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WO1995021527A1 - Greffe de cellules souches - Google Patents

Greffe de cellules souches Download PDF

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Publication number
WO1995021527A1
WO1995021527A1 PCT/US1994/001616 US9401616W WO9521527A1 WO 1995021527 A1 WO1995021527 A1 WO 1995021527A1 US 9401616 W US9401616 W US 9401616W WO 9521527 A1 WO9521527 A1 WO 9521527A1
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WO
WIPO (PCT)
Prior art keywords
recipient
cells
mammal
donor
treatment
Prior art date
Application number
PCT/US1994/001616
Other languages
English (en)
Inventor
David H. Sachs
Megan Sykes
Original Assignee
The General Hospital Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The General Hospital Corporation filed Critical The General Hospital Corporation
Priority to PCT/US1994/001616 priority Critical patent/WO1995021527A1/fr
Priority to AU62681/94A priority patent/AU6268194A/en
Priority to PCT/US1994/005527 priority patent/WO1994026289A1/fr
Publication of WO1995021527A1 publication Critical patent/WO1995021527A1/fr
Priority to US09/710,971 priority patent/US6911220B1/en
Priority to US09/874,512 priority patent/US20020168348A1/en
Priority to US11/151,744 priority patent/US20060147428A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins

Definitions

  • the invention relates to tissue and organ transplantation and to the engraftment of stem cells.
  • the engraftment of exogenously supplied hematopoietic stem cells can be promoted by treating the recipient of the cells so as to induce hematopoietic space in the recipient.
  • Hematopoietic space is commonly induced by radiation, but can also be induced by anti class I antibodies or by myelosuppressive drugs.
  • WBC white blood cell
  • the invention features, a method of determining if a myelosuppressive or hematopoietic-space inducing treatment is sufficient to create hematopoietic space.
  • the method includes administering a myelosuppressive treatment to a recipient, and determining the level of white blood cells in the recipient, e.g., by determining the WBC count of the recipient, a depression in the level of white blood cells being indicative of the presence or induction of hematopoietic space.
  • the white blood cell level is compared to a white blood cell determination made prior to administration of myelosuppressive treatment; white blood cell levels are taken at two, three, four, or five time points after treatment; subsequent to determining the white blood cell level, hematopoietic stem cells are administered to the recipient; the stem cells are administered during the period in which the white blood cell level is depressed.
  • the recipient and the stem cell donor are from the same species; the recipient and the stem cell donor are from different, e.g., concordant or discordant, species; the recipient and the stem cell donor are from the same species; the recipient is a mammal, (preferably a mammal other than a mouse), e.g., a primate, e.g., a human; the donor is a mammal, (preferably a mammal other than a mouse), e.g., a primate, e.g., a non-human primate, or a swine.
  • the invention features, a method of promoting the engraftment of exogenously administered hematopoietic stem cells in a recipient.
  • the method includes: administering myelosuppressive or hematopoietic-space-inducing treatment to the recipient, determining the white blood cell level of the recipient, administering the hematopoietic stem cells to the recipient (preferably the stem cells are administered during the period in which the white blood cell level is depressed), and, optionally, implanting in the recipient, a graft from a donor.
  • the recipient and the stem cell donor are from the same species; the recipient and the stem cell donor are from different, e.g.
  • the recipient and the stem cell donor are from the same species;
  • the recipient is a mammal, (preferably a mammal other than a mouse), e.g., a primate, e.g., a human;
  • the donor is a mammal, (preferably a mammal other than a mouse), e.g., a primate, e.g., a non-human primate, or a swine.
  • the graft is from the same individual which donates the stem cells; the graft is from an individual which is syngeneic with the individual which donates the stem cells; the graft is from an individual which is MHC matched with the individual which donates the stem cells.
  • the invention features a method of inducing tolerance in a recipient mammal (preferably a mammal other than a mouse), e.g., a primate, e.g., a human, of a first species, to a graft from a mammal (preferably a mammal other than a mouse), e.g., a swine, e.g., a miniature swine, of a second species, which graft expresses a major histocompatibility complex (MHC) antigen.
  • the method includes inserting DNA encoding an MHC antigen of the second species into a hematopoietic stem cell of the recipient mammal and allowing the MHC antigen encoding DNA to be expressed in the recipient.
  • the method includes the step of, prior to administering the stem cells, administering a hematopoietic space inducing or myelosuppressive treatment, e.g., irradiation, e.g., whole body irradiation, to the recipient, monitoring the white cell level of said recipient after the administration of the space inducing treatment, and administering the stem cells to the recipient while the white cell level is depressed.
  • a hematopoietic space inducing or myelosuppressive treatment e.g., irradiation, e.g., whole body irradiation
  • space for said hematopoietic stem cells is created by irradiating the recipient mammal with low dose whole body irradiation.
  • the low dose whole body irradiation is more than 100 rads and less than 400 rads.
  • space can be created by administering to the recipient mammal anti- MHC class I antibodies or myelosuppressive drugs.
  • levels of white blood cells are monitored by white blood cell counts.
  • white blood cell counts in said recipient mammal are monitored for less than two weeks following creation of space within the recipient mammal. More preferably, white blood cell counts are monitored for less than one week.
  • Another embodiment includes the further step of selecting a time for administration of said hematopoietic stem cells when white blood cells levels are below normal in said recipient mammal.
  • the time for administering hematopoietic stem cells is within 4 or within 7 days following creation of space in the recipient mammal.
  • hematopoietic stem cells can be administered within 14 or within 21 days following creation of space in the recipient mammal.
  • the method includes administering to the recipient a short course of help reducing treatment, e.g., a short course of high dose cyclosporine treatment; the duration of the short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments the duration is approximately equal to or is less than two, three, four, five, or ten times the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • help reducing treatment e.g., a short course of high dose cyclosporine treatment
  • the duration of the short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments the duration is approximately equal to or is less than two, three, four, five, or ten times the period required for a mature T cell of the
  • the short course of help reducing treatment is administered in the absence of a treatment which stimulates the release of a cytokine by mature T cells in the recipient, e.g., in the absence of Prednisone or similar compounds.
  • the help reducing treatment is begun before or at about the time the graft is introduced; the short course is perioperative; or the short course is postoperative.
  • Preferred embodiments include those in which: the cell is removed from the recipient mammal prior to the DNA insertion and returned to the recipient mammal after the DNA insertion; the DNA is obtained from the individual mammal from which the graft is obtained; the DNA is obtained from an individual mammal which is syngeneic with the individual mammal from which the graft is obtained; the DNA is obtained from an individual mammal which is MHC matched, and preferably
  • the DNA includes an MHC class I gene; the DNA includes an MHC class II gene; the DNA is inserted into the cell by transduction, e.g., by a retrovirus, e.g., by a Moloney-based retrovirus; and the DNA is expressed in bone marrow cells and or peripheral blood cells of the recipient for at least 14, preferably 30, more preferably 60, and most preferably 120 days, after the DNA is introduced into the recipient.
  • the invention features a method of inducing tolerance in a recipient mammal, (preferably a mammal other than a mouse), e.g., a primate, e.g., a human, to a graft obtained from a donor of the same species, which graft expresses an MHC antigen.
  • a recipient mammal preferably a mammal other than a mouse
  • the method includes: inserting DNA encoding an MHC antigen of the donor into a bone marrow hematopoietic stem cell of the recipient; allowing the MHC antigen encoding DNA to be expressed in the recipient.
  • the method includes the step of, prior to administering the stem cells, administering a hematopoietic space inducing or myelosuppressive treatment, e.g., irradiation, e.g., whole body irradiation, to the recipient, monitoring the white cell level of said recipient after the administration of the space inducing treatment, and administering the stem cells to the recipient while the white cell level is depressed.
  • a hematopoietic space inducing or myelosuppressive treatment e.g., irradiation, e.g., whole body irradiation
  • space for said hematopoietic stem cells is created by irradiating the recipient mammal with low dose whole body irradiation.
  • the low dose whole body irradiation is more than 100 rads and less than 400 rads.
  • space can be created by administering to the recipient mammal anti- MHC class I antibodies or myelosuppressive drugs.
  • levels of white blood cells are monitored by white blood cell counts.
  • white blood cell counts in said recipient mammal are monitored for less than two weeks following creation of space within the recipient mammal. More preferably, white blood cell counts are monitored for less than one week.
  • Another embodiment includes the further step of selecting a time for administration of said hematopoietic stem cells when white blood cells levels are below normal in said recipient mammal.
  • the time for administering hematopoietic stem cells is within 4 or within 7 days following creation of space in the recipient mammal.
  • hematopoietic stem cells can be administered within 14 or within 21 days following creation of space in the recipient mammal.
  • the method further includes administering to the recipient a short course of help reducing treatment, e.g., a short course of high dose cyclosporine: the duration of the short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments the duration is approximately equal to or is less than two, three, four, five, or ten times the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • a short course of help reducing treatment e.g., a short course of high dose cyclosporine
  • the short course of help reducing treatment is administered in the absence of a treatment which stimulates the release of a cytokine by mature T cells in the recipient, e.g., in the absence of Prednisone or similar compounds.
  • the help reducing treatment is begun before or at about the time the graft is introduced; the short course is perioperative, the short course is postoperative; or the donor and recipient are class I matched.
  • Preferred embodiments include those in which: the cell is removed from the recipient prior to the DNA insertion and returned to the recipient after the DNA insertion; the DNA includes a MHC class I gene; the DNA includes a MHC class II gene; the DNA is inserted into the cell by transduction, e.g. by a retrovirus, e.g., by a Moloney-based retrovirus; and the DNA is expressed in bone marrow cells and/or peripheral blood cells of the recipient at least 14, preferably 30, more preferably 60, and most preferably 120 days, after the DNA is introduced into the recipient.
  • the invention features a method of inducing tolerance in a recipient mammal of a first species, (preferably a mammal other than a mouse) e.g., a primate, e.g., a human, to a graft obtained from a mammal of a second, preferably a concordant or discordant species, e.g., a swine, e.g., a miniature swine, or a discordant primate species.
  • a first species e.g., a mammal other than a mouse
  • a primate e.g., a human
  • a graft obtained from a mammal of a second
  • a concordant or discordant species e.g., a swine, e.g., a miniature swine, or a discordant primate species.
  • the method includes: prior to or simultaneous with transplantation of the graft, introducing, e.g., by intravenous injection, into the recipient mammal hematopoietic stem cells, e.g., bone marrow cells or fetal liver or spleen cells, of the second species (preferably the hematopoietic stem cells home to a site in the recipient mammal); and (optionally) inactivating the natural killer cells of said recipient mammal, e.g., by prior to introducing the hematopoietic stem cells into the recipient mammal, introducing into the recipient mammal an antibody capable of binding to natural killer cells of said recipient mammal.
  • hematopoietic stem cells e.g., bone marrow cells or fetal liver or spleen cells
  • the second species preferably the hematopoietic stem cells home to a site in the recipient mammal
  • inactivating the natural killer cells of said recipient mammal e.g., by prior
  • the method includes the step of, prior to administering the stem cells, administering a hematopoietic space inducing or myelosuppressive treatment, e.g., irradiation, e.g., whole body irradiation, to the recipient, monitoring the white cell level of said recipient after the administration of the space inducing treatment, and administering the stem cells to the recipient while the white cell level is depressed.
  • a hematopoietic space inducing or myelosuppressive treatment e.g., irradiation, e.g., whole body irradiation
  • space for said hematopoietic stem cells is created by irradiating the recipient mammal with low dose whole body irradiation.
  • the low dose whole body irradiation is more than 100 rads and less than 400 rads.
  • space can be created by administering to the recipient mammal anti- MHC class I antibodies or myelosuppressive drugs.
  • levels of white blood cells are monitored by white blood cell counts.
  • white blood cell counts in said recipient mammal are monitored for less than two weeks following creation of space within the recipient mammal. More preferably, white blood cell counts are monitored for less than one week.
  • Another embodiment includes the further step of selecting a time for administration of said hematopoietic stem cells when white blood cells levels are below normal in said recipient mammal.
  • the time for administering hematopoietic stem cells is within 4 or within 7 days following creation of space in the recipient mammal.
  • hematopoietic stem cells can be administered within 14 or within 21 days following creation of space in the recipient mammal.
  • the method includes administering to the recipient a short course of help reducing treatment, e.g., a short course of high dose cyclosporine; the duration of the short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments the duration is approximately equal to or is less than two, three, four, five, or ten times, the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • a short course of help reducing treatment e.g., a short course of high dose cyclosporine
  • the duration of the short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen
  • the duration is approximately equal to or is less than two, three, four, five, or ten times, the period required for a mature T cell of
  • the short course of help reducing treatment is administered in the absence of a treatment which stimulates the release of a cytokine by mature T cells in the recipient, e.g., in the absence of Prednisone or similar compounds.
  • the help reducing treatment is begun before or at about the time the graft is introduced; or the short course is perioperative, the short course is postoperative.
  • the hematopoietic cells prepare the recipient for the graft that follows, by inducing tolerance at both the B-cell and T-cell levels.
  • hematopoietic cells are fetal liver or spleen, or bone marrow cells, including immature cells (i.e., undifferentiated hematopoietic stem cells; these desired cells can be separated out of the bone marrow prior to administration), or a complex bone marrow sample including such cells can be used.
  • immature cells i.e., undifferentiated hematopoietic stem cells; these desired cells can be separated out of the bone marrow prior to administration
  • a complex bone marrow sample including such cells can be used.
  • anti-NK antibody is anti-human thymocyte polyclonal anti-serum.
  • a second, anti-mature T cell antibody can be administered as well, which lyses T cells as well as NK cells. Lysing T cells is advantageous for both bone marrow and xenograft survival.
  • Anti-T cell antibodies are present, along with anti-NK antibodies, in anti-thymocyte anti-serum. Repeated doses of anti-NK or anti-T cell antibody may be preferable.
  • Monoclonal preparations can be used in the methods of the invention.
  • kits for introducing into the recipient mammal include: the step of introducing into the recipient mammal, donor species-specific stromal tissue, preferably hematopoietic stromal tissue, e.g., fetal liver or thymus.
  • the stromal tissue is introduced simultaneously with, or prior to, the hematopoietic stem cells; the hematopoietic stem cells are introduced simultaneously with, or prior to, the antibody.
  • Other preferred embodiments include those in which: the same mammal of the second species is the donor of one or both the graft and the hematopoietic cells; and the antibody is an anti-human thymocyte polyclonal anti-serum, obtained, e.g., from a horse or pig.
  • Other preferred embodiments include: the step of prior to hematopoietic stem cell transplantation, irradiating the recipient mammal with low dose, e.g., between about 100 and 400 rads, whole body irradiation to deplete or partially deplete the bone marrow of said recipient; and the step of prior to hematopoietic stem cell transplantation, inactivating T cells in the thymus, by, e.g., administration of anti-T cell antibodies or by irradiating the recipient mammal with, e.g., about 700 rads of thymic irradiation.
  • kits for prior to hematopoietic stem cell transplantation include: the step of prior to hematopoietic stem cell transplantation, depleting natural antibodies from the blood of the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney, obtained from a mammal of the second species. (In organ hemoperfusion antibodies in the blood bind to antigens on the cell surfaces of the organ and are thus removed from the blood.)
  • the method further includes, prior to hematopoietic stem cell transplantation, introducing into the recipient an antibody capable of binding to mature T cells of said recipient mammal.
  • Other preferred embodiments further include the step of introducing into the recipient a graft obtained from the donor, e.g., a graft which is obtained from a different organ than the hematopoietic stem cells, e.g., a liver or a kidney.
  • the invention features a method of inducing tolerance in a recipient mammal, (preferably a mammal other than a mouse) e.g., a primate, e.g., a human, to a graft obtained from a donor, e.g., of the same species.
  • the method includes: prior to or simultaneous with transplantation of the graft, introducing, e.g., by intravenous injection, into the recipient hematopoietic stem cells, e.g.
  • bone marrow cells or fetal liver or spleen cells, of a mammal preferably the donor (preferably the hematopoietic stem cells home to a site in the recipient); and (optionally) inactivating the natural killer cells or T cells (or both) of the recipient, e.g., by prior to introducing the hematopoietic stem cells into the recipient, introducing into the recipient an antibody capable of binding to natural killer cells or T cells (or both) of the recipient.
  • the method includes the step of, prior to administering the stem cells, administering a hematopoietic space inducing or myelosuppressive treatment, e.g., irradiation, e.g., whole body irradiation, to the recipient, monitoring the white cell level of said recipient after the administration of the space inducing treatment, and administering the stem cells to the recipient while the white cell level is depressed.
  • a hematopoietic space inducing or myelosuppressive treatment e.g., irradiation, e.g., whole body irradiation
  • space for said hematopoietic stem cells is created by irradiating the recipient mammal with low dose whole body irradiation.
  • the low dose whole body irradiation is more than 100 rads and less than 400 rads.
  • space can be created by administering to the recipient mammal anti- MHC class I antibodies or myelosuppressive drugs.
  • levels of white blood cells are monitored by white blood cell counts.
  • white blood cell counts in said recipient mammal are monitored for less than two weeks following creation of space within the recipient mammal. More preferably, white blood cell counts are monitored for less than one week.
  • Another embodiment includes the further step of selecting a time for administration of said hematopoietic stem cells when white blood cells levels are below normal in said recipient mammal.
  • the time for administering hematopoietic stem cells is within 4 or within 7 days following creation of space in the recipient mammal.
  • hematopoietic stem cells can be administered within 14 or within 21 days following creation of space in the recipient mammal.
  • the method includes administering to the recipient a short course of help reducing treatment, e.g., a short course of high dose cyclosporine: the duration of the short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments the duration is approximately equal to or is less than two, three, four, five, or ten times the period required for a mature T cell of the recipient species, to initiate re ⁇ ection of an antigen after first being stimulated by the antigen.
  • a short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments the duration is approximately equal to or is less than two, three, four, five, or ten times the period required for a mature T cell of the recipient species, to initiate re ⁇ ection of an antigen after first being stimulated by the antigen
  • the short course of help reducing treatment is administered in the absence of a treatment which stimulates the release of a cytokine by mature T cells in the recipient, e.g., in the absence of Prednisone or similar compounds.
  • the help reducing treatment is begun before or at about the time the graft is introduced; the short course is perioperative, the short course is postoperative; the donor and recipient are class I matched.
  • the hematopoietic stem cells are introduced simultaneously with, or prior to administration of the antibody; the antibody is an anti- human thymocyte polyclonal anti-serum; and the anti-serum is obtained from a horse or pig.
  • Other preferred embodiments include: the further step of, prior to hematopoietic stem cell transplantation, introducing into the recipient mammal an antibody capable of binding to mature T cells of the recipient mammal; and those in which the same individual is the donor of both the graft and the bone marrow.
  • Other preferred embodiments include: further including the step of prior to hematopoietic stem cell transplantation, irradiating the recipient with low dose, e.g., between about 100 and 400 rads, whole body irradiation to completely or partially deplete the bone marrow of the recipient; and further including the step of prior to hematopoietic stem cell transplantation, inactivating T cells in the thymus, by, e.g., administration of anti-T cell antibodies or by irradiating the recipient with, e.g., about 700 rads of, thymic irradiation.
  • kits for treating diseases and conditions include: the further step prior of to bone marrow transplantation, absorbing natural antibodies from the blood of the recipient by hemoperfusing an organ, e.g., the liver, or a kidney, obtained from the donor.
  • Preferred embodiments include: the step of introducing into the recipient mammal, donor speciesspecific stromal tissue, preferably hematopoietic stromal tissue, e.g., fetal liver or thymus.
  • Other preferred embodiments further include the step of introducing into the recipient, a graft obtained from the donor, e.g., a graft which is obtained from a different organ than the hematopoietic stem cells, e.g., a liver or a kidney.
  • the invention further provides a method for reducing or eliminating immature or mature thymic T cells without thymic irradiation in a recipient mammal comprising administering to the recipient mammal at least one T cell ablative or T cell-depleting antibody at a dose sufficient to reduce or eliminate immature or mature thymic T cells.
  • T cell ablative antibodies are administered to the recipient prior to administering an allograft to the recipient.
  • Preferred T cell ablative antibodies are anti-CD4 and anti-CD8 antibodies.
  • the anti-CD4 and anti-CD8 antibodies are coadministered to the recipient.
  • Help reduction means the reduction of T cell help by the inhibition of the release of at least one cytokine, e.g. , any of IL-2, IL-4, IL-6, gamma interferon, or TNF, from T cells of the recipient at the time of the first exposure to an antigen to which tolerance is desired.
  • the inhibition induced in a recipient's T cell secretion of a cytokine must be sufficient such that the recipient is tolerized to an antigen which is administered during the reduction of help.
  • help reducing agent is an agent, e.g., an immunosuppressive drug, which results in the reduction of cytokine release.
  • help reducing agents are cyclosporine, FK-506, and rapamycin.
  • Anti-T cell antibodies because they can eliminate T cells, are not preferred for use as help reducing agents.
  • a help reducing agent must be administered in sufficient dose to give the level of inhibition of cytokine release which will result in tolerance.
  • the help reducing agent should be administered in the absence of treatments which promote cytokine, e.g., IL-2, release.
