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WO1996006642A1 - Transplantation allogenique et xenogenique - Google Patents

Transplantation allogenique et xenogenique Download PDF

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Publication number
WO1996006642A1
WO1996006642A1 PCT/US1995/010598 US9510598W WO9606642A1 WO 1996006642 A1 WO1996006642 A1 WO 1996006642A1 US 9510598 W US9510598 W US 9510598W WO 9606642 A1 WO9606642 A1 WO 9606642A1
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WO
WIPO (PCT)
Prior art keywords
recipient
tolerance
donor
cells
thymus
Prior art date
Application number
PCT/US1995/010598
Other languages
English (en)
Inventor
David H. Sachs
Pierre Gianello
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 AU34094/95A priority Critical patent/AU3409495A/en
Publication of WO1996006642A1 publication Critical patent/WO1996006642A1/fr

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Classifications

    • 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
    • A61K38/16—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
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4

Definitions

  • the invention relates to tissue and organ transplantation. SUMMARY OF THE INVENTION
  • the invention provides several methods of prolonging or promoting the acceptance or survival of a graft, e.g., by inducing tolerance to foreign antigens on allogeneic or xenogeneic organ grafts. These methods can be used individually or in combination with one another. For example, maintenance or enhancement of thymus function can prolong survival of the graft.
  • the thymus function-maintaining or -enhancing methods of the invention can be combined with one or more other methods of prolonging graft acceptance or survival.
  • the invention features, a method of promoting, in a recipient mammal, e.g., a primate, e.g., a human, acceptance of an allograft, from a donor mammal, including: inducing tolerance to the allograft in the recipient mammal; (preferably) implanting the donor allograft in the recipient; and maintaining or enhancing thymus function in the recipient.
  • thymus function is maintained or enhanced: by administering growth hormone, e.g., recombinant human growth hormone, or (if the recipient is a male) administering a substance, e.g., a drug which mimics orchiemectomy.
  • thymus function is maintained by not removing recipient thymic tissue.
  • the thymus function which is enhanced or maintained is that of: the recipient's thymus, a donor thymus, or both the recipient's thymus and a donor thymus.
  • Tolerance can be induced by a method known to those skilled in the art, or by a method described in one of the above recited U.S. patent applications, e.g., by the administration of a short course of help reducing treatment, as is described in USSN 08/220,371, filed March 29, 1994.
  • tolerance is induced by the administration of a short course of help reducing treatment to, e.g., induce tolerance to unmatched antigens on the graft.
  • the recipient can be mismatched at a first locus which affects graft rejection, e.g.. an MHC class I or II locus, or a minor antigen locus, and matched, or tolerant of a mismatch, at a second locus which affects graft rejection, e.g., an MHC class I or II locus, or a minor antigen locus.
  • Matching at the second locus can be achieved by selection of a recipient or donor of the appropriate genotype.
  • tolerance is induced by a short course of help reducing treatment and: the recipient and donor are matched at a class II locus and the short course of help reducing treatment induces tolerance to unmatched class I and/or minor antigens on the graft.
  • tolerance is induced by a short course of help reducing treatment and: tolerance to a class II antigen is induced by a method other than a short course of a help reducing treatment, and the short course of help reducing treatment induces tolerance to unmatched class I and minor antigens on the graft.
  • tolerance is induced by a short course of help reducing treatment and: 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 to initiate rejection of an antigen after first being stimulated by the antigen (in humans this is usually 8- 12 days, preferably about 10 days); 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 mature T cells of the recipient to initiate rejection 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 a steroid drug in a sufficient concentration to counteract the desired effect of the help reducing treatment, e.g., in the absence of Prednisone (17, 21-dihydroxypregna-l, 4-diene-3, 1 1, 20-trione) at a concentration which stimulates the release of a cytokine by mature T cells in the recipient.
  • the short course of help reducing treatment is administered in the absence of a steroid drug, e.g., in the absence of Prednisone.
  • 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; or the donor and recipient are class I matched.
  • Tolerance can be also be induced by the implantation of donor hematopoietic stem cells, e.g., by a method described in one or more of the above recited U.S. patent applications, e.g., in USSN 07/838,595, filed February 19, 1992.
  • tolerance is induced by preferably prior to or simultaneous with introduction of the graft, implanting, e.g., by intravenous injection, into the recipient, donor hematopoietic stem cells, e.g., bone marrow, hematopoietic stem cells, (preferably the hematopoietic stem cells home to a site in the recipient mammal).
  • donor hematopoietic stem cells e.g., bone marrow, hematopoietic stem cells, (preferably the hematopoietic stem cells home to a site in the recipient mammal).
  • the graft is obtained from a different organ than the hematopoietic stem cells, e.g., a liver or a kidney.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting recipient natural killer (NK) cells, e.g., by introducing into the recipient an antibody capable of binding to NK cells of the recipient, to prevent NK mediated rejection of the swine graft.
  • NK natural killer
  • One source of anti-NK antibody is anti-human thymocyte polyclonal anti-serum.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting host T cell function, e.g., by introducing into the recipient an antibody capable of binding to T cells of the recipient; (preferably prior to or at the time of introducing the thymic tissue into the recipient) depleting, inactivating or inhibiting host CD4 + cell function, e.g., by introducing into the recipient an antibody capable of binding to CD4, or CD4 + cells of the recipient.
  • Repeated doses of anti-NK or anti-T cell antibody may be preferable.
  • Monoclonal preparations can be used in the methods of the invention.
  • Other preferred embodiments include: the step of creating hematopoietic space, e.g., by one or more of, irradiating the recipient with low dose, e.g., between about 100 and 400 rads, whole body irradiation, administering a myelosuppressive drug to the recipient, or administering anti-class I antibodies to the recipient, to deplete or partially deplete the bone marrow of the recipient; the method includes the a step which creates hematopoietic space and the step is performed prior to introducing the hematopoietic stem cells into the recipient.
  • Other preferred embodiments include inactivating thymic T cells by one or more of:
  • irradiating the recipient mammal with, e.g., about 700 rads of thymic irradiation; administering one, or preferably two or more, doses of an anti-T cell antibody; or administering to the recipient a short course of an immunosuppressant as described in USSN 08/220,371, filed March 29, 1994.
  • Other preferred embodiments include: the step of depleting or otherwise inactivating natural antibodies in the blood of the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney, (obtained preferably from the donor) or administering a drug, e.g., deoxyspergualin (DSG) which inactivates or depletes natural antibodies; the method includes a step which depletes or otherwise inactivates natural antibodies in the blood of the recipient and the step is performed prior to hematopoietic stem cell transplantation.
  • a drug e.g., deoxyspergualin
  • Tolerance can be induced by the implantation of transduced bone marrow cells to induce tolerance to an antigen, e.g., the methods described in one or more of the above recited U.S. patent applications, e.g., in USSN 08/266,427, filed June 27, 1994.
  • the invention features a method of promoting, in a recipient mammal, e.g., a primate, e.g., a human, acceptance of a xenograft from a donor mammal, e.g., a swine, e.g., a miniature swine, including: inducing tolerance to the xenograft; (preferably) implanting the donor xenograft in the recipient; and enhancing thymus function in the recipient.
  • a recipient mammal e.g., a primate, e.g., a human
  • a swine e.g., a miniature swine
  • thymus function is maintained or enhanced: by administering growth hormone, e.g., recombinant human growth hormone, or (if the recipient is a male) administering a substance, e.g., a drug which mimics orchiemectomy.
  • growth hormone e.g., recombinant human growth hormone
  • a substance e.g., a drug which mimics orchiemectomy.
  • thymus function is maintained by not removing recipient thymic tissue.
  • thymus function which is enhanced or maintained is that of: the recipient's thymus, a donor thymus, or both the recipient's thymus and a donor thymus.
  • Tolerance can be induced by a method known to skilled in the art or by a method described in one of the above-recited U.S. patent applications, e.g., by the implantation of donor hematopoietic stem cells, e.g., the methods described in one or more of the above recited U.S. patent applications, e.g., in USSN 07/838,595, filed February 19, 1992.
  • tolerance is induced by: preferably prior to or simultaneous with introduction of the graft, implanting, e.g., by intravenous injection, into the recipient, donor hematopoietic stem cells, e.g., bone marrow, cord blood or fetal spleen or liver hematopoietic stem cells (preferably, the hematopoietic stem cells home to a site in the recipient mammal).
  • donor hematopoietic stem cells e.g., bone marrow, cord blood or fetal spleen or liver hematopoietic stem cells (preferably, the hematopoietic stem cells home to a site in the recipient mammal).
  • the recipient is a primate, e.g., a human, and the swine graft is obtained from a miniature swine.
  • the graft is obtained from a different organ than the hematopoietic stem cells, e.g., a liver or a kidney.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting recipient natural killer (NK) cells, e.g., by introducing into the recipient an antibody capable of binding to NK cells of the recipient, to prevent NK mediated rejection of the swine graft.
  • NK natural killer
  • One source of anti-NK antibody is anti-human thymocyte polyclonal anti-serum.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting host T cell function, e.g., by introducing into the recipient an antibody capable of binding to T cells of the recipient; (preferably prior to or at the time of introducing the thymic tissue into the recipient) depleting, inactivating or inhibiting host CD4 + cell function, e.g., by introducing into the recipient an antibody capable of binding to CD4, or CD4 + cells of the recipient.
  • Repeated doses of anti-NK or anti-T cell antibody may be preferable.
  • Monoclonal preparations can be used in the methods of the invention.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting recipient natural killer (NK) cells, e.g., by introducing into the recipient an antibody capable of binding to NK cells of the recipient, to prevent NK mediated rejection of the swine graft.
  • NK natural killer
  • One source of anti-NK antibody is anti-human thymocyte polyclonal anti-serum.
  • Other preferred embodiments include: the step of creating hematopoietic space, e.g., by one or more of, irradiating the recipient with low dose, e.g., between about 100 and 400 rads, whole body irradiation, administering a myelosuppressive drug to the recipient, or administering anti-class I antibodies to the recipient, to deplete or partially deplete the bone marrow of the recipient; the method includes the a step which creates hematopoietic space and the step is performed prior to introducing the swine hematopoietic stem cells into the recipient.
  • Other preferred embodiments include inactivating thymic T cells by one or more of:
  • irradiating the recipient with, e.g., about 700 rads of thymic irradiation; administering one, or preferably two or more, doses of an anti-T cell antibody; or administering to the recipient a short course of an immunosuppressant as described in USSN 08/220,371 , filed March 29, 1994.
  • Other preferred embodiments include: the step of depleting or otherwise inactivating natural antibodies in the blood of the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney, obtained from the donor species, e.g., from a pig, or administering a drug, e.g., deoxyspergualin (DSG) which inactivates or depletes natural antibodies; the method includes a step which depletes or otherwise inactivates natural antibodies in the blood of the recipient and the step is performed prior to hematopoietic stem cell transplantation.
  • a drug e.g., deoxyspergualin
  • stromal tissue e.g., swine stromal tissue, preferably hematopoietic stromal tissue, e.g., fetal liver tissue or thymus tissue.
  • the stromal tissue is introduced simultaneously with, or prior to, the hematopoietic stem cells; the bone marrow cells are introduced simultaneously with, or prior to, any anti-NK or T cell antibody.
  • the donor is a pig and 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.
  • Tolerance can be also be induced by the implantation of transduced bone marrow cells to induce tolerance to an antigen, e.g., the methods described in one or more of the above recited U.S. patent applications, e.g., in USSN 08/266,427, filed June 27, 1994.
  • tolerance is induced by inserting DNA encoding an MHC antigen of the donor species into a hematopoietic stem cell, e.g., a bone marrow hematopoietic stem cell, of the recipient; and allowing the MHC antigen encoding DNA to be expressed in the recipient.
  • a hematopoietic stem cell e.g., a bone marrow hematopoietic stem cell
  • 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 identical, with the individual mammal from which the graft is obtained; 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.
  • kits further include the step of introducing into the recipient a graft obtained from the donor, e.g., a liver or a kidney.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting recipient natural killer (NK) cells, e.g., by introducing into the recipient an antibody capable of binding to NK cells of the recipient, to prevent NK mediated rejection of the swine graft.
  • NK natural killer
  • One source of anti-NK antibody is anti-human thymocyte polyclonal anti-serum.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting host T cell function, e.g., by introducing into the recipient an antibody capable of binding to T cells of the recipient; (preferably prior to or at the time of introducing the thymic tissue into the recipient) depleting, inactivating or inhibiting host CD4 + cell function, e.g., by introducing into the recipient an antibody capable of binding to CD4, or CD4 + cells of the recipient.
  • Repeated doses of anti-NK or anti-T cell antibody may be preferable.
  • Monoclonal preparations can be used in the methods of the invention.
  • Other preferred embodiments include: the step of creating hematopoietic space, e.g., by one or more of, irradiating the recipient with low dose, e.g., between about 100 and 400 rads, whole body irradiation, administering a myelosuppressive drug to the recipient, or administering anti-class I antibodies to the recipient, to deplete or partially deplete the bone marrow of the recipient; the method includes the a step which creates hematopoietic space and the step is performed prior to introducing the genetically engineered hematopoietic stem cells into the recipient.
  • thymic T cells include inactivating thymic T cells by one or more of: (preferably prior to hematopoietic stem cell transplantation) irradiating the recipient mammal with, e.g., about 700 rads of thymic irradiation; administering one, or preferably two or more, doses of an anti-T cell antibody; or administering to the recipient a short course of an immunosuppressant as described in USSN 08/220,371 , filed March 29, 1994.
  • Other preferred embodiments include: the step of depleting or otherwise inactivating natural antibodies in the blood of the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney, obtained from the donor species, e.g., from a pig, or administering a drug, e.g., deoxyspergualin (DSG) which inactivates or depletes natural antibodies; the method includes a step which depletes or otherwise inactivates natural antibodies in the blood of the recipient and the step is performed prior to hematopoietic stem cell transplantation.
  • a drug e.g., deoxyspergualin
  • the invention features a method of promoting, in a recipient mammal, e.g., a primate, e.g., a human, acceptance of an allograft, e.g., a heart allograft, from a donor mammal, including: (preferably) inducing tolerance to the allograft; implanting into the recipient the allograft from the donor; and maintaining thymus function in the recipient.
  • a recipient mammal e.g., a primate, e.g., a human
  • an allograft e.g., a heart allograft
  • all or part of the thymus is generally removed.
  • thymus function is maintained by not removing recipient thymic tissue.
  • the recipient thymus function is enhanced.
  • thymus function is maintained or enhanced: by administering growth hormone, e.g., recombinant human growth hormone, or (if the recipient is a male) administering a substance, e.g., a drug which mimics orchiemectomy.
  • growth hormone e.g., recombinant human growth hormone
  • a substance e.g., a drug which mimics orchiemectomy.
  • Tolerance can be induced by a method known to those skilled in the art, or by a method described in one of the above recited U.S. patent applications, e.g., by the administration of a short course of help reducing treatment, as is described in USSN 08/220,371, filed March 29, 1994.
  • tolerance is induced by the administration of a short course of help reducing treatment to, e.g., induce tolerance to unmatched antigens on the graft.
  • the recipient can be mismatched at a first locus which affects graft rejection, e.g., an MHC class I or II locus, or a minor antigen locus, and matched, or tolerant of a mismatch, at a second locus which affects graft rejection, e.g., an MHC class I or II locus, or a minor antigen locus.
  • Matching at the second locus can be achieved by selection of a recipient or donor of the appropriate genotype.
  • tolerance is induced by a short course of help reducing treatment and: the recipient and donor are matched at a class II locus and the short course of help reducing treatment induces tolerance to unmatched class I and/or minor antigens on the graft.
  • tolerance is induced by a short course of help reducing treatment and: tolerance to a class II antigen is induced by a method other than a short course of a help reducing treatment, and the short course of help reducing treatment induces tolerance to unmatched class I and minor antigens on the graft.
  • tolerance is induced by a short course of help reducing treatment and: 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 to initiate rejection of an antigen after first being stimulated by the antigen (in humans this is usually 8- 12 days, preferably about 10 days); 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 mature T cells of the recipient to initiate rejection 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 a steroid drug in a sufficient concentration to counteract the desired effect of the help reducing treatment, e.g., in the absence of Prednisone (17, 21-dihydroxypregna-l, 4-diene-3, 11, 20-trione) at a concentration which stimulates the release of a cytokine by mature T cells in the recipient.
  • the short course of help reducing treatment is administered in the absence of a steroid drug, e.g., in the absence of Prednisone.
  • 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; or the donor and recipient are class I matched.
  • Tolerance can be also be induced by the implantation of donor hematopoietic stem cells, e.g., by a method described in one or more of the above recited U.S. patent applications, e.g., in USSN 07/838,595, filed February 19, 1992.
  • tolerance is induced by preferably prior to or simultaneous with introduction of the graft, implanting, e.g., by intravenous injection, into the recipient, donor hematopoietic stem cells, e.g., bone marrow, hematopoietic stem cells, (preferably the hematopoietic stem cells home to a site in the recipient mammal).
  • donor hematopoietic stem cells e.g., bone marrow, hematopoietic stem cells, (preferably the hematopoietic stem cells home to a site in the recipient mammal).
  • the graft is obtained from a different organ than the hematopoietic stem cells, e.g., a liver or a kidney.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting recipient natural killer (NK) cells, e.g., by introducing into the recipient an antibody capable of binding to NK cells of the recipient, to prevent NK mediated rejection of the swine graft.
  • NK natural killer
  • One source of anti-NK antibody is anti-human thymocyte polyclonal anti-serum.
  • the method includes: (preferably prior to or at the time of introducing the hematopoietic stem cells into the recipient) depleting, inactivating or inhibiting host T cell function, e.g., by introducing into the recipient an antibody capable of binding to T cells of the recipient; (preferably prior to or at the time of introducing the thymic tissue into the recipient) depleting, inactivating or inhibiting host CD4 + cell function, e.g., by introducing into the recipient an antibody capable of binding to CD4, or CD4 + cells of the recipient.
  • Repeated doses of anti-NK or anti-T cell antibody may be preferable.
  • Monoclonal preparations can be used in the methods of the invention.
  • Other preferred embodiments include: the step of creating hematopoietic space, e.g., by one or more of, irradiating the recipient with low dose, e.g., between about 100 and 400 rads, whole body irradiation, administering a myelosuppressive drug to the recipient, or administering anti-class I antibodies to the recipient, to deplete or partially deplete the bone marrow of the recipient; the method includes the a step which creates hematopoietic space and the step is performed prior to introducing the hematopoietic stem cells into the recipient.
  • thymic T cells include inactivating thymic T cells by one or more of: (preferably prior to hematopoietic stem cell transplantation) irradiating the recipient mammal with, e.g., about 700 rads of thymic irradiation; administering one, or preferably two or more, doses of an anti-T cell antibody; or administering to the recipient a short course of an immunosuppressant as described in USSN 08/220,371, filed March 29, 1994.
  • Other preferred embodiments include: the step of depleting or otherwise inactivating natural antibodies in the blood of the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney, (obtained preferably from the donor) or administering a drug, e.g., deoxyspergualin (DSG) which inactivates or depletes natural antibodies; the method includes a step which depletes or otherwise inactivates natural antibodies in the blood of the recipient and the step is performed prior to hematopoietic stem cell transplantation.
  • a drug e.g., deoxyspergualin
  • Tolerance can be induced by the implantation of transduced bone marrow cells to induce tolerance to an antigen, e.g., the methods described in one or more of the above recited U.S. patent applications, e.g., in USSN 08/266,427, filed June 27, 1994.
  • “Enhancing thymus function”, as used herein, refers to increasing the ability of recipient or donor thymus to mediate the induction or maintenance of tolerance.
  • “Maintaining thymus function”, as used herein, refers to procedures which maintain the ability of recipient or donor thymus to mediate the induction or maintenance of tolerance.
  • 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 can involve humoral, cellular, or both humoral and cellular responses.
  • Hematopoietic stem cell refers to a cell, e.g., a bone marrow 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.
  • An immunosuppressive agent capable of inactivating thymic or lymph node T cells is an agent, e.g., a chemical agent, e.g., a drug, which, when administered at an appropriate dosage, results in the inactivation of thymic or lymph node T cells.
  • agents e.g., cyclosporine, FK-506, and rapamycin.
  • Anti-T cell antibodies because they are comparatively less effective at inactivating thymic or lymph node T cells, are not preferred for use as agents.
  • An agent should be administered in sufficient dose to result in significant inactivation of thymic or lymph node T cells which are not inactivated by administration ofan anti-T cell antibody, e.g., an anti-ATG preparation.
  • Putative agents, and useful concentrations thereof, can be prescreened by in vitro or in vivo tests, e.g., by administering the putative agent to a test animal, removing a sample of thymus or lymph node tissue, and testing for the presence of active T cells in an in vitro or in vivo assay. Such prescreened putative agents can then be further tested in transplant assays.
  • "Short course of a immunosuppressive agent" means a transitory non- chronic course of treatment.
  • the treatment should begin before or at about the time the treatment to induce tolerance is begun, e.g., at about the time, xenogeneic, allogeneic, genetically engineered syngeneic, or genetically engineered autologous stem cells are introduced into the recipient, e.g., the short course can begin on the day the treatment to induce tolerance is begun, e.g., on the day, xenogeneic, allogeneic, genetically engineered syngeneic, or genetically engineered autologous stem cells are introduced into the recipient or the short course can begin within 1, 2, 4, 6, 8, or 10 days before or after the treatment to induce tolerance is begun, e.g., within 1, 2, 4, 6, 8, or 10 days before or after xenogeneic, allogeneic, genetically engineered syngeneic, or genetically engineered autologous stem cells are introduced into the recipient.
  • the short course can last for: a period equal to or less than about 8-12 days, preferably about 10 days, or a time which is approximately equal to or is less than two, three, four, five, or ten times the 8-12 or 10 day period. Optimally, the short course lasts about 30 days.
  • the dosage should be sufficient to maintain a blood level sufficient to inactivate thymic or lymph node T cells. A dosage of approximately 15 mg/kg/day has been found to be effective in primates.
  • Lymph node or thymic T cell refers to T cells which are resistant to inactivation by traditional methods of T cell inactivation, e.g., inactivation by a single intravenous administration of anti-T cell antibodies, e.g., anti-bodies, e.g., ATG preparation.
  • 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.
  • the level of reduction is one which substantially eliminates the initial burst of IL-2 which accompanies the first recognition of a foreign antigen but which does not eliminate all mature T cells, which cells may be important in educating and producing tolerance.
  • a 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 ofan 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 ofan 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 10 mg/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.
  • the same blood level, 500-1,000 ng/ml is sufficient to induce tolerance in pigs.
  • Long-term administration of 5mg/kg prevents rejection (by long term immune suppression) but does not result in tolerance. 0
  • 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”, as used herein, refers to wholly or partially inbred animal.
  • “Graft”, as used herein, 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.
  • “Stromal tissue”, as used herein, refers to the supporting tissue or matrix of an organ, as distinguished from its functional elements or parenchyma. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
  • FIG. 1A shows creatinine levels from animal #10348 (o) which was not treated with cyclosporin A (CyA). This animal rejected the kidney on POD 8, while non-thymectomized CyA-treated animal #10349 ( ⁇ ) went on to tolerate the kidney long-term.
  • the sham thymectomized animal #10418 ( ⁇ ) maintained a normal renal function during the entire followup.
  • FIGS. 2A-2D show the results from cell-mediated lymphocytotoxicity assays performed as follows: SLA dd PBL from each experimental animal were stimulated by outbred cells (Yucatan) and tested on SLA Yucatan target cells (•) as positive control and on SLA dd target cells as negative control ( ⁇ ). SLA dd PBL of the same animals were stimulated by SLA88 matched cells and tested on SLA88 target cells (o).
  • Figure 2A shows representative CML for a non-thymectomized CyA-treated animal #10349.
  • Figure 2B shows representative CML for a sham-thymectomized animal (#10418).
  • Figure 2C shows representative CML for a partially thymectomized animal #10310 and
  • Figure 2D shows representative CML for a completely thymectomized animal #10549.
  • Figures 3A-3B show an assessment of cytokine expression in serial kidney biopsies from sham, partially and totally thymectomized animals.
  • Figure 3A shows a Northern blot of mRNA derived from kidney biopsy tissue from partially (#10233) and totally (#10770) thymectomized animals on the PODs indicated and developed with probes for IFN ⁇ and the housekeeping gene GAPDH.
