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WO1996014853A1 - Implantation intrathymique de cellules souches - Google Patents

Implantation intrathymique de cellules souches Download PDF

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WO1996014853A1
WO1996014853A1 PCT/US1995/014773 US9514773W WO9614853A1 WO 1996014853 A1 WO1996014853 A1 WO 1996014853A1 US 9514773 W US9514773 W US 9514773W WO 9614853 A1 WO9614853 A1 WO 9614853A1
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cells
donor
graft
hematopoietic cells
positive hematopoietic
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PCT/US1995/014773
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English (en)
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Margaret D. Allen
Robert G. Andrews
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University Of Washington
Fred Hutchinson Cancer Research Center
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Priority to AU42362/96A priority Critical patent/AU4236296A/en
Publication of WO1996014853A1 publication Critical patent/WO1996014853A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K35/14—Blood; Artificial blood
    • 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
    • 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
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • 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
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • 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

Definitions

  • Hematopoietic chimerism can currently be produced in humans by allogeneic bone marrow transplantation following myeloablation.
  • Concomitant allogenic marrow and solid organ transplantation has been proposed as a method of producing chimerism and, perhaps, tolerance to the solid organ graft. This would entail the risks of myeloablative therapy and/or graft versus host disease.
  • the excellent early graft survival rates in cardiac transplantation are such that the risks incurred by reproducing full marrow transplant myelosuppressive strategies might override the benefits of chimerism.
  • the present invention provides compositions and methods for enhancing engraftment of a graft in a transplant recipient.
  • the graft is obtained from a donor who is different from the transplant recipient, i.e., allogeneic or xenogeneic.
  • CD34-positive hematopoietic cells obtained from the donor are implanted in the thymus of the transplant recipient in amount sufficient to establish tolerance to the graft, thereby enhancing engraftment of the graft in the recipient.
  • the CD34-positive hematopoietic cells may be obtained from the bone marrow or peripheral blood of the graft donor, and preferably are at least about 50% pure for CD34- positive cells, more preferably at least approximately 98% pure.
  • CD34-positive hematopoietic cells are implanted in the thymus of the transplant recipient.
  • the implantation can be by way of injection into at least one lobe of the thymus following thoracotomy, thoraacotamyor by injection using a thoracoscope, CT-guided or ultrasound guided percutaneous injection, catheter injection under fluoroscope guidance, or mediastinoscopy.
  • the CD34-positive hematopoietic cells of the donor can be implanted in the transplant recipient prior to transplanting the graft, concurrently with transplanting the graft, or subsequent to the transplantation. Periodic maintenance administration to the thymus may be necessary to maintain adequate levels of tolerance in some patients.
  • the graft which can be transplanted is a solid organ, tissue or cell collection.
  • the solid organ can be a heart, lung, heart-lung, kidney, pancreas, intestine or liver.
  • a suitable tissue for transplantation according to the present methods is a vessel, heart valve, connective tissue or skin.
  • the transplanted cell collection can comprise CD34-positive hematopoietic cells or cells which express a disease- associated gene product of interest. A microchimerism of donor hematopoietic cells may be detectable in the blood of the transplant recipient for a substantial time after transplant according to the methods described herein.
  • the invention provides a method for producing a gene product of interest in a host mammal in need of said gene product.
  • This aspect comprises implanting in the thymus of the host CD34-positive hematopoietic progenitor cells which encode the gene product of interest.
  • the CD34-positive hematopoietic cells can be genetically engineered to produce the gene product of interest in the recipient host.
  • prior to implanting the modified cells in the host the CD34-positive hematopoietic cells are obtained from the recipient host mammal and genetically engineered to produce the gene-product of interest.
  • the CD34-positive hematopoietic cells can be those obtained from a donor of the same or different species as the recipient and which cells produce the gene product in said donor.
  • the present invention provides methods and compositions for intrathymic administration of CD34-positive donor cells into a transplant recipient to produce lymphohematopoietic microchimerism that results in prolonged survival of transplanted grafts or organs with or without conventional immunosuppression.
  • the invention provides methods and compositions for inducing tolerance to a donor cell, tissue or organ in a recipient individual.
  • tolerance is meant to refer to an immune response to an allogeneic or xenogeneic graft that is smaller than the immune response that would be observed in an animal which has not received the thymic implant of nonautologous CD34-positive hematopoietic cells, thereby resulting in an extension of graft survival time.
  • the engraftment of donor material into transplant recipients can be accomplished safely and substantially without the risk of graft versus host disease.
  • implantation of donor CD34-positive hematopoietic cells into the thymus provides a level of tolerance in the recipient host to the donor tissue that is sufficient to permit engraftment.
  • the direct intrathymic injection of CD34-positive hematopoietic donor fractions produces lymphohematopoietic microchimerism and thus a tolerance which prolongs survival of the graft.
  • the nature of the graft intended for transplantation into a recipient host varies widely.
  • the grafts include, for example, organ transplants, such as heart, heart-lung, lung, kidney, kidney-pancreas, liver, pancreas. intestines, etc.
  • the grafts can also include tissue transplants, such as of skin, connective tissue, vessels, heart valves, etc.
  • the grafts can be of cells from a donor, such as bone marrow cells, including the CD34-positive hematopoietic cells which are used to induce tolerance, wherein the CD34-positive hematopoietic cells include stem (progenitor) cells.
  • Other suitable cell collections include pancreatic islet cells, hepatic cells, bone marrow or stem cells.
  • the grafts can also be CD34- positive hematopoietic cells which express a desired protein or chemical or which could be genetically engineered to produce a desired protein or chemical as can be used in treatment of individuals with genetic or acquired deficiencies.
  • the donor CD34-positive hematopoietic cells and grafts can also be xenografts, i.e., from a different species than the recipient, for example, a non-human primate or pig graft can be implanted into a human.
  • graft is meant to include the implant in an individual of any non-autologous organ, tissue or cells, unless the context specifically indicates otherwise.
  • Engraftment of the CD34-positive hