,853,697).
  • Antibody Fragments of antibodies which maintain their ability to bias an immune response towards a Th2 profile are also included in the term "antibody.” Methods of making such antibodies and fragments are known in the art (e.g., Harlow and Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.) and can readily be screened e.g., for their ability to bind to the antigen to which they were raised using standard methods, e.g., using in an ELISA assay.
  • the antibodies can be administered orally or parenterally in a pharmaceutically acceptable carrier to human subjects, suitable carriers for use in the present invention include, but are limited to, pyrogen-free saline.
  • suitable carriers for use in the present invention include, but are limited to, pyrogen-free saline.
  • a sterile solution or suspension is prepared in a vehicle, e.g., saline, that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected, e.g., into subcutaneous or intramuscular tissues.
  • the antibodies may be microencapsulated with either a natural or synthetic polymer into microparticles which produce a sustained release of antibody (Eldrige et al. 1989. Cur. Topics in Microbiol. and Immunol.
  • antibodies can be administered at a dosage readily determined by one of ordinary skill in the art.
  • the antibodies can be administered in a single dosage, e.g., between 10 mg and 20 mg/kg of body weight.
  • patients can be given a dosage of 10 mg to 20 mg/kg weekly until colitis symptoms subside.
  • 500 mg to 1000 can be given.
  • 10 mg to 20 mg/kg of body weight can be administered as a single or as a weekly intravenous injection.
  • the skilled clinician will consider the age, weight, and condition of the individual in determining a final dose.
  • 500 mg to 100 mg can be microencapsulated as described for slow release over a prolonged period.
  • cells can be directly administered to the site of an organ in the recipient.
  • the cells are administered systemically.
  • the cells in order to achieve systemic administration, the cells are injected intraperitoneally, (Wilson, J. et al. 1991. Clin. Biotech. 3(l):21-25).
  • the cells in order to achieve systemic administration the cells are injected intravenously.
  • the cells can be injected into the portal vein (Kay, M. 1993. Cell Trans. 2:405-406; Tejera, J.L. et al. 1992. Transplan. Proc. 24(1): 160- 161 ; Wiederledge, J.C. et al. 1990.
  • Transplantation 50(3):466-476 or the mesenteric vein (Grossman, M. et al. 1994. Nature Gen. 6:335- 341; Wilson, J.M. et al. 1990. Proc. Natl. Acad. Sci. 87:8437-8441).
  • Intrasplenic injection of the cells (Rhim, J.A. et al. 1994. Science 263:1149-1152; Kay, M.A. 1993. Cell Trans. 2:405-406; Wiederledge, J.C. et al. 1990. Transplantation 50(3):466-476), or infusion of the cells into the splenic artery can also be performed.
  • the cells for use in the claimed methods can be delivered to multiple sites in the recipient.
  • the cells can be delivered directly to an organ and can also be administered systemically.
  • cells for transplantation can be bound to microcarrier beads such as collagen-coated dextran beads (Pharmacia, Uppsala, Sweden) (Wilson, J. et al. 1991. Clin. Biotech. 3(l):21-25) prior to transplantation.
  • Cells can be administered in a pharmaceutically acceptable carrier or diluent as described herein.
  • Administration of the compositions and/or agents described herein can be in any pharmacological form that includes a therapeutically active amount of an agent and a pharmaceutically acceptable carrier.
  • Administration of a therapeutically active amount of the subject agents and/or compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve treatment of the disorder in the case of the transplant, and to inhibit rejection of the transplant in the case of an agent which alters a cytokine profile.
  • a therapeutically active amount of an agent or composition may vary, for example, depending upon such factors as the disease state, age, sex or weight of the recipient, the type of cell transplanted, the site in the donor to which the transplanted cells were administered, and the reason for administration. Such an amount can be readily determined by one of ordinary skill in the art.
  • the optimal course of administration of the agents and/or compositions of the invention may also vary depending upon the subject to be treated.
  • a subject will be transplanted and treated with an agent that alters a cytokine profile at the same time.
  • staggered administration may be desirable to achieve optimal alteration in a cytokine profile or optimal inhibition of the recipient immune response to the graft.
  • an agent which alters a cytokine profile can be administered alone prior to transplantation, or can be administered alone after transplantation.
  • Cytokine profile altering agents can be administered systemically or locally using art recognized methods. A dosage regime may be adjusted to provide the optimum therapeutic response for each subject without undue experimentation.
  • the cytokine profile of the recipient can be measured (e.g., levels of cytokines can be measured in serum or by in situ staining or nucleic acid hybridization in the graft) to determine whether or not a shift in cytokine profile has occurred in response to administration of the agent.
  • graft rejection can be monitored in the recipient, e.g., by detecting the presence of cells or assaying for their continued function.
  • cells e.g. mixed or purified population of cells
  • the recipient e.g. , from periferal blood or from a biopsy specimen
  • the cytokine production profile of the patient will be determined prior to transplantation and comparisions can be made to post transplantation cytokine production profiles.
  • comparisons can be made to cytokine profiles of untransplanted, control individuals. Given the results of such assays, the skilled clinician will be capable of determining whether any of these tests indicates that treatment with a cytokine profile altering agent should be modified.
  • An agent or composition of the present invention may be administered to an individual in an appropriate carrier or diluent, co- administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al, (1984) J.
  • the active agent or composition may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating active composition 0 or agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (e.g., agent or composition) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the protein may be orally administered, for example, with an inert diluent or an o assimilable edible carrier.
  • pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the agent that alters a cytokine profile, it can be used in the therapeutic compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active agent or composition for the treatment of individuals.
  • a cytokine profile altering agent is administered to a subject transiently, pre-transplantation, post-transplantation, or both.
  • the cytokine profile altering agent is administered chronically, i.e., over a prolonged period of time post-transplantation.
  • Administration of the cytokine profile altering agent to the subject can begin prior to transplantation of the cells into the subject. For example, initiation of agent administration can be a few days (e.g., one to fourteen days) before transplantation. Alternatively, agent administration can begin the day of transplantation or a few days after transplantation.
  • administration of the agent is continued for sufficient time such that a measurable change in cytokine secretion profile occurs in the recipient or until graft acceptance is promoted in the recipient.
  • Administration of the agent can continue chromically in order to maintain graft acceptance.
  • donor cells will not be rejected by the recipient when administration of the agent ceases.
  • the agent can be administered for as short as three days or longer than three months following transplantation or over the lifetime of the recipiant.
  • the cytokine profile altering agent is administered for at least one week following transplantation.
  • Induction of tolerance to the transplanted cells in a subject can be measured by the lack of rejection of the transplanted cells after administration of the cytokine profile altering agent has ceased, e.g., by measuring continued graft function or presence of donor cells in the recipient.
  • the cells and/or compositions of the invention can be inserted into a delivery device which facilitates introduction of the cells and/or compositions into the subject.
  • delivery devices include tubes, e.g., catheters, for infusing or injecting cells and fluids into the body of a recipient subject.
  • the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location.
  • the cells (and compositions containing the cells) of the invention can be inserted into such a delivery device, e.g., a syringe, e.g., syringe pump, in different forms.
  • the cells can be suspended in a solution or embedded in a support matrix when contained in such a delivery device.
  • solution includes a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable.
  • Pharmaceutically acceptable carriers and diluents include sterile saline and aqueous buffer solutions. The use of such carriers and diluents is well known in the art.
  • the solution is preferably sterile and fluid to the extent that easy syringability exists.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Solutions of the invention can be prepared by incorporating cells and/or agent as described herein in a sterile pharmaceutically acceptable carrier or diluent and, as required, other ingredients.
  • Support matrices in which the cells can be inco ⁇ orated or embedded include matrices which are recipient-compatible and which degrade into products which are not harmful to the recipient. Natural and/or synthetic biodegradable matrices are examples of such matrices. Natural biodegradable matrices include collagen matrices. Synthetic biodegradable matrices include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid. These matrices can provide support and protection for certain types of cells in vivo. Treatment Of Disorders Using Cells
  • the subject cells can be transplanted into subjects having a disorder that would benefit by the transplantation of such a cell.
  • the cells of the invention can be introduced into animal transplantation models of various disorders.
  • the survival and/or function of transplanted cells can be monitored. For example, the ability of the cells to synthesize a protein which is not produced in active form in the recipient can be determined. This can be done, for example, by detecting the presence of the protein itself or an activity of the protein, e.g., an enzymatic activity. Additionally or alternatively, the cells themselves can be detected, for example, by taking biopsy specimens and detecting the transplanted cells.
  • additional agents in addition to the administration of cytokine profile altering agents, additional agents can be administered to the subject to further inhibit the immune response of the recipient subject and facilitate acceptance of the xenograft.
  • additional agents include:
  • a soluble form of a costimulatory molecule can additionally be administered to the transplant recipient. Soluble forms of costimulatory molecules have been found to block the transduction of a costimulatory signal in a T cell.
  • the soluble form of a costimulatory molecule is a soluble form of CTLA4.
  • DNA sequences encoding the human and murine CTLA4 protein are known in the art, see e.g., Dariavich, et al. (1988) Eur. J. Immunol. 18(12), 1901-1905; Brunet, J.F., et al. (1987) supra; Brunet, J.F. et al. (1988) Immunol. Rev.
  • the soluble CTLA4 protein comprises the entire CTLA4 protein.
  • a soluble CTLA4 protein comprises the extracellular domain of a CTLA4 protein.
  • a soluble, recombinant form of the extracellular domain of CTLA4 has been expressed in yeast (Gerstmayer et al. 1997. FEBS Lett. 407:63).
  • the soluble CTLA4 proteins comprise at least a portion of the extracellular domain of CTLA4 protein which retains the ability to bind to B7-1 and/or B7-2.
  • the soluble CTLA4 protein or portion thereof is a fusion protein comprising at least a portion of CTLA4 which binds to B7-1 and/or B7-2 and at least a portion of a second non-CTLA4 protein.
  • the CTLA4 fusion protein comprises a CTLA4 extracellular domain which is fused at the amino terminus to a signal peptide, e.g., from oncostatin M (see e.g., WO93/00431).
  • a soluble form of CTLA4 is a fusion protein comprising the extracellular domain of CTL A4 fused to a portion of an immunoglobulin molecule.
  • a fusion protein, CTLA4Ig can be made using methods known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2:221; Linsley WO 93/00431 and U.S. Patent 5,770,197).
  • the soluble form of B7-1 or B7-2 or a combination of B7-1 and B7-2 can additionally be administered to the transplant recipient.
  • DNA sequences encoding B7 proteins are known in the art, see e.g., B7-2 (Freeman et al. 1993 Science. 262:909 or GenBank Accession numbers P42081 or A48754); B7-1 (Freeman et al. J. Exp. Med. 1991. 174:625 or GenBank Accession numbers P33681 or A45803.
  • the soluble B7 protein comprises an entire B7 protein.
  • a soluble B7 protein comprises the extracellular domain of a B7 protein.
  • soluble, recombinant form of the extracellular domain of CTLA4 has been expressed in yeast (Gerstmayer et al. 1997. FEBS Lett. 407:63).
  • the soluble B7 proteins comprise at least a portion of the extracellular domain of B7 protein which retains the ability to bind to CTLA4 and/or CD28.
  • the soluble B7 protein or portion thereof is a fusion protein comprising at least a portion of B7 which binds to CD28 and/or CTLA4 and at least a portion of a second non-B7 protein.
  • the B7 fusion protein comprises a B7 extracellular domain which is fused at the amino terminus to a signal peptide, e.g., from oncostatin M (see e.g., WO93/00431).
  • a soluble form of B7 is a fusion protein comprising the extracellular domain of B7 fused to a portion of an immunoglobulin molecule.
  • a fusion protein a B7Ig
  • Such a fusion protein, a B7Ig can be made using methods known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2:221; Linsley WO 93/00431, U.S. Patent 5,770,197, and U.S. Patent 5,580,756).
  • administration of a population of lymphocytes taken from the same donor or a related donor can accompany administration of a cytokine profile altering agent.
  • the population of lymphocytes comprises T lymphocytes.
  • the population of lymphocytes is a xenogeneic population of lymphocytes.
  • the lymphocytes are administered systemically.
  • the lymphocytes are administered intravenously.
  • the lymphocytes are administered intraperitoneally.
  • the lymphocytes are administered intrasplenically.
  • the lymphocytes are administered into the portal vasculature of the subject (Morita et al. 1998. Proc. Natl Acad. Sci. 95:6947; Hirakawa et al. 1993. Transplant. Proc. 25:346; Zhang et al. 1994. Eur. J. Immunol. 24:1558; Yu et al. 1994. surgery. 116:229).
  • the administration of the cells of the invention into this vessel can be accompanied by a step which involves maintaining portal blood pressure.
  • a "maintaining portal blood pressure" of a subject refers to maintaining a relatively stable blood pressure level in a subject during and after transplantation of the cells of the invention relative to the portal blood pressure level of the subject prior to transplantation of the cells, e.g., lymphocytes, of the invention. This may involve temporarily decreasing portal blood pressure so that the infusion of cells does not increase portal blood pressure.
  • the portal blood pressure of a subject is decreased or maintained through the use of an transjugular intra-hepatic porto-systemic shunt (TIPS). See e.g., Rossle, M. and Ring, E.J. in Progress in Liver Disease (Saunders, 1994) Vol. XI 177-189; Ochs, A.
  • the TIPS procedure includes the passage of a tube e.g., needle, catheter, which acts as a shunt, from the jugular vein into a hepatic vein and then advancing the tube through the liver parenchyma into a portal vein branch.
  • This shunt allows blood flowing through the portal venous system to pass directly from the portal vein into the hepatic vein, thereby bypassing the liver parenchyma.
  • Bypass of the liver parenchyma results in a decrease or at least a maintenance of the portal blood pressure in a subject after transplantation of the cells of the invention.
  • the step of decreasing or maintaining the portal venous blood pressure in a subject can be performed prior to, during, or after transplantation of the cells of the invention.
  • the cells of the invention are administered to the subject via the TIPS catheter into the portal vein, thereby eliminating the need for providing an additional route of administration for the cells.
  • the step of decreasing or maintaining the portal venous blood pressure in a subject is performed after transplantation of the cells of the invention.
  • cells of the invention are transplanted and the portal blood pressure is decreased or maintained by performance of the following steps: 1) transjugular cannulation of the portal vein; 2) transplantation of cells into the liver via the portal vein, e.g., through the use of the cannula in the portal vein; and 3) placement of the TIPS within the liver.
  • the subject method further comprises administration of an immunosuppressive drug or general immunosuppelich.
  • a preferred agent for use in inhibiting T cell activity in a recipient subject is an immunosuppressive drug.
  • the term "immunosuppressive drug or agent” is intended to include pharmaceutical agents which inhibit or interfere with normal immune function.
  • a preferred immunsuppressive drug is cyclosporin A.
  • Other immunosuppressive drugs which can be used include FK506, RS-61443, and deoxyspergualin.
  • the immunosuppressive drug is administered in conjunction with at least one other therapeutic agent.
  • Additional therapeutic agents which can be administered include steroids (e.g., glucocorticoids such as prednisone, methyl prednisolone and dexamethasone) and chemotherapeutic agents (e.g., azathioprine and cyclosphosphamide).
  • steroids e.g., glucocorticoids such as prednisone, methyl prednisolone and dexamethasone
  • chemotherapeutic agents e.g., azathioprine and cyclosphosphamide
  • Suitable immunosuppressive drugs are commercially available (e.g., cyclosporin A is available from Sandoz, Co ⁇ ., East Hanover, NJ).
  • An immunsuppressive drug is administered in a formulation which is compatible with the route of administration. Suitable routes of administration include intravenous injection (either as a single infusion, multiple infusions or as an intravenous drip over time), intraperitoneal injection, intramuscular injection
  • the drug can be dissolved in a physiologically acceptable carrier or diluent (e.g., a buffered saline solution) which is sterile and allows for syringability.
  • a physiologically acceptable carrier or diluent e.g., a buffered saline solution
  • Dispersions of drugs can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
  • Convenient routes of administration and carriers for immunsuppressive drugs are known in the art.
  • cyclosporin A can be administered intravenously in a saline solution, or orally, intraperitoneally or intramuscularly in olive oil or other suitable carrier or diluent.
  • An immunosuppressive drug is administered to a recipient subject at a dosage sufficient to achieve the desired therapeutic effect (e.g., inhibition of rejection of transplanted cells).
  • Dosage ranges for immunosuppressive drugs, and other agents which can be coadministered therewith are known in the art (See e.g., Freed et al. New Engl. J. Med. 1992. 327:1549: Spencer et al. 1992. New Engl J. Med. 327:1541; Widner et al. 1992. New Engl. J. Med. 327:1556; Lindvall et al. 1992. Ann. Neurol 31 :155; and Lindvall et al. 1992.
  • a preferred dosage range for immunosuppressive drugs, suitable for treatment of humans, is about 1-30 mg/kg of body weight per day.
  • a preferred dosage range for cyclosporin A is about 1-10 mg/kg of body weight per day, more preferably about 1-5 mg/kg of body weight per day.
  • Dosages can be adjusted to maintain an optimal level of the immunosuppressive drug in the serum of the recipient subject. For example, dosages can be adjusted to maintain a preferred serum level for cyclosporin A in a human subject of about 100-200 ng/ml. It is to be noted that dosage values may vary according to factors such as the disease state, age, sex, and weight of the individual.
  • an immunsuppressive drug is administered to a subject transiently after transplantation of the subject.
  • Administration of the drug to the subject can begin prior to transplantation of the cells into the subject.
  • initiation of drug administration can be a few days (e.g., one to fourteen days) before transplantation.
  • drug administration can begin the day of transplantation or a few days after transplantation.
  • administering is continued for sufficient time such that, in combination with the cyokine profile altering agent, aceptance of the graft is promoted.
  • donor specific tolerance to the graft is induced.
  • Acceptance of the graft is indicated by the presence of the transplanted cells after administration of the immunosuppressive drug has ceased. Tolerance can be determined using standard methods, e.g., failure of host immune cells to respond to donor antigen (e.g., by proliferating or in a cellular cytotoxicity assay).
  • Acceptance of transplanted tissue can be determined mo ⁇ hologically (e.g., with biopsies of liver) or by assessment of the functional activity of the graft.
  • Another type of agent which can be used to inhibit the anti-graft immune response in a subject is an antibody, or fragment or derivative thereof, e.g., which depletes or sequesters immune cells in a recipient.
  • antibodies which are capable of depleting or sequestering T cells in vivo when administered to a subject can be given. Such antibodies are known in the art. Typically, these antibodies bind to an antigen on the surface of a T cell. Polyclonal antisera can be used, for example anti-lymphocyte serum. Alternatively, one or more monoclonal antibodies can be used.
  • Preferred T cell-depleting antibodies include monoclonal antibodies which bind to CD2, CD3, CD4 or CD8 on the surface of T cells.
  • Antibodies which bind to these antigens are known in the art and are commercially available (e.g., from American Type Culture Collection).
  • a preferred monoclonal antibody for binding to CD3 on human T cells is OKT3 (ATCC CRL 8001).
  • the binding of an antibody to surface antigens on a T cell can facilitate sequestration of T cells in a subject and/or destruction of T cells in a subject by endogenous mechanisms.
  • a T cell- depleting antibody which binds to an antigen on a T cell surface can be conjugated to a toxin (e.g., ricin) or other cytotoxic molecule (e.g., a radioactive isotope) to facilitate destruction of T cells upon binding of the antibody to the T cells. See WO 95/26740, published on 12 October 1995, for further details concerning the generation of antibodies which can be used in the present invention.
  • an antibody which can be used to inhibit T cell activity in a recipient subject is an antibody which inhibits T cell proliferation.
  • an antibody directed against a T cell growth factor, such as IL-2, or a T cell growth factor receptor, such as the IL-2 receptor can inhibit proliferation of T cells (See e.g., DeSilva, D.R. et al. 1991. J. Immunol 147:3261-3267).
  • an anti-IL-2 or an anti-IL-2 receptor antibody can be administered to a recipient to inhibit rejection of a transplanted cell (see e.g. Wood et al. 1992. Neuroscience 49:410).
  • both an anti-IL-2 and an anti-IL-2 receptor antibody can be coadministered to inhibit T cell activity or can be administered with another antibody (e.g., which binds to a surface antigen on T cells).
  • An antibody which depletes, sequesters or inhibits T cells within a recipient can be administered at a dose and for an appropriate time to inhibit rejection of cells upon transplantation when administered in conjunction with a cytokine profile altering agent.
  • Antibodies are preferably administered intravenously in a pharmaceutically acceptable carrier or diluent (e.g., a sterile saline solution).
  • Antibody administration can begin prior to transplantation (e.g., one to five days prior to transplantation) and can continue on a daily basis after transplantation to achieve the desired effect (e.g., up to fourteen days after transplantation).
  • a preferred dosage range for administration of an antibody to a human subject is about 0.1-0.3 mg/kg of body weight per day.
  • a single high dose of antibody e.g., a bolus at a dosage of about 10 mg/kg of body weight
  • a single high dose of antibody can be administered to a human subject on the day of introduction of the cells and/or agent that alters a cytokine secretion profile into the subject.
  • the effectiveness of antibody treatment in depleting T cells from the peripheral blood can be determined by comparing T cell counts in blood samples taken from the subject before and after antibody treatment. Dosage regimes can be adjusted over time to provide the optimum therapeutic response according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • the cytokine secretion profile of a recipient can be determined pre and/or post xenotransplantation.
  • cells of the recipient e.g., peripheral blood mononuclear cells
  • the cells are cocultured in vitro with cells from the same species as the donor.
  • the cells are from the same donor (e.g., are cells that were cryopreserved at the time of transplantation), or are from another donor that is genetically related to the donor of the transplanted cells.
  • the donor cells can be irradiated or fixed prior to coculturing them with the recipient responder cells using standard methods.
  • the lymphokines secreted by the recipient cells can be measured from the in vitro coculture supernatants using standard techniques, e.g., as described in the appended examples and as known in the art.
  • changes in cytokine levels can be determined using a commercially available ELISA kit (R&D systems Quantikine kit, Minneapolis, Minn.) or by bioassay using a cytokine dependent cell line, e.g., CTLL-2 (ATCC, Rockville, MD).
  • IFN-gamma can be measured using a kit available from Endogen (Cambridge, MA).
  • cytokine production can be measured using an ELISPOT assay (e.g., Williams et al. 1994. J. Infect. Disease.
  • Cytokine genes can also be measured (e.g., using PCR, RNA protection assays, or northern blot analysis). Cytokine production can be measured in either primary or secondary cocultures, e.g., as is known in the art and is described in Examples.
  • the polyclonal response of the recipient T cells can be measured.
  • Polyclonal T cell stimulators include, e.g., anti-CD3 antibody, and phorbol myristate acetate and ionomycin.
  • limiting dilution analysis can be performed to Thl and Th2 cells isolated and a determination of the precursor frequency of each type of cell can be calculated pre and post transplantation as an indication of the cytokine secretion profile of the recipient and whether or not the secretion profile has been altered towards a Th2 phenotype.
  • the cytokines produced by the recipient can also be measured in situ, e.g., by removing peripheral blood mononuclear cells or by taking a biopsy sample from the recipient and staining the cells for cytokines.
  • the cytokines can also be measured in situ, e.g., by removing peripheral blood mononuclear cells or by taking a biopsy sample from the recipient and staining the cells for cytokines.
  • fluorescence-conjugated antibodies to cell surface antigens using standard methods (e.g., U.S. patent 5,767,097).
  • the cells can be treated with Brefeldin A and stained with PE-labeled anti-IL-2 antibody and then washed in permeabilization buffer prior to FACS analysis.
  • nucleic acid molecules can be isolated from recipient cells, e.g., from recipient peripheral blood lymphocytes or from infiltrates into a graft site. mRNA levels can be measured or reverse transcribed into cDNA for amplification using the polymerase chain reaction prior to quantitation. PCR can be performed using parameters which have been optimized for the detection of cytokines. Primers for human cytokines are commercially available, e.g., from Stratagene, La Jolla, CA. Alternatively, mRNA encoding a particular cytokine can be measured by in situ hybridization.
  • Th2 and/or Thl cytokines to be detected are known in the art and can be found, for example, on GenBank or in the references set forth supra.
  • primers for amplifying these sequences and/or oligonucleotide probes that specifically bind to Thl and Th2 cytokine sequences are known in the art and can e.g., be chemically synthesized.
  • many are commercially available.
  • RNA protection assay demonstrated a clear inhibition of IL-15 message during anti-class I masking conditions both in the primary and secondary cultures. Since activated monocytes and dendritic cells are the only known producers of the potent T cell growth factor IL-15, our findings support a direct role in inhibiting not only the pathway of direct recognition but the indirect pathway which requires the presentation of donor antigen peptide on responder APC.
  • RNAzolTM B Following the manufacture's procedure (Tel-Test, Friends wood, TX). The first strands of cDNA from mRNA were synthesized using the Advantage RT-for-PCR TM following the manufacture's procedure (Clontech, Palo Alto, CA).
  • the cDNA of H2D ⁇ was amplified by PCR using a 5' primer containing an Xho I linker tail (CGA TCT CGA GAT GGG GGC GAT GGC TCC GCG CAC) (SEQ ID NO:l) and a 3' primer containing an Hind III linker tail (ATC GAA GCT TTC ACA CTT TAC AAT CTG GGA GAG) (SEQ ID NO:2) to facilitate cloning into pGEM-7Zf (Promega, Madison, WI).
  • the H2D ⁇ clone was sequenced to assure reliable amplification.
  • the PD1 gene was used as a template for the chimeric constructs.
  • the MMP construct: H2D ⁇ /pGEM-7Zf was linearized with Bsm I and partially digested with BsrD I in order to excise exon 4 portion of this gene.
  • the exon 4 of pig PD1 corresponding to the ⁇ 3 domain was amplified by PCR using primers that contained the junctions of mouse exon 3/pig exon 4 and pig exon 4/mouse exon 5.
  • the 5' primer also contains the Bsm I site for ligation to the mouse exon 3 (TCG ATC GAA TGC TAC GCT GCT GCG CAC AGA CCT TCC AAA GAC ACA TG) (SEQ ID NO:3) and the 3' primer contains the BsrD I site for ligation to the mouse exon 5 (A AC AGC AAT GAT TAC TGT GTT AGT CTT GGT GGA TGA AGG AGG CTC CCA TCT CAG GGT GAG) (SEQ ID NO:4).
  • the resulting PCR product was digested with Bsm I and BsrD I.
  • H2Dd/pGEM-7Zf The fragment was then ligated into the linearized H2Dd/pGEM-7Zf vector.
  • the presence of exon 4 of pig in this vector added a new restriction site (Bgl II) that was used for screening.
  • a clone containing the pig insert was sequenced and the new chimeric gene was transferred to the expression vector pcDNA3.TK (Invitrogen co ⁇ oration, Carlsbad, CA) at the Xho I and Hind III sites.
  • Exons 2-3 of PDl corresponding to the ⁇ l and ⁇ 2 domains were amplified by PCR using the 5' primer representing the junction mouse exon 1/pig exon 2 and containing the Not I site for insertion into H2Dd/pGEM-7Zf (GAT CGA TCG CGG CCG CCC TGG GTC CGA CTC AGA CCC GCG CTG GTC CCC ACT CCC TGA) (SEQ ID NO:5) and the 3' primer representing the junction pig exon 3/mouse exon 4 containing the site BstY I for ligation (TCG ATC GGA TCT GCG CGC TGC AGC GTG TCC TTC CCC) (SEQ ID NO:6).
  • the PCR product was treated in the same manner as for the MMP construct. Table 1 shows the structure of the chimeric proteins.
  • Stable transfectants were selected with G418 (Gibco/BRL, Grand Island, NY) at a concentration of 1,200 ⁇ g/ml for Balb/c fibroblasts and 800 ⁇ g/ml for C1498 for 2 weeks. Magnetic bead separation was performed on the PD I and MMP transfectants using monoclonal antibody 9-3 as the primary Ab, previously determined to be pig MHC Class I ⁇ 3 domain specific. Dynabeads M-450 coated with goat antimouse IgG (Dynal, Oslo, Norway) were used to select cells expressing either protein. Magnetic bead separation was performed as well on the H2D ⁇ and PPM transfectants by using 34-2-12 (PharMingen, San Diego, CA) as the primary Ab.
  • Clones were selected by limiting dilution in a 96-well plate. The selected cells were analyzed by flow cytometry using the Becton Dickinson FACScan. A variety of primary Abs were analyzed for their domain specificity on MHC class I using FITC conjugated goat anti-mouse IgG (H+L) secondary reagent for detection (Jackson Immunoresearch laboratory, Bar Harbor, ME).
  • SMC porcine smooth muscle cells
  • endothelial cell layer was removed and small pieces of tissue containing the SMC layer was cut and digested with equal volumes of collagenase-P (0.8 mg/ml) and trypsin-versene (BioWhittaker) at 37°C for 60 minutes.
  • the cell layer was washed several times and cultured with DMEM supplemented with 10% FCS in 100 mm tissue 5 culture dishes.
  • cells were harvested and replated at a 1 to 3 dilution. Based on mo ⁇ hology, greater than 98% of the cells used were SMC. Experiments were conducted using cells between passage 12 and 20.
  • peripheral blood mononuclear cells PBMC
  • PBMC peripheral blood mononuclear cells
  • Purified T cells, CD4+ and CD8+ T cells were enriched by negative selection using magnetic beads (Dynal, Oslo, Norway).
  • B cells, macrophages, and NK cells were removed by incubating the PBMC with mouse anti-human CD 14, CD 19, CD56, CD 16 followed by goat anti mlg magnetic beads.
  • T cells were incubated with anti-CD4 or anti-CD8 mAbs (5 ⁇ g/ml) at 4°C for 45 minutes.
  • Goat anti mouse IgG magnetic beads (Dynal, Oslo, Norway) were added at 10: 1 (beadxell) ratio and discarded unwanted cells.
  • the negatively enriched T cells, CD4+ or CD8+ T cell subsets were greater than 95% pure as determined by FACS analysis.
  • SMC proliferation assay SMC were plated in 96-well flat bottom microtiter plate at a density of 3x10 ⁇ cells/well at 37°C. Cells were incubated with media alone or with the blocking antibody (lO ⁇ g/ml) 1 hour at 4°C. Hybridomas producing the anti-porcine MHC class I antibodies PT-85 were purchased from VMRD, Inc. (Pullman WA; Davis et al. 1987. Vet. Immunol. Immunopathol 15:337).
  • Monoclonal antibody 9.3 was raised against porcine PBL as previously described (Oettinger et al. 1997. Xenotransplantation 4:252).
  • Hybridoma producing the anti- human MHC class I monoclonal antibody, W6/32, was obtained from ATCC (Barnstable. 1978. Cell 14:9).
  • Human PBMC were then added at 3xl0 5 cells/well, whereas human CD4 or CD8 T cells were added at lxl 0 ⁇ cells/well. All experiments were done in AIM V media (Gibco) supplemented with 5% heat inactivated FCS.
  • cells were harvested and restimulated with fresh SMC or immobilized anti human CD3 antibody (5 ⁇ g/ml) in 96-well flat bottom plates for the times indicated in text. Supernatants were harvested from appropriate cultures at indicated times and frozen for cytokine detection.
  • cells were pulsed with ⁇ H-thymidine ( 1 ⁇ Ci/well) for 20 hours, and harvested with a cell harvester (Packard Instruments, Meriden, CT)at indicated time points. The thymidine inco ⁇ oration was determined by counting the filter plate using a microplate scintillation counter (Packard Instrument, Model #B9906). FACS analysis.
  • Fluorescein isothiocyanate (FITC) or R-phycoerythrin (PE) conjugated monoclonal antibodies (mAb) to CD4, CD8, CD 14 and CD 19 were purchased from PharMingen (San Diego, CA). Cells (5x10 ⁇ ) were incubated with FITC or PE conjugated mAb for 60 minutes at 4°C. Cells were washed and analyzed by two-color flow cytometric analysis using a FACScan (Becton Dickson, Mountain View, CA). Viable cells were gated using propidium iodide (PI) at 2.5 ng/ml.
  • PI propidium iodide
  • the antibodies used in the IFN- ⁇ , IL-2, IL-4 and IL-10 assays were purchased from PharMingen (San Diego, CA). All assays were performed according to the manufacturer's protocol. Briefly, plates were coated with respective anti-cytokine mAb and culture samples were captured on plates, and detected by the respective biotin- labeled second anti-IFN- ⁇ anti-IL-2, anti-IL-4 or anti-IL-10 mAbs. The assay was developed by using strepavidin-horseradish peroxidase and substrate. The color reaction was stopped by addition of equal volume of 1M H2SO4. The absorbance of the assay plate was read at 450 nm using a microplate reader (Model 3550, Bio-Rad Labs,
  • Recombinant human IFN- ⁇ , IL-2, IL-4 and IL-10 cytokines were used as standards, respectively.
  • the sensitivity of the assay was determined to be between 1 pg/ml to 10 ng/ml for all cytokines.
  • RNase protection assay was performed using the RiboQuant multi-probe RNase protection assay kit according to the manufacturer's protocol (PharMingen). Briefly, the multi template probe was labeled with ( ⁇ -32P)UTP (3000 Ci/mmol, NEN) for 1 hour at 37°C. After DNase treatment and phenol-chloroform extraction, the probe was hybridized with sample RNAs overnight at 56°C. After RNase and proteinase K treatment and phenol-chloroform extraction, samples were precipitated with ammonium acetate and ethanol, and run on a 5% acrylamide gel (19:1 acrylamide/bis). The protected probes was resolved by exposing the gel to a X-ray film for an optimal time, and developed by a film processor. RESULTS:
  • Proliferation of human PBMC to class I positive cells Inhibition with SLA-class I reactive PT85 antibody.
  • SLA-class I reactive PT85 antibody Inhibition with SLA-class I reactive PT85 antibody.
  • a proliferation assay was established that measured human T cell response to porcine cells that expressed only MHC class I antigens.
  • Human T cell responses were measured using primary SMC from two partially inbred miniature pigs and isolated embryonic brain cells (EBC) from outbred pigs. Proliferation of human T cells against porcine SMC or EBC showed similar primary (day 7) profiles that were inhibited with the anti-class I PT85 antibody (Figure la).
  • the human PBMC responses to SMC from the SLA aa and SLA ⁇ d an d to EBC from outbred pigs were inhibited with PT85 by more than 50% compared to no antibody, control antibody or anti-human HLA class I W6/32 antibody.
  • restimulation as measured in a 3 day assay without additional antibody added showed hyporesponsiveness in the previously PT85 blocked cell group relative to controls ( Figure lb).
  • the HLA class I specific W6/32 mAb group was included as control in our model to help delineate anti-class I reactivity directly effecting the responding human T cells.
  • PBMC from a number of different donors were stimulated with porcine SMC with or without the blocking antibodies.
  • Primary stimulation was consistently inhibited by more than 50% in the human anti-porcine T cell response when blocked with the anti-class I PT85 F(ab')2 antibody but not with the W6/32 or mouse IgG F(ab')2 control antibodies in all twelve human blood donors ( Figure 2a).
  • Figure 2b In a secondary stimulation with porcine SMC in the absence of any blocking antibody, there was greater than 60% inhibition in proliferation by day 3 for the initially masked group.
  • the PBMC response was inhibited with PT85 by 50% and less so with 9.3 and 74-11-10 mAbs.
  • PT85 When isolated CD8+ T cells from different donors were tested, consistent proliferation was observed in response to porcine SMC that were inhibited with anti-class I mAb.
  • the CD8 + T cell response was completely inhibited with PT85 and 9.3 F(ab')2 as well as several other anti-porcine SLA class IlgG antibodies ( Figure 3).
  • the human HLA class I specific W6/32 F(ab')2 antibody did not inhibit the CD8 + T cell response.
  • CD4 + T cells were dependent on the CD8 + T cells for a direct response to the class I + SMC.