  • Putative agents help reducing agents can be prescreened by in vitro or in vivo tests, e.g., by contacting the putative agent with T cells and determining the ability of the treated T cells to release a cytokine, e.g., IL-2. The inhibition of cytokine release is indicative of the putative agent's efficacy as a help reducing agent. Such prescreened putative agents can then be further tested in a, kidney transplant assay.
  • a putative help reducing agent is tested for efficacy by administering the putative agent to a recipient monkey and then implanting a kidney from a Class II matched Class I and minor antigen mismatched donor monkey into the recipient. Tolerance to the donor kidney (as indicated by prolonged acceptance of the graft) is indicative that the putative agent is, at the dosage tested, a help reducing agent.
  • “Short course of a help reducing agent”, as used herein, means a transitory non-chronic course of treatment.
  • the treatment should begin before or at about the time of transplantation of the graft.
  • the treatment can begin before or at about the time of the recipient's first exposure to donor antigens.
  • the treatment lasts for a time which is approximately equal to or less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • the duration of the treatment can be extended to a time approximately equal to or less than two, three, four, five, or ten times, the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • the duration will usually be at least equal to the time required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • about 12 days of treatment is sufficient.
  • Experiments with cyclosporine A (10 mg/kg) in pigs show that 6 days is not sufficient.
  • Other experiments in monkeys show that IL-2 administered on day 8, 9, or 10 of cyclosporine A treatment will result in rejection of the transplanted tissue. Thus, 8, 9, or 10 days is probably not sufficient in pigs.
  • a dose of lOmg/kg cyclosporine with a blood level of about 500-1,000 ng/ml is sufficient to induce tolerance to Class II matched Class I and minor antigen mismatched kidneys.
  • Tolerance refers to the inhibition of a graft recipient's immune response which would otherwise occur, e.g., in response to the introduction of a nonself MHC antigen into the recipient. Tolerance can involve humoral, cellular, or both humoral and cellular responses. The term tolerance refers to states characterized by total or partial inhibition of the immune response.
  • Hematopoietic stem cell refers to a cell which is capable of developing into a mature myeloid and/or lymphoid cell.
  • Stem cells derived from the cord blood of the recipient or the donor can be used in methods of the invention. See U.S. Patent 5,192,553, hereby incorporated by reference, and U.S. Patent 5,004,681, hereby incorporated by reference.
  • Other sources of stem cells are bone marrow, fetal liver, fetal spleen, neonatal liver, and neonatal spleen.
  • MHC antigen refers to a protein product of one or more MHC genes; the term includes fragments or analogs of products of MHC genes which can evoke an immune response in a recipient organism.
  • MHC antigens include the products (and fragments or analogs thereof) of the human MHC genes, i.e., the HLA genes.
  • MHC antigens in swine, e.g., miniature swine include the products (and fragments and analogs thereof) of the SLA genes, e.g., the DRB gene.
  • Miniature swine refers to wholly or partially inbred animal.
  • Graft refers to a body part, organ, tissue, or cells.
  • Grafts may consist of organs such as liver, kidney, heart or lung; body parts such as bone or skeletal matrix; tissue such as skin, intestines, endocrine glands; or progenitor stem cells of various types.
  • a discordant species combination refers to two species in which hyperacute rejection occurs when a graft is grafted from one to the other. Generally, discordant species are from different orders, while non-discordant species are from the same order. For example, rats and mice are non-discordant species, i.e. their MHC antigens are substantially similar, and they are members of the same order, rodentia.
  • Hematopoietic space refers to the capacity of a mammal to accept engraftment of exogenously supplied hematopoietic stem cells. Hematopoetic space is an induced condition and it exists when a treatment renders the recipient more receptive to engraftment of exogenously administered hematopoietic stem cells.
  • Myelosuppressive treatment refers to any treatment which renders a recipient more susceptible to engraftment of exogenously administered stem cells or which kills or inhibits the maintenance, growth, or proliferation of stem cells. Irradiation, antibodies, or drugs or chemicals can be used as myelosuppressive agents.
  • Figure 1 A is a graph depicting the number of white blood cells in the peripheral blood of irradiated mice on days following irradiation.
  • Cell counts are from the following mice: (•) Untreated B6; ( ⁇ ) Untreated B6.Ly-5.2; ( ) B6 mice injected with 1.5 x 10 7 B6.Ly-5.2 BMC; (_• ⁇ _) B6 mice irradiated with 0.5 Gy and injected with 1.5 x 10? B6.Ly-5.2 BMC;
  • Figure IB is a graph depicting hemoglobin levels in the peripheral blood of irradiated mice on days following irradiation. Mice are as described in Figure IA.
  • Figure IC is a graph depicting platelet counts in the peripheral blood of irradiated mice on days following irradiation. Mice are as described in Figure IA. Overview
  • the invention provides several methods of inducing tolerance to foreign antigens, e.g., to antigens on allogeneic or xenogeneic tissue or organ grafts. These methods can be used individually or in combination.
  • a depression in WBC corresponds to a window for stem cell engraftment
  • myelosuppressive treatment e.g., whole body irradiation (WBI)
  • WBI whole body irradiation
  • WBI whole body irradiation
  • hematopoietic stem cells can be successfully transplanted within this "window" of time when space for donor hematopoietic stem cells persists in the recipient.
  • white blood cell counts decrease from normal levels following WBI of the recipient during the permissive period when bone marrow engraftment can be successfully accomplished.
  • White blood cells counts in the recipient gradually increase back to normal levels following WBI, although the window for bone marrow engraftment can persists for a period of time after WBC counts have returned to normal in the recipient.
  • a minimal dose of WBI which can still prepare the recipient for bone marrow engraftment is used.
  • a preferred low dose of irradiation is between about 100 to 400 rads WBI.
  • Successful bone marrow engraftment can be achieved if bone marrow transplantation is performed on the same day as WBI (day 0).
  • high level, long term (i.e., stable) donor-type repopulation can also be achieved if bone marrow transplantation is performed as many as about seven days following WBI (i.e., bone marrow transplantation can be performed between day 0 and day 7 following WBI).
  • Somewhat less successful repopulation i.e., mixed chimerism
  • bone marrow transplantation is performed up to 14 days following WBI.
  • Somewhat less successful repopulation i.e., mixed chimerism
  • bone marrow transplantation is performed up to 21 days following WBI.
  • White blood cell counts in the recipient can be used as an indicator of the permissive period for bone marrow engraftment following WBI. It has been found that WBC counts in the recipient decline between about day 2 and day 7 following WBI at a dose sufficient to allow engraftment, whereas WBI at a lower dose does not result in decreased WBC counts following irradiation or successful engraftment. Accordingly, WBC counts can serve as a marker for the presence of space for HSC engraftment and can be monitored to monitor space for HSC engraftment in the recipient. Preferably, WBC counts are monitored within about two weeks following irradiation, since counts in the recipient return to normal levels by about day 14 following irradiation.
  • engraftment can generally be achieved at just after irradiation. It may, however, be desirable to delay administration of stem cells.
  • WBC counts can be monitored by obtaining a blood sample from the recipient and counting WBCs within the sample by standard techniques. WBCs can be counted manually (e.g., with a hemocytometer) or more preferably are counted using an automated cell counter (e.g., System 9000; Serono-Baker Diagnostics Inc., Allentown, PA). WBC counts can be compared to a known standard to determine whether the WBC count at a particular time following WBI (or other suitable procedure to create space) is normal, below normal or above normal. Normal WBC counts for different species are known in the art.