  • Figure 3B shows densitometer readings for each of the IFN ⁇ bands, corrected for the amount of RNA loaded onto the gel as determined by the intensity of GAPDH labeling. (*) this RNA sample was degraded.
  • Figures 4A-4C show densitomer readings, determined as illustrated in Figures 3A and 3B, plotted for labeling of northern blots of mRNA from serial kidney biopsy tissues with probes for IL-10 and IFN ⁇ (compared to the GAPDH bands in each case). (*) no sample.
  • Figures 4A, 4B, and 4C correspond to data from three separate autoradiographs with various signal intensities, thus leading to different scale for Expression Indices. The Importance of Thvmus Function in Graft Acceptance
  • Tolerance to renal allografts in miniature swine occurs spontaneously in about one- third of animals selectively matched for class II antigens and mismatched for class I antigens at a single MHC haplotype (Pescovitz, M.D. et al., (1984) J. Exp. Med. 160: 1495-1508).
  • Treatment with a short course of Cyclosporine A (CyA, 10-13 mg/kg for the first 12 postoperative days) extended tolerance induction to animals mismatched for class I antigens at both haplotypes and raised the percentage of tolerance induction to 100% (Rosengard, B.R. et al. (1992) Transplantation 54:490-497).
  • the thymus plays an important role in the induction of central tolerance in numerous systems (Kappler, J.W. et al. (1987) Cell 49:273-280; Schwartz, R.H. et al. (1989) Cell
  • Kidney transplants were performed between swine bearing a two-haplotype class I plus minor antigen disparity (SLABS to SLA dd ). The details of the surgical procedures have been described elsewhere (Kortz, E.O. et al. (1990) Transplantation 49:1 142-1149). Placement ofan indwelling central venous silastic catheter into an external jugular vein facilitated CyA administration and frequent blood sampling for in vitro assays and for monitoring of renal function (BUN, plasma creatinine) and whole blood CyA levels. Thymectomy was performed 3 weeks prior to the SLABS kidney transplant in two SLA ⁇ d pigs (#10233, #10310) through a low transverse cervicotomy.
  • the pre-tracheal muscles were retracted and the trachea exposed from the cervico-thoracic junction to the mandibula, exposing the thymus, which was then dissected and excised. Due to the fact that residual thymic tissue was found at the time of sacrifice of these two animals, we performed subsequent thymectomies through a combined cervicotomy and partial sternotomy approach. Using this exposure, two SLA dd pigs (#10549, #10770) underwent extensive neck and upper chest dissection. In the neck, the thymus is surrounded by a capsule which simplifies the thymic dissection of the cervical portion.
  • Immunosuppression was performed as follows. An intravenous preparation of CyA (Sandimmune, i.v.) was generously provided by Sandoz Pharmaceuticals Corporation, Hanover, NJ. CyA was given each morning as a single daily infusion at a dose of 10-13 mg/kg adjusted to maintain trough levels of 500-800 ng/ml for 12 consecutive days postransplant. The first dose was administered peri-operatively prior to the unclamping of the kidney allograft. Whole blood trough levels were determined by monoclonal radioimmunoassay techniques, and the results were expressed in ng/ml.
  • Allograft rejection monitoring was performed as follows. The clinical endpoints were survival beyond 100 days or terminal renal failure. Animals that were moribund with profound uremia (Creatinine > 10 mg/dl) were euthanized in accordance with the N.I.H. Guidelines for Animal Care and Use. All animals underwent a complete postmortem examination. A careful examination of the renal vascular anastomoses was made in cases of acute rejection to eliminate possible technical failure. A kidney graft was considered rejected when edema, cyanosis and interstitial hemorrhage were present without any signs of vascular thrombosis due to technical failure, concomitantly with a markedly elevated serum creatinine level (> 10 mg/dl).
  • kidney allograft acceptance the animals were sacrificed at one year. At autopsy, the chest and neck were carefully inspected, and 6-9 biopsies in the upper mediastinum were performed for pathological detection of residual thymic tissue. The kidney allograft was harvested and also examined histologically. Histology Serial kidney biopsies on POD 4,8,11,18, and 30 were performed in all experimental animals. The control animals treated with CyA only or sham thymectomy showed similar histology. An interstitial infiltrate of mononuclear cells was observed as early as POD 4 (5.10% of parenchyma), and this rapidly increased to a 50-60% interstitial infiltrate by POD 8.
  • the infiltrate began to diminish, and decreased slowly to a level of 5-10%, which remained present for more than a year.
  • Focal or patchy tubular epithelial infiltration was also often see during the early postoperative period, but likewise disappeared during long-term follow-up.
  • Vascular changes were absent or minimal, consisting of attachment of few mononuclear cells to the endothelium but without intra-endothelial penetration or vasculitis.
  • the two partially thymectomized animals (#10233 and #10310) showed a similar histological picture to CyA-treated animals during the two first postoperative weeks.
  • the interstitial infiltrate increased (70-90%) but then declined progressively during long-term follow-up.
  • pig #10233 showed a similar histological pattern to that of non-thymectomized CyA-treated animals, while animal #10310 showed persistent interstitial infiltrate and evidence of interstitial fibrosis (5-20%) and glomerular hypercellularity, suggesting the effects of previous damage during the rejection crisis. These latter signs were absent in CyA-treated tolerant animals and in the sham-thymectomized animal.
  • the two completely thymectomized pigs showed clear histological evidence of acute cellular rejection during the first postoperative month, with a significantly higher and persistent interstitial mononuclear infiltrate (70-80%).
  • the most significant difference from the other animals was the presence of severe vasculitis and glomerular hemorrhage in the late specimens (POD 18 and 30).
  • the tubular infiltration was more marked, with notable tubular injury and interstitial edema. Increased glomerular cellularity was also present, and one animal (#10549) also showed interstitial fibrosis (20%).
  • the three animals which underwent thymectomy during the maintenance phase of tolerance did not show any histologic evidence of rejection during the entire follow-up. At the time of thymectomy and later, a low (5-20%) interstitial infiltrate of mononuclear cells were observed, but no vascular lesions were noted.
  • donor class I plus third party (TP) class II mismatched skin grafts were rejected almost as promptly in LTT animals (9.5 +/- 1.8 days) as in naive animals (7.8 +/- 1.0 days).
  • the partially thymectomized animals and the sham thymectomized control rejected TP class II mismatched skin grafts in 8, 10 and 12 days, respectively.
  • LTT animal #10511 and #10719 were challenged by donor class I plus third party class II (SLA CC ) skin grafts.
  • Skin graft techniques were performed as follows. Split-thickness skin grafts (4x4cm) were harvested from donors with a Zimmer dermatome and placed on a graft bed, also prepared with a dermatome, on the dorsum of recipients. Grafts were protected with an occlusive dressing for the first three postoperative days. The time of rejection was defined as the day on which less than 10% of the skin was viable by criteria of warmth, color and texture.
  • the two partially thymectomized animals developed low anti-donor CML reactivity (8 to 15 % specific lysis) during the rejection crisis, but became specifically unresponsive thereafter (Figure 2C).
  • the two completely thymectomized animals developed strong, specific antidonor reactivity during the rejection crisis, as has previously been seen in untreated rejector pigs, and anti-third party reactivity was maintained (Figure 2D).
  • HBSS Hank's buffered saline solution
  • LSM Separation Medium
  • CML assays were performed as follows.
  • Media for CML assays consisted of RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 6% fetal calf serum (FCS), lOOU/ml penicillin, 135 ⁇ g/ml streptomycin, 50, ⁇ g/ml gentamicin, 10 mM HEPES, 2mM 1-glutamine, ImM sodium pyruvate, and 5x10" ⁇ M ⁇ -2 mercaptoethanol.
  • Media used for the effector phase of CML assays consisted of Basal Medium Eagle (GIBCO) supplemented with 6% serum replacement media (Sigma).
  • lymphocyte cultures containing 4x10 ⁇ responder and 4x10" irradiated (25 Gy) stimulator peripheral blood lymphocytes (PBL) in 2 ml of medium were incubated for 6 days at 37°C in 7% C02 and 100% humidity.
  • Bulk cultures were harvested, and effector cells were tested on ⁇ C ⁇ labeled blasts. The tests were run at serially diluted ratios (100:1 ; 50:1; 25:1; 12:1).
  • Humoral responses were determined as follows. Animals were tested for the presence of anti-donor or third-party cytotoxic antibody (Ab) by a two-stage complement dependent lymphocytotoxicity assay as previously described (Pescovitz, M.D. et al. (1984) J. Exp. Med. 160:1495-1508).
  • the medium used consisted of medium 199 (GIBCO) with 1% FCS added as a protein source. Targets matched to the MHC of the donor kidney were used to test for the presence of Ab. Briefly, 25 ⁇ l serial dilutions of sera were performed in 96-well round-bottomed plates.
  • target cells fresh PBL labeled with 51Cr at a concentration of 2xl ⁇ 6/ml
  • a positive control alloantiserum
  • a normal pig serum to control for non-specific complement cytotoxicity.
  • Figure 3 A shows an example of the bands obtained for IFN ⁇ and GAPDH probes on northern blots of kidney biopsy tissues for one partially thymectomized (#10233) and one totally thymectomized (#10770) animal at several time points post-transplant.
  • a plot of the densitometry readings for these bands, calculated as expression indices for IFN ⁇ relative to GAPDH is shown in Figure 3B.
  • IL-10 expression was relatively high in the partially thymectomized animal #10310 on POD 8,11,18 and 30.
  • IL- 10 message was not measured beyond day 11 in animals #10233 (partially thymectomized) and 10418 (sham thymectomized), but both of these animals showed high IL-10 expression relative to IFN ⁇ expression up to day 1 1.
  • Animal #10770 (complete thymectomy) expressed a high level of IL-10 on POD 8, but this expression was transient and fell to very low levels over the following week.
  • the sham-thymectomized animal (#10418) expressed a strong signal on POD 8 and 1 1, with very little expression of IFN ⁇ .
  • IL-10 probe was a cDNA fragment of 0.18 kb generated by PCR from pig DNA using primers chosen from the most homologous portions of human and mouse IL-10 sequences.
  • Northern blot analysis was performed as described (Meinkoth, J. et al. (1984) Anal. Biochem. 138:267). Briefly, 20 ⁇ g samples of RNA were loaded on 1.2% formaldehyde agarose gels, run overnight at 22 Volts, washed in distilled water twice for 10 min and then incubated in 10X SSC for one hour at room temperature.
  • the gels were then blotted onto Zetaprobe membranes (Bio-Rad) and cross-linked in a UV Stratalinker (Stratagene) by being exposed to 1200 ⁇ joules.
  • Probes were labeled with 32p ATP and dCTP to a specific activity of 10 ⁇ cpm/ ⁇ g by random priming (Promega). Filters were prehybridized for at least 4 hours in 10% Dextran Sulfate, 4X SSC, 40% Formaldehyde, 5X Denhardt's, 0.5 % SDS, 20 ug/ml salmon sperm DNA, and then hybridized to 32p labeled probes in the same solution at 42°C for 14 hours and developed as autoradiographs.
  • Another potential route by which donor antigen could reach the thymus after a renal allograft might be by indirect presentation of that antigen on the surface of host antigen presenting cells which could pick up antigen in the transplant and then carry it to the thymus.
  • this possibility is particularly attractive for the CD4 helper T cell pathway, since host and donor share the same class II antigens.
  • induction of tolerance to host class II plus allogeneic peptides might produce tolerance to the same allogeneic determinants which would be encountered on donor antigen presenting cells in the graft.
  • Thl clones producing IL-2 and IFN ⁇ would be immunostimulatory with respect to transplant immunity
  • Th2 clones secreting the cytokines IL-4 and IL-10 would be immunosuppressive of the same reactions
  • Th2 cells can inhibit IFN ⁇ and IL-2 production by Thl clones, the net result of a low Thl/Th2 ratio might be local suppression of transplant rejection.
  • the activity (or number or concentration) of the cells and/or thymic products responsible for the promotion of tolerance can be maintained, or enhanced, to promote tolerance. This may be particularly important in older subjects.
  • the following procedure was designed to lengthen the time an implanted organ (a xenograft) survives in a xenogeneic host prior to rejection.