  • a mixing experiment using purified CD4 + and CD8 + T cells. No increase above the CD8 response was evident indicating that CD4 cells if contributing to the PBMC response were responding to antigen presented on human APCs by the indirect pathway.
  • the CD8+ T cell response was completely inhibited with PT-85 and 9.3 but not with W6/32 mAb F(ab')2.
  • several other mAbs including the 74-1 1-10 IgG completely inhibited the CD8+ T cell response.
  • this proliferation was completely inhibited with the anti-CD8 (OKT8), but not with the anti-CD4 (OKT4)mAb.
  • the purified CD8+ T cells(lxl0 5 ) responded to porcine MHC class I + SMC, in most experiments, the proliferation was lower than that seen with PBMC (3x10 ⁇ ). None of the primary PBMC response were inhibited completely. In contrast, purified CD4+ T cells showed no detectable proliferation.
  • CD8+ T cells may be the initial responders and that blocking these cells with anti-porcine MHC class I antibody prevents further stimulation mediated by the indirect pathway.
  • Domain specificity of the anti-MHC class I antibodies In order to delineate differences between several anti-MHC class I mAbs, porcine and murine "exon-shuffled" MHC class I transfected mouse cell lines were established to examine domain specificities of these antibodies.
  • the porcine and mouse MHC class I chimeric molecules were generated using ⁇ l, ⁇ 2 and ⁇ 3 domains from pig PDl (Sullivan et al. 1997. J. Immunol. 159:2318) and mouse H-2 D genes.
  • the recombinant genes were transfected and expressed in C1498 cells and Balb/c fibroblasts. Stable transfected lines were tested for reactivity with anti-porcine MHC class I mAbs. Only mAb 9.3 required the PDl ⁇ 3 domain for reactivity, while mAb PT85, 74.11.10 and 2.27.3 did not require the ⁇ 3 domain for reactivity.
  • These three Abs may require both the ⁇ l and/or ⁇ 2 domains for reactivities.
  • mAb 7.34.1 required the pig ⁇ 2 microglobulin for proper recognition of pig MHC class I molecules since it only reacted with PD 1 transfected mouse cells in the presence of pig, ⁇ 2-microglobulin found in pig serum.
  • Blocking with PT85 F(Ab')? antibody inhibits IL-2 and IFN- ⁇ and induces IL-4 and IL- 10 production To assess whether inhibition of proliferation in the primary and secondary human anti-porcine T cell responses were correlated with changes in cytokine production, the culture supernatants were examined for IL-2, IFN- ⁇ , IL-4 and IL-10 cytokine levels. Primary and secondary stimulation conditions were set up to harvest and measure the cytokine production profile of the different cultures. Tissue culture supernatants generated from human anti-porcine SMC stimulation assays from day 2 to day 6 were collected. Supernatants from at least five different donor PBMC were monitored for cytokine production by ELISA.
  • IL-2 production was evident in the supernatants of the unmasked human anti-porcine responses between days 2 to 5 (Figure 4).
  • the level of IL-2 was decreased by days 6 and 7 during culture primarily due to consumption by T cell proliferation.
  • supernatants from PT85 masked cell cultures showed decreased levels of IL-2 from day 2 to day 5. While there were differences in the levels of IL-2 detected from the different PBMC donors, all had a striking decrease in IL-2 levels.
  • the level of IFN- ⁇ production was inhibited in cultures that were PT85 masked ( Figure 5). Five individual donor PBMC were tested in response to porcine SMC with or without the PT85 blocking.
  • IL- 10 levels in the day 6 supernatant ranged between 15 pg/ml to 100 pg/ml depending on donor PBMC, no decrease in levels was observed. In fact, two of the samples showed moderate IL-10 increases during the primary stimulation ( Figure 6; top 2 panels). IL-4 levels were also tested in supernatants but found very low or undetectable levels in these primary cultures. In contrast to no antibody conditions, the control mouse IgG F(ab')2 or the anti-HLA class I W6/32 F(ab')2 antibodies showed little or no effect on all cytokines measured.
  • cytokines IL-2, IFN- gamma, IL-4 and IL-10 in day 2 culture conditions in a secondary stimulation assay using either 1) fresh porcine SMC, 2) anti-CD3 antibody or 3) anti-CD3/CD28 antibodies. These conditions were selected to examine both antigen specific and antigen non-specific secondary responses using anti-CD3 or anti-CD3 and anti-CD28 mAbs.
  • PBMC from four different donors were stimulated for 7 days (primary) with porcine SMC in the presence or absence of the masking PT85 antibody.
  • IL- 10 and IL-4 levels in the secondary stimulation increased in the previously PT85 masked group relative to the control groups.
  • Levels of IL-10 in 48 hour 0 culture supernatants using fresh identical porcine SMC were increased in 3 of 4 donors tested ( Figure 8).
  • increased levels of IL-10 and IL-4 were detected when secondary cultures were generated using anti-CD3/CD28 stimulatory antibodies.
  • IL-10 levels for donor 1 PT85 relative to control group increased from 200 pg/ml to 800 pg/ml with anti-CD3 restimulation and 600 pg/ml to 1200 pg/ml with anti- 5 CD3/CD28 restimulation (Figure 9).
  • the results from the secondary stimulation more strongly suggest a shift in the type 1/type 2 cytokine ratio shift since IL-2 and IFN-gamma decreased whereas IL-4 and IL-10 both maintained or increased their levels.
  • the levels of IL-4 were also increased in 3 of 3 donors when 5 restimulated with anti-CD3/CD28 mAbs and to a lesser degree in 4 of 4 donors when restimulated with anti-CD3 mAb alone ( Figure 10). All donors showed differences in IL-4 production between the masked and control treatments.
  • the results from secondary stimulation strongly suggest a shift in the typel/type2 cytokine ratio since 11-2 and IFN-gamma decreased whereas levels of IL-10 and IL-4 were maintained or 0 increased.
  • RNA protection assay confirms cytokine production profiles: RNA was isolated from primary and secondary cultures that were initially stimulated with SMC in the presence or absence of PT85. While RNA protection assay revealed significantly reduced levels of IL-2 and IFN-gamma message in the PT85 masking conditions, the change in the message levels of IL-4, IL-5 and IL-10 were not detected in the 48 hr time point tested. However, the level of IL-15 was reduced in the PT85 treatment conditions. Since IL-15 is a strong T cell growth factor and produced only by activated myeloid cells these data indicate that human antigen presenting cells (macrophages and dendritic cells) may be prevented from being activated in the presence of the PT85 masking.