  • a WBC count for the recipient can be determined prior to WBI, and if necessary prior to other treatments of the recipient which could affect WBC counts (e.g., administration of ablative antibodies), and this value can be used as a normal WBC count standard against which other WBC counts can be compared.
  • Procedures alternative to WBI can be used to create "space" for donor b hematopoietic stem cells within a recipient.
  • space can be created by treating the recipient with a monoclonal antibody against MHC class I antigens expressed by the recipient (see e.g., Voralia, M. et al. (1987) Transplantation 44:487) or space can be created by treating the recipient with myelosuppressive drugs (see e.g., Lapidot, T. et al. (1990) Proc. Natl. Acad. Sci. USA £2:4595).
  • space created within the recipient for bone marrow transplantation by other mechanisms e.g., anti-MHC class I treatment or myelosuppressive drugs
  • the method of the invention further comprises introducing into the recipient mammal at least one antibody capable of binding to mature or immature T cells of the recipient mammal at a dose sufficient to induce tolerance to a graft without thymic irradiation of the recipient mammal.
  • the preferred T cell ablative antibody treatment procedure comprises the combined use of antibodies directed against CD4 and CD8 molecules on the surface of T cells (i.e., combined administration of an anti-CD4 antibody and an anti-CD8 antibody).
  • T cell ablative antibodies to be used instead of thymic irradiation can be determined by treating a recipient mammal with increasing dosages of one or more T cell ablative antibodies (e.g., an anti-CD4 antibody and an anti-CD8 antibody used in combination) in the absence of thymic irradiation and determining the dose of antibody needed to induce tolerance to an allograft, using transplantation procedures disclosed in the Examples.
  • T cell ablative antibodies e.g., an anti-CD4 antibody and an anti-CD8 antibody used in combination
  • zero time is defined as the moment that the arterial and venous cannulas of the recipient are connected to the liver to be perfused (note zero time is chosen to fall within the window of white blood cell level, as described herein).
  • ATG horse anti-human anti-thymocyte globulin
  • ATG eliminates mature T cells and natural killer cells that would otherwise cause rejection of the bone marrow cells used to induce tolerance.
  • the recipient is anesthetized, an IV catheter is inserted into the recipient, and 6 ml of heparinized whole blood are removed before injection.
  • the ATG preparation is then injected (50 mg/kg) intravenously.
  • Six ml samples of heparinized whole blood are drawn for testing at time points of 30 min., 24 hrs and 48 hrs.
  • Blood samples are analyzed for the effect of antibody treatment on natural killer cell activity (testing on K562 targets) and by FACS analysis for lymphocyte subpopulations, including CD4, CD8, CD3, CDUb, and CD16.
  • Preliminary dat ⁇ from both assays indicate that both groups of cells are eliminated by the administration of ATG. If mature T cells and NK cells are not eliminated, ATG can be re-administered at later times in the procedure, both before and after organ transplantation.
  • Sublethal irradiation is administered to the recipient between days -1 and -8. Irradiation is necessary to eliminate enough of the recipient's endogenous BMC to stimulate hematopoiesis of the newly introduced foreign BMC. Sublethal total body irradiation is sufficient to permit engraftment with minimal toxic effects to the recipient.
  • Whole body radiation 150 Rads
  • TRBC bilateral cobalt teletherapy unit at 10 Rads/min.
  • the recipients white blood cell count is determined on a daily basis. A depression in the count corresponds to the onset of the window for stem cell engraftment.
  • Natural antibodies are a primary cause of organ rejection.
  • an operative absorption of natural antibodies is performed, using a miniature swine liver, as follows. At -90 minutes the swine donor is anesthetized, and the liver prepared for removal by standard operative procedures. At -60 minutes the recipient monkey is anesthetized. A peripheral IV catheter is inserted, and a 6 ml sample of whole blood is drawn. Through mid-line incision, the abdominal aorta and the vena cava are isolated. Silastic cannulas containing side ports for blood sampling are inserted into the blood vessels.
  • the liver is perfused in situ until it turns pale, and then removed from the swine donor and placed into cold Ringers Lactate. The liver is kept cold until just prior to reperfusion in the monkey. A liver biopsy is taken. At -10 minutes the liver is perfused with warm albumin solution until the liver is warm (37 degrees). At 0 time the arterial and venous cannulas of the recipient are connected to the portal vein and vena cava of the donor liver and perfusion is begun. Liver biopsies are taken at 30 minutes and 60 minutes, respectively. Samples of recipient blood are also drawn for serum at 30 minutes and 60 minutes respectively. At 60 minutes the liver is disconnected from the cannulas and the recipient's large blood vessels are repaired.
  • the liver having served its function of absorbing harmf l natural antibodies from the recipient monkey, is discarded. Additional blood samples for serum are drawn from the recipient at 2, 24, and 48 i hours. When this procedure was performed on two sequential perfusions of swine livers, the second liver showed no evidence of mild ischemic changes during perfusion. At the end of a 30 minute perfusion the second liver looked grossly normal and appeared to be functioning, as evidenced by a darkening of the venous o ⁇ tflow blood compared to the arterial inflow blood in the two adjacent cannulas. Tissue sections from the livers were normal, but immunofluorescent stains showed IgM on endothelial cells. Serum samples showed a decrease in natural antibodies.
  • donor bone marrow cells are administered to the recipient to form chimeric bone mairow.
  • the presence of donor antigens in the bone marrow allows newly developing B cells, and newly sensitized T cells, to recognize antigens of the donor as self, and thereby induces tolerance for the implanted organ from the donor.
  • donor stromal tissue in the form of tissue slices of fetal liver, thymus, and/or fetal spleen are transplanted under the kidney capsule of the recipient.
  • Stromal tissue is preferably implanted simultaneously with, or prior to, administration of hematopoietic stem cells, e.g., BMC, or a fetal liver cell suspension.
  • BMC can in turn be injected either simultaneously with, or preceding, organ transplant. Bone marrow is harvested and injected intravenously (7.5 x 108/kg) as previously described (Pennington et al., 1988, transplantation 45:21-26). Should natural antibodies be found to recur before tolerance is induced, and should these antibodies cause damage to the graft, the protocol can be modified to permit sufficient time following BMT for humoral tolerance to be established prior to organ grafting.
  • the approaches described above are designed to synerglst-cally prevent the problem of transplant rejection.
  • a kidney is implanted into a cynomolgus monkey following liver absorption of natural antibodies, without use of bone marrow transplantation to induce tolerance, renal functions continued for 1-2 days before rejection of the kidney.
  • foru steps of the procedure were performed (absorption of natural antibodies by liver perfusion, administration of ATG, sublethal irradiation and bone marrow infusion, followed by implant of a procine kidney into a primate recipient), the kidney survived 7 days before rejection. Despite rejection of the transplanted organ, the recipient remained healthy.
  • the methods of the invention may be employed in combination, as described, or in part.
  • the method of introducing bone marrow cells may be altered, particularly by (1) increasing the time interval between injecting hematopoietic stem cells and implanting the graft; (2) increasing or decreasing the amount of hematopoietic stem cells injected; (3) varying the number of hematopoietic stem cell injections; (4) varying the method of delivery of hematopoietic stem cells; (5) varying the tissue source of hematopoietic stem cells, e.g., a fetal liver cell suspension may be used; of (6) varying the donor source of hematopoeitic stem cells.
  • tissue source of hematopoietic stem cells e.g., a fetal liver cell suspension may be used
  • hematopoietic stem cells may be obtained from other individuals or species, or from genetically- engineered inbred donor strains, or from in vitro cell culture.
  • Methods of preparing the recipient for transplant of hematopoeitic stem cells Amy be varied. For instance, the recipient may undergo a splenectomy or a thymectomy. The latter would preferably by administered prior to the non- myeloablative regimen, e.g., at day -14.
  • Hemoperfusion of natural antibodies may: (1) make use of other vascular organs, e.g., liver, kidney, intestines; (2) make use of multiple sequential organs; (3) vary the length of time each organ is perfused; (4) vary the donor of the perfused organ.
  • Irradiation of the recipient may make use of: (1) varying the absorbed dose of whole body radiation below the sublethal range; (2) targeting different body parts (e.g., thymus, spleen); (3) varying the rate of irradiation (e.g., 10 rads/min, 15 rads/min); or (4) varying the time interval between irradiation and transplant of hematopoeitic stem cells; any time interval between 1 and 14 days can be used, and certain advantages may flow from use of a time interval of 4-7 days.
  • Antibodies introduced prior to hematopoietic cell transplant may be varied by: (1) using monoclonal antibodies to T cell subsets or NK cells (e.g., anti-NKHl _ , as described by United States Patent No. 4,772,552 to Hercend, et al., hereby incorporated by reference); (2) preparing anti-human ATB in other mammalian hosts (e.g., monkey, pig, rabbit, dog); or (3) using anti-monkey ATG prepared in any of the above mentioned hosts.
  • monoclonal antibodies to T cell subsets or NK cells e.g., anti-NKHl _ , as described by United States Patent No. 4,772,552 to Hercend, et al., hereby incorporated by reference
  • preparing anti-human ATB in other mammalian hosts e.g., monkey, pig, rabbit, dog
  • anti-monkey ATG prepared in any of the above mentioned hosts.
  • the methods of the invention may be employed with other mammalian recipients (e.g., rhesus monkeys) and may use other mammalian donors (e.g., primates, sheep, or dogs).
  • mammalian recipients e.g., rhesus monkeys
  • mammalian donors e.g., primates, sheep, or dogs
  • host antibodies can be depleted by administration of an excess of hematopoietic cells.
  • WBI whole body irradiation
  • HSC hematopoietic stem cell
  • Ly-5 is a leukocyte common antigen expressed on all hematopoietic lineages
  • the origin (donor versus host) of myeloid and lymphoid cells can be followed over time and thus pluripotent HSC engraftment can be evaluated.
  • Ly-5 alleles have been shown not to elicit alloresistance or graft rejection (Sykes, M. et al. (1989) J. Immunol. 143:3503), so engraftment can be evaluated in the absence of alloreactivity. Engraftment was examined following various doses of WBI. Additionally, the time period ("window") for engraftment following WBI was examined.
  • mice Female C57BL/6NCR (B6;h-2 b , Ly-5.2) and Ly-5 congenic B6.Ly-5.2 (Ly-5.1) mice were obtained from the Frederick Cancer Research Facility, Frederick, MD. Ly-5 alleles are described according to the nomenclature of Morse et al. (Immunogenetics (1987) 25:71). All mice were housed in sterilized microisolator cages, in which they received autoclaved food and autoclaved acidified drinking water. Recipients were age-matched and were used at 12-16 weeks of age.
  • BMT was performed as previously described (Sykes, M. et al. (1993) J. Immunol. 150:197). Briefly, recipient B6 mice were irradiated with various doses (0.5-9.5 Gy) (137 Cs source, approximately 1.0 Gy/min) and reconstituted with T cell-depleted (TCD) Ly-5.1 BMC (1.5 x 10 7 unless indicated otherwise), obtained from the tibiae and femora of sex-matched B6.Ly-5.2 donors aged 6-14 weeks. T cell- depletion was performed as described (Morse et al. (1987) Immunogenetics 25:71) using anti-CD4 (Dialynas, D.P. et al. (1983) J. Immunol. 131:2445) and CD8 (Sarmiento, M. (1980) J. Immunol. 125:2665) mAb and C.
  • Phenotyping of chimeras Phenotyping was performed at various times beginning 2 weeks following BMT. Animals were tail bled and white blood cells (WBC) were prepared by hypotonic shock. Suspensions of spleen cells, thymocytes, BMC and bone marrow colonies were also analyzed. Staining with both donor- specific and recipient-specific mAb was performed on each chimera and control animal. Cells were incubated with 20 ⁇ l undiluted culture supernatant of A20-1.7 (anti-Ly-5.1 mAb; mouse IgG2a) or 104-2.1 (anti Ly-5.2 mAb; mouse IgG2a) for 30 minutes at 4°C, then washed twice.
  • WBC white blood cells
  • the percentage of cells staining with each mAb was determined from one-color fluorescence histograms and comparison with those obtained from normal donor and host-type animals, which were used as positive and negative controls.
  • the percentage of cells considered positive after staining with a mAb was determined using a cutoff chosen as the fluorescence level at the beginning of the positive peak for the positive control strain, and by subtracting the percentage of cells stained with an irrelevant mAb (non-reactive IgG2a mAb HOPC1 plus FITC- conjugated anti-mouse IgG2a mAb).
  • the relative percent staining of a chimera with mAb was calculated using the formula:
  • Ly5.1 + and Ly5.2 + mice For test cell populations in which staining with an anti-Ly5 mAb was less than that of the negative control, and the calculated percent chimerism was therefore less than 0, the values are reported as 0. Using this method of calculation, less than 0.1% contaminating Ly5.1 + cells could be detected in artificial Ly5.2(99.9%)/Ly5.1(0.1%) mixtures. However, a visible positive peak was not detectable in artificial mixtures containing 0.1% or fewer Ly5.1 + cells, but was visible with 1% contaminating Ly5.1 + cells (data not shown). All hematopoietic lineages stained strongly with anti-Ly-5-mAblO.
  • lymphocyte FSC- and SSC-low population
  • granulocyte SSC-high population
  • monocyte FSC-high but SSC-low population
  • All SSC-high cells in the granulocyte gate stained with FITC- conjugated anti-mouse granulocyte mAb (Gr-1) (data not shown). Dead cells were excluded by gating out low FSC/high propidium iodide-retaining cells.
  • Colony-forming unit Thirty three thousand BMC were cultured in 1 ml of Iscove's 2.3% methylcellulose (HCC-410; Terry Fox Laboratory, British Columbia, Canada) medium, supplemented with 5% mouse IL-3 -containing supernatant (WEHI-3 supernatant, Lot No. 906852; Beckton Dickinson, Bedford, MA), 30% FCS (HyClone, Logan, UT), 5x10$ M 2-ME, 15% Iscove's modified Dulbecco's medium (Mediatech, Washington, D.C.), and antibiotics (50 U/ml penicillin and 50 ⁇ l/mh streptomycin) in a culture flask (Cat. #171099, Nunc, Inc., Naperville, IL). Eight days later, colonies were enumerated, and were then harvested and stained for phenotyping by FCM, as described above.
  • WBC decreased in proportion to the WBI dose administered.
  • WBC numbers did not decrease, but increased above normal from days 14 to 29, then gradually decreased to normal levels by day 42.
  • WBC counts decreased to an average of 3.3 x 10- ⁇ /m ⁇ r by day 2, then gradually increased until day 29. Only animals that received BMT showed an "overshoot" in WBC counts at day 29.
  • Ly-5.1 + cells increased to 54 and 66% of WBC by 12 weeks after WBI and remained constant for the entire follow-up period of 30 weeks. Similar levels of Ly-5.1 reconstitution were observed for lymphocytes, granulocytes and monocytes of each animal. In the remaining 3 mice, however, Ly-5.1 + WBC did not increase between 2 and 6 weeks post-BMT, and began to decrease at 12 weeks post-BMT. By 30 weeks, donor cells were undetectable in one of these three mice. Although Ly-5.1 + lymphocytes remained detectable in all three mice for at least 20 weeks, granulocyte chimerism was no longer detectable by 6 weeks following BMT.
  • Chimerism was not detectable in WBC, spleen cells, thymocytes, BMC, or BM progenitors of any of six B6 mice injected with Ly-5.1 BMC with or without 0.5 Gy WBI.
  • SUBSTTTUTE SHEET (RULE 26) 5.2 + , 5% Ly-5.1 + ) confirmed this prediction, as long-term chimerism was close to 5% in all recipients of 10 ⁇ BMC (Group 2, Table II), but ranged from 0-28% in recipients of smaller numbers (10 ⁇ of the same cell mixture (Group 1, table II).
  • Ly-5.1 + cells were not detected in WBC of Ly-5.2 mice irradiated with 9.5 Gy and injected with either 10 5 or 10 6 TCD BMC from Ly-5.2 mice (mouse numbers 2544 and 2548) which had been irradiated with 0.5 Gy and injected with Ly-5.1 + BMC 43 weeks earlier.
  • Ly5.1 + WBC are Not Detectable in Secondary, 9.5 Gy-Irradiated Recipients of TCD BMC from Ly5.2+ Recipients of 0.5 Gy WBI and Ly5.1 + BMC 43 Weeks earlier
  • Ly5.1+ BMC Ly5.1+ BMC
  • Recipient B6 mice were irradiated with supralethal dose of 9.5 Gy and reconstituted with 1 or 10 x 10 5 TCD BMC.
  • Donor animal was untreated or Mouse number 2544 and 2548 were treated with 0.5 Gy WBI followed by injection of 1.5 x 10 7 TCD BMC from untreated B6.Ly-5.2 mice 43 weeks earlier.
  • Stromal tissue introduced prior to hematopoietic cell transplant may be varied by: (1) administering the fetal liver and thymus tissue as a fluid cell suspension; (2) administering fetal liver or thymus stromal tissue but not both; (3) placing a stromal implant into other encapsulated, well-vascularized sites, or (4) using adult thymus or fetal spleen as a source of stromal tissue.
  • the methods described herein for inducing tolerance to an allogeneic antigen or allogeneic graft can be used where, as between the donor and recipient, there is any degree of mismatch at MHC loci or other loci which influence graft rejection.
  • class I and class II MHC loci the donor and recipient can be: matched at class I and mismatched at class II; mismatched at class I and matched at class II; mismatched at class I and mismatched at class II; matched at class I, matched at class II.
  • mismatched at MHC class I means mismatched for one or more MHC class I loci, e.g., in the case of humans, mismatched at one or more of HLA-A, HLA-B, or HLA-C, or in the case of swine, mismatch at one or more SLA class I loci, e.g., the swine A or B loci.
  • Mismatched at MHC class II means mismatched at one or more MHC class II loci, e.g., in the case of humans, mismatched at one or more of a DP ⁇ , a DP ⁇ , a DQ ⁇ , a DQ ⁇ , a DR ⁇ , or a DR ⁇ , or in the case of swine, mismatch at one or SLA class II loci, e.g., mismatch at DQ ⁇ or ⁇ , or DR ⁇ or ⁇ .
  • the methods described herein for inducing tolerance to an allogeneic antigen or allogeneic graft can be used where, as between the donor and recipient, there is any degree of reactivity in a mixed lymphocyte assay, e.g., wherein there is no, low, intermediate, or high mixed lymphocyte reactivity between the donor and the recipient.
  • mixed lymphocyte reactivity is used to define mismatch for class II, and the invention includes methods for performing allogenic grafts between individuals with any degree of mismatch at class II as defined by a mixed lymphocyte assay.
  • Serological tests can be used to determine mismatch at class I or II loci and the invention includes methods for performing allogenic grafts between individuals with any degree of mismatch at class I and or II as measured with serological methods.
  • the invention features methods for performing allogeneic grafts between individuals which, as determined by serological and or mixed lymphocyte reactivity assay, are mismatched at both class I and class II.
  • the methods of the invention are particularly useful for replacing a tissue or organ afflicted with a neoplastic disorder, particularly a disorder which is resistant to normal modes of therapy, e.g., chemotherapy or radiation therapy.
  • Methods of the invention can be used for inducing tolerance to a graft,. e.g., an allograft, e.g., an allograft from a donor which is mismatched at one or more class I loci, at one or more class II loci, or at one or more loci at each of class I and class II.
  • the graft includes tissue from the digestive tract or gut, e.g., tissue from the stomach, or bowel tissue, e.g., small intestine, large intestine, or colon; the graft replaces a portion of the recipient's digestive system e.g., all or part of any of the digestive tract or gut, e.g., the stomach, bowel, e.g., small intestine, large intestine, or colon.
  • Tolerance refers not only to complete immunologic tolerance to an antigen, but to partial immunologic tolerance, i.e., a degree of tolerance to an antigen which is greater than what would be seen if a method of the invention were not employed.
  • the inventor has discovered that it is possible to induce mixed chimerism with less radiation toxicity by fractionating the radiation dose, i.e., by delivering the radiation in two or more exposures or sessions.
  • the radiation can either be delivered in a single exposure, or more preferably, can be fractionated into two or more exposures or sessions.
  • the sum of the fractionated dosages is preferably equal, e.g., in rads or Gy, to the radiation dosage which can result in mixed chimerism when given in a single exposure.
  • the fractions are preferably approximately equal in dosage.
  • a single dose of 700 rads can be replaced with, e.g., two fractions of 350 rads, or seven fractions of 100 rads.
  • Hyperfractionation of the radiation dose can also be used in methods of the invention.
  • the fractions can be delivered on the same day, or can be separated by intervals of one, two, three, four, five, or more days. Whole body irradiation, thymic irradiation, or both, can be fractionated.
  • the inventor has also discovered that much or all of the preparative regimen can be delivered or administered to a recipient, e.g., an allograft or xenograft recipient, within a few days, preferably within 72, 48, or 24 hours, of transplantation oftolerizing stem cells and or the graft. This is particularly useful in the case of humans receiving grafts from cadavers.
  • the treatment(s) can be administered, within a few days, preferably within 72, 48, or 24 hours, of transplantation of the stem cells and/or the graft.