  • the organ can be any organ, e.g., a liver, e.g., a kidney, e.g., a heart.
  • the main strategies are elimination of natural antibodies by organ perfusion, transplantation of tolerance-inducing bone marrow, optionally, the implantation of donor stromal tissue, and the administering growth hormone, to enhance host or donor thymus function (and optionally, in the case of a male recipient administration of a drug which mimics orchiemectomy).
  • Preparation of the recipient for transplantation includes any or all of these steps. Preferably they are carried out in the following sequence.
  • a preparation of horse anti-human thymocyte globulin (ATG) is intravenously injected into the recipient.
  • the antibody preparation eliminates mature T cells and natural killer cells. If not eliminated, mature T cells would promote rejection of both the bone marrow transplant and, after sensitization, the xenograft itself.
  • the ATG preparation also eliminates natural killer (NK) cells. NK cells probably have no effect on the implanted organ, but would act immediately to reject the newly introduced bone marrow.
  • Anti-human ATG obtained from any mammalian host can also be used, e.g., ATG produced in pigs, although thus far preparations of pig ATG have been of lower titer than horse-derived ATG.
  • ATG is superior to anti-NK monoclonal Antibodies, as the latter are generally not lytic to all host NK cells, while the polyclonal mixture in ATG is capable of lysing all host NK cells.
  • Anti-NK monoclonal antibodies can, however, be used.
  • the presence of donor antigen in the host thymus during the time when host T cells are regenerating post-transplant is critical for tolerizing host T cells. If donor hematopoietic stem cells are not able to become established in the host thymus and induce tolerance before host T cells regenerate repeated doses of anti-recipient T cell antibodies may be necessary throughout the non-myeloablative regimen. Continuous depletion of host T cells may be required for several weeks. Alternatively, e.g. if this approach is not successful, and tolerance (as measured by donor skin graft acceptance, specific cellular hyporesponsiveness in vitro, and humoral tolerance) is not induced in these animals, the approach can be modified to include host thymectomy and implantation of donor thymus..
  • thymectomized recipients host T cells do not have an opportunity to differentiate in a host thymus, but must differentiate in the donor thymus. If this is not possible, then the animal has to rely on donor T cells developing in the donor thymus for immunocompetence. Immunocompetence can be measured by the ability to reject a non-donor type allogeneic donor skin graft, and to survive in a pathogen-containing environment.
  • the recipient is administered low dose radiation in order to create hematopoietic space for newly injected bone marrow cells.
  • natural antibodies are absorbed from the recipient's blood by hemoperfusion of a liver of the donor species.
  • Pre-formed natural antibodies are the primary agents of graft rejection.
  • Natural antibodies bind to xenogeneic endothelial cells and are primarily of the IgM class. These antibodies are independent of any known previous exposure to antigens of the xenogeneic donor.
  • B cells that produce these natural antibodies tend to be T cell- independent, and are normally tolerized to self antigen by exposure to these antigens during development. The mechanism by which newly developing B cells are tolerized is unknown.
  • the liver is a more effective absorber of natural antibodies than the kidney.
  • the fourth step in the non-myeloablative procedure is to implant donor stromal tissue, preferably obtained from fetal liver, thymus, and/or fetal spleen, into the recipient, preferably in the kidney capsule.
  • donor stromal tissue preferably obtained from fetal liver, thymus, and/or fetal spleen
  • Stem cell engraftment and hematopoiesic across disparate species barriers is enhanced by providing a hematopoietic stromal environment from the donor species.
  • the stromal matrix supplies species-specific factors that are required for interactions between hematopoietic cells and their stromal environment, such as hematopoietic growth factors, adhesion molecules, and their ligands.
  • fetal liver can also serve as an alternative to bone marrow as a source of hematopoietic stem cells.
  • the thymus is the major site of T cell maturation.
  • Each organ includes an organ specific stromal matrix that can support differentiation of the respective undifferentiated stem cells implanted into the host.
  • adult thymus may be used, fetal tissue obtained sufficiently early in gestation is preferred because it is free from mature T lymphocytes which can cause GVHD. Fetal tissues also tend to survive better than adult tissues when transplanted.
  • thymic stromal tissue can be irradiated prior to transplantation, e.g., irradiated at 1000 rads.
  • fetal liver cells can be administered in fluid suspension.
  • BMC bone marrow cells
  • another source of hematopoietic stem cells e.g., a fetal liver suspension
  • Donor BMC home to appropriate sites of the recipient and grow contiguously with remaining host cells and proliferate, forming a chimeric lymphohematopoietic population.
  • BMC bone marrow cells
  • newly forming B cells (and the antibodies they produce) are exposed to donor antigens, so that the transplant will be recognized as self.
  • Tolerance to the donor is also observed at the T cell level in animals in which hematopoietic stem cell, e.g., BMC, engraftment has been achieved.
  • Recipient (or donor) thymic activity can be enhanced by the administration of growth hormone or (if the recipient is a male), administering a substance, e.g., a drug which mimics orchiemectomy. Administration can begin prior to, at the time of, or after, implantation of donor tissue.
  • a useful dosage and course of administration of an administered substance can be determined by trials in a model system (e.g., one of the systems for the induction of tolerance described in one of the above referenced U.S. patent applications) in which the substance is administered at various doses and at various times. While any of these procedures may aid the survival of an implanted organ, best results are achieved when all steps are used in combination.
  • Methods of the invention can be used to confer tolerance to allogeneic grafts, e.g., wherein both the graft donor and the recipient are humans, and to xenogeneic grafts, e.g., wherein the graft donor is a nonhuman animal, e.g., a swine, e.g., a miniature swine, and the graft recipient is a primate, e.g., a human.
  • the donor of the implant and the individual that supplies either the tolerance-inducing hematopoietic cells or the liver to be perfused should be the same individual or should be as closely related as possible.
  • the methods of the invention may be employed with other mammalian recipients
  • host antibodies can be depleted by administration of an excess of hematopoietic cells.
  • 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, or promoting the acceptance of, 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.
  • 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 allogeneic 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 allogeneic 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.
  • Some of the methods described herein use lethal irradiation to create hematopoietic space, and thereby prepare a recipient for the administration of xenogeneic, stem cells.
  • the use of sublethal levels of bone marrow depletion allows the generation of mixed chimerism in the recipient.