  • CD8+ cells can secret low levels of IL-2 in addition to high levels of IFN- ⁇ (Fong, T.A. et al. 1990. J Immunol. 144:1744-52). It has also been demonstrated that CD8 + cells are capable of producing other regulatory cytokines including IL-4 and IL-10. Thus, if the early CD8 + T cell response is blocked in cellular xenotransplantation, it may influence the overall immune response and graft survival by altering the cytokine profile.
  • preventing or blocking CD 8 activation may have profound effect on the subsequent CD4+ T cell activation towards a type 2 phenotype (Williams, N.S. et al. J Immunol. 159:2091-9).
  • the alterations in cytokine profile seen in this study were comprised of a decrease in IFN- ⁇ and IL-2 production and an increased or sustained IL-10 secretion in the human anti-porcine response when PT-85, an anti-porcine MHC class I antibody, was used to block the response.
  • Such a change is consistent with a type 1 to type 2 shift, including an increase in IL-4 production although this was less easily measured than IL-10.

Abstract

La présente invention concerne un procédé d'amélioration d'acceptation de greffe chez un receveur par l'administration d'un agent modificateur du profile de la cytokine. Les procédés comprennent une modification de la réaction de la cytokine chez le receveur en la transformant en une réaction dominée par les cytokines Th2 soit en accroissant la production de la cytokine Th2 soit en réduisant la production de la cytokine Th1 par l'administration d'une cellule isolée et un agent modificateur du profile de la cytokine au sujet favorisant ainsi l'acceptation de la greffe chez un sujet.
PCT/US2000/005647 1999-03-05 2000-03-02 Procede d'amelioration d'acceptation de greffe chez un receveur par l'administration d'un agent modificateur du profile de la cytokine WO2000051630A2 (fr)

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CA002364279A CA2364279A1 (fr) 1999-03-05 2000-03-02 Procede d'amelioration d'acceptation de greffe chez un receveur par l'administration d'un agent modificateur du profile de la cytokine
EP00921362A EP1158999A2 (fr) 1999-03-05 2000-03-02 Procede d'amelioration d'acceptation de greffe chez un receveur par l'administration d'un agent modificateur du profile de la cytokine
AU41698/00A AU4169800A (en) 1999-03-05 2000-03-02 Methods for improving graft acceptance in a recipient by administration of a cytokine profile altering agent

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US26301199A 1999-03-05 1999-03-05
US09/263,011 1999-03-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022262496A1 (fr) * 2021-06-17 2022-12-22 Suzhou Fuse Biosciences Limited Molécules d'immunoconjugué et procédés et compositions associés

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140115728A1 (en) 2012-10-24 2014-04-24 A. Joseph Tector Double knockout (gt/cmah-ko) pigs, organs and tissues
US20180184630A1 (en) * 2015-06-26 2018-07-05 Indiana University Research & Technology Corporation Transgenic pigs with genetic modifications of sla

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5624823A (en) * 1991-11-22 1997-04-29 The General Hospital Corporation DNA encoding procine interleukin-10
WO1998042838A1 (fr) * 1997-03-25 1998-10-01 Morphogenesis, Inc. Cellules souches universelles
WO2000039294A1 (fr) * 1998-12-24 2000-07-06 Novartis Ag Cellules porcines incapables d'exprimer l'antigene cd40, et destinees a des xenogreffes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5624823A (en) * 1991-11-22 1997-04-29 The General Hospital Corporation DNA encoding procine interleukin-10
WO1998042838A1 (fr) * 1997-03-25 1998-10-01 Morphogenesis, Inc. Cellules souches universelles
WO2000039294A1 (fr) * 1998-12-24 2000-07-06 Novartis Ag Cellules porcines incapables d'exprimer l'antigene cd40, et destinees a des xenogreffes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BESSIS N ET AL: "Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-alpha)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13." CLINICAL AND EXPERIMENTAL IMMUNOLOGY, vol. 111, no. 2, February 1998 (1998-02), pages 391-396, XP000952993 ISSN: 0009-9104 *
EDGE A S ET AL: "Xenogeneic cell therapy: current progress and future developments in porcine cell transplantation." CELL TRANSPLANTATION, (1998 NOV-DEC) 7 (6) 525-39. REF: 181 , XP000952985 *
EDGE A S: "Current applications of cellular xenografts." TRANSPLANTATION PROCEEDINGS, (2000 AUG) 32 (5) 1169-71. REF: 37 , XP000952982 *
KUNZENDORF ULRICH ET AL: "Immunomodulation in experimental and clinical nephrology using chimeric proteins." KIDNEY & BLOOD PRESSURE RESEARCH, vol. 19, no. 3-4, 1996, pages 201-204, XP000952996 ISSN: 1420-4096 *
PAN, L. (1) ET AL: "Anti-MHC class I Ab inhibits the human anti- porcine response and shifts responder cells toward a type 2 cytokine profile." FASEB JOURNAL, (MARCH 15, 1999) VOL. 13, NO. 5 PART 2, PP. A1129. MEETING INFO.: ANNUAL MEETING OF THE PROFESSIONAL RESEARCH SCIENTISTS ON EXPERIMENTAL BIOLOGY 99 WASHINGTON, D.C., USA APRIL 17-21, 1999 FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENT, XP002150756 *
VANHOVE B ET AL: "GENETIC ENGINEERING IN THE PIG GENE KNOCKOUT AND ALTERNATIVE TECHNIQUES" ANNALS OF THE NEW YORK ACADEMY OF SCIENCES,US,NEW YORK ACADEMY OF SCIENCES, NEW YORK, NY, vol. 862, 30 December 1998 (1998-12-30), pages 28-36, XP000906935 ISSN: 0077-8923 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022262496A1 (fr) * 2021-06-17 2022-12-22 Suzhou Fuse Biosciences Limited Molécules d'immunoconjugué et procédés et compositions associés

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CA2364279A1 (fr) 2000-09-08

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