  • primate e.g., human
  • recipients of allografts can be given any or all of treatments to inactivate or deplete host antibodies, treatments to inactivate host T cells or NK cells, or irradiation, within a few days, preferably within 72, 48, or 24 hours, of transplantation of stem cells and or the graft.
  • treatment to deplete recipient T cells and/or NK cells e.g., administration of ATG
  • stem cell e.g., bone marrow stem cells, administered on day 0.
  • the graft e.g., a renal allograft, is transplanted on day 0).
  • window there is a permissible time period ("window") for hematopoietic stem cell engraftment following creation of space for the donor hematopoietic stem cell (e.g., by whole body irradiation) in a recipient. It has further been discovered that space created for hematopoietic stem cell engraftment can be monitored over time by monitoring white blood cell levels in a recipient.
  • transplantion can be performed during the permissible window for engraftment following creation of space for the hematopoietic stem cell, as provided by the invention.
  • white blood cell levels can be monitored to monitor space for the donor hematopoietic stem cells (i.e., to assess the permissible window for engraftment).
  • Examples of procedures involving hematopoietic stem cell transplantation include 1) conditioning of a recipient for an allo- or xenograft, as described herein, in which hematopoietic stem cell transplantation is performed in conjunction with transplantation of another allo- or xenograft; 2) treatment of various hematopoietic disorders, including leukemias, lymphomas and other hematopoietic malignancies and genetic hematopoietic disorders (e.g., adenosine deaminase deficiency, bare lymphocyte syndrome and other congenital immunodeficiency diseases) in which hematopoietic stem cell transplantation is performed therapeutically; and 3) transplantation of genetically modified hematopoietic stem cells (e.g., genetically modified autologous hematopoietic stem cells) to deliver a gene product to a recipient (e.g., as gene therapy).
  • genetically modified hematopoietic stem cells e.g
  • SUBSTTTUTE SHEET monitor this window by monitoring white blood cell levels in the recipient, can be applied to any type of bone marrow transplantation situation, e.g., autologous, syngeneic, allogeneic or xenogeneic transplantation.
  • syngeneic bone marrow transplantation is the preferred method for gene therapy using hematopoietic stem cells (i.e., an individuals own stem cells are isolated, genetically modified and reintroduced into the individual) whereas allogeneic or xenogeneic bone marrow transplantation is necessary for conditioning of a recipient of an allograft or xenograft.
  • the ability to monitor space for donor hematopoietic stem cell within a recipient allows for selection of a suitable time for administration of donor bone marrow and ensures that the optimal window for engraftment is not missed.
  • thymic irradiation of a recipient can be replaced by treatment of the recipient with (mature and immature) T cell ablative antibodies. Accordingly, in any method calling for thymic irradiation in order to deplete mature and immature T cells within the recipient, administration of T cell ablative antibodies can be substituted for thymic irradiation.
  • Procedures involving thymic irradiation can include preparative treatment of a recipient for an allograft in conjunction with hematopoietic stem cell (as described herein) or preparative treatment of a recipient of a hematopoietic stem cell transplant alone (e.g., as therapy for a hematopoietic disorder or for gene therapy purposes, as discussed above).
  • the ability to replace thymic irradiation with administration of T cell ablative antibodies as a preparative step for a transplant recipient reduces the amount of irradiation to which a recipient need be exposed. This is an advantageous improvement considering the possible unwanted side effects (e.g., mutations, toxicity) which can occur from excessive irradiation.

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Abstract

L'invention concerne une méthode améliorée de greffe de cellules souches (hématopoïétiques) exogènes chez un receveur, consistant à appliquer un traitement myélodépressif à celui-ci, afin de créer un espace hématopoïétique, à déterminer le taux de ses globules blancs, et à lui administrer les cellules hématopoïétiques durant une période de temps où le taux des globules blancs dans son sang est diminué.
PCT/US1994/001616 1992-02-19 1994-02-14 Greffe de cellules souches WO1995021527A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
PCT/US1994/001616 WO1995021527A1 (fr) 1994-02-14 1994-02-14 Greffe de cellules souches
AU62681/94A AU6268194A (en) 1994-02-14 1994-02-14 Stem cell engraftment
PCT/US1994/005527 WO1994026289A1 (fr) 1992-02-19 1994-05-16 Transplantation allogenique et xenogenique
US09/710,971 US6911220B1 (en) 1992-02-19 2000-11-09 Allogeneic and xenogeneic transplantation
US09/874,512 US20020168348A1 (en) 1992-02-19 2001-06-05 Allogeneic and and xenogeneic transplantation
US11/151,744 US20060147428A1 (en) 1992-02-19 2005-06-13 Allogeneic and xenogeneic transplantation

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PCT/US1994/001616 WO1995021527A1 (fr) 1994-02-14 1994-02-14 Greffe de cellules souches

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US15073993A Continuation 1992-02-19 1993-11-10

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US21222894A Continuation 1992-02-19 1994-03-14
US08/451,210 Continuation US6296846B1 (en) 1992-01-08 1995-05-26 Induced tolerance to xenografts
US08/458,720 Continuation-In-Part US5876708A (en) 1992-02-19 1995-06-01 Allogeneic and xenogeneic transplantation

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WO1995021527A1 true WO1995021527A1 (fr) 1995-08-17

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Publication number Priority date Publication date Assignee Title
EP0699073A4 (fr) * 1993-05-17 1997-09-10 Gen Hospital Corp Transplantation allogenique et xenogenique
EP1005866A3 (fr) * 1993-05-17 2001-08-16 The General Hospital Corporation Transplantation allogénique et xénogénique
US6521448B1 (en) 1997-08-19 2003-02-18 Diacrin, Inc. Porcine MHC class I genes and uses thereof
US6911220B1 (en) 1992-02-19 2005-06-28 The General Hospital Corporation Allogeneic and xenogeneic transplantation

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Title
HUMAN IMMUNOLOGY, Volume 28, issued 1990, D.H. SACHS et al., "Immunology of Xenograft Rejection", pages 245-251. *
THE JOURNAL OF EXPERIMENTAL MEDICINE, Volume 169, issued February 1989, Y. SHARABI et al., "Mixed Chimerism and Permanent Specific Transplantation Tolerance Induced by a Nonlethal Preparative Regimen", pages 493-502. *
TRANSPLANTATION PROCEEDINGS, Volume 21, Number 1, issued February 1989, J.C. MADSEN et al., "Induction of Immunological Unresponsiveness Using Recipient Cells Transfected With Class I or Class II MHC Genes", page 477. *
TRANSPLANTATION, Volume 54, Number 3, issued September 1992, B.R. ROSENGARD et al., "Induction of Specific Tolerance to Class I-Disparate Renal Allografts in Miniature Swine With Cyclosporine", pages 490-497. *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6911220B1 (en) 1992-02-19 2005-06-28 The General Hospital Corporation Allogeneic and xenogeneic transplantation
EP0699073A4 (fr) * 1993-05-17 1997-09-10 Gen Hospital Corp Transplantation allogenique et xenogenique
EP1005866A3 (fr) * 1993-05-17 2001-08-16 The General Hospital Corporation Transplantation allogénique et xénogénique
US6521448B1 (en) 1997-08-19 2003-02-18 Diacrin, Inc. Porcine MHC class I genes and uses thereof

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