  • Mixed chimerism is generally preferable to total or lethal ablation of the recipient bone marrow followed by complete reconstitution of the recipient with administered stem cells.
  • any method of the invention calling for the irradiation of a recipient, e.g., a primate, e.g., a human, recipient, of a xenograft or allograft, the radiation can either be delivered in a single exposure, or more preferably, can be fractionated into two or more exposures or sessions.
  • a recipient e.g., a primate, e.g., a human, recipient
  • 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.
  • 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 of tolerizing 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, can be given on day -2, -1, and 0, and
  • WBI thymic irradiation
  • stem cell e.g., bone marrow stem cells
  • the graft e.g., a renal allograft, is transplanted on day 0.
  • Methods of the invention can include recipient splenectomy.
  • hemoperfusion e.g., hemoperfusion with a donor organ
  • Other methods for depleting or otherwise inactivating natural antibodies can be used with any of the methods described herein.
  • drugs which deplete or inactivate natural antibodies e.g., deoxyspergualin (DSG) (Bristol), or anti-IgM antibodies
  • DSG deoxyspergualin
  • anti-IgM antibodies can be administered to the recipient of an allograft or a xenograft.
  • DSG or similar drugs
  • anti-IgM antibodies can be used to deplete or otherwise inactivate recipient natural antibodies in methods of the invention.
  • DSG at a concentration of 6 mg/kg/day, i.v. has been found useful in suppressing natural antibody function in pig to cynomolgus kidney transplants.
  • 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 other procedures can replace or reduce the need for WBI.
  • 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.
  • thymic T cells are also included in embodiments of the invention. Some of the methods described herein include the administration of thymic irradiation to inactivate host thymic-T cells or to otherwise diminish the host's thymic-T cell mediated responses to donor antigens.
  • thymic irradiation called for in allogeneic or xenogeneic methods of the invention can be supplemented with, or replaced by, other treatments which diminish (e.g., by depleting thymic-T cells and/or down modulating one or more of the T cell receptor (TCR), CD4 co- receptor, or CD8 co-receptor) the host's thymic-T cell mediated response.
  • TCR T cell receptor
  • CD4 co- receptor CD4 co- receptor
  • CD8 co-receptor CD8 co-receptor
  • anti-T cell antibodies e.g., anti- CD4 and/or anti-CD8 monoclonal antibodies administered a sufficient number of times, in sufficient dosage, for a sufficient period of time, to diminish the host's thymic-T cell mediated response.
  • anti-T cell antibodies should be administered repeatedly.
  • anti-T cell antibodies can be administered one, two, three, or more times prior to donor bone marrow transplantation.
  • a pre-bone marrow transplantation dose of antibodies will be given to the patient about 5 days prior to bone marrow transplantation. Additional, earlier doses 6, 7, or 8 days prior to bone marrow transplantation can also be given. It may be desirable to administer a first treatment then to repeat pre-bone marrow administrations every 1 -5 days until the patient shows excess antibodies in the serum and about 99% depletion of peripheral T cells and then to perform the bone marrow transplantation.
  • Anti-T cell antibodies can also be administered one, two, three, or more times after donor bone marrow transplantation.
  • a post-bone marrow transplant treatment will be given about 2-14 days after bone marrow transplantation.
  • the post bone marrow administration can be repeated as many times as needed. If more than one administration is given the administrations can be spaced about 1 week apart. Additional doses can be given if the patient appears to undergo early or unwanted T cell recovery.
  • anti-T cell antibodies are administered at least once (and preferably two, three, or more times) prior to donor bone marrow transplantation and at least once (and preferably two, three, or more times) after donor bone marrow transplantation.
  • the myelosuppressive treatment sufficient to create hematopoietic space generally results in a reduction in white blood cell (WBC) levels (as revealed, e.g., by WBC counts) and the WBC reduction serves as a marker for the presence o hematopoietic space.
  • WBC white blood cell
  • the marker is a conservative one since WBC counts may recover at a time when space is still present in an animal. Accordingly, in any method which involves hematopoietic stem cell transplantation, and thus also requires the creation of hematopoietic space in a recipient, transplantation can be performed during the permissible window for engraftment following creation of space for the hematopoietic stem cells.
  • white blood cell levels can be followed 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 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., genetically modified
  • methods of the invention include 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.

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Abstract

L'invention concerne un procédé permettant de promouvoir, chez un mammifère receveur, l'acceptation d'un greffon provenant d'un mammifère donneur. Ce procédé consiste à induire une tolérance au greffon chez le receveur, à implanter le greffon chez ce dernier et à renforcer la fonction du thymus chez ledit receveur.
PCT/US1995/010598 1994-08-26 1995-08-18 Transplantation allogenique et xenogenique WO1996006642A1 (fr)

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US6197294B1 (en) 1998-10-26 2001-03-06 Neurotech S.A. Cell surface molecule-induced macrophage activation

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EUROPEAN JOURNAL OF IMMUNOLOGY, Volume 6, issued 1976, ASHERSON et al., "Adult Thymectomy Prevention of the Appearance of Suppressor T Cells Which Depress Contact Sensitivity to Picryl Chloride and Reversal of Adult Thymectomy Effect by Thymus Extract", pages 699-703. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 87, issued June 1990, LAPIDOT et al., "Enhancement of Bone Marrow Allografts from Nude Mice Into Mismatched Recipients by T Cells Void of Graft-Versus Host Activity", pages 4595-4599. *
THE JOURNAL OF IMMUNOLOGY, Volume 135, No. 3, issued 03 September 1985, UHTEG et al., "Cyclosporine-Induced Transplantation Unresponsiveness is Rat Cardiac Allograft Recipients: in Vitro Determination of Helper and Suppressor Activity", pages 1800-1805. *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197294B1 (en) 1998-10-26 2001-03-06 Neurotech S.A. Cell surface molecule-induced macrophage activation
US6225448B1 (en) 1998-10-26 2001-05-01 Neurotech S.A. 1gG /transferrin receptor fusion protein
US6506891B2 (en) 1998-10-26 2003-01-14 Neurotech S.A. Cell surface molecule-induced macrophage activation
US7189837B2 (en) 1998-10-26 2007-03-13 Neurotech S.A. Cell surface molecule-induced macrophage activation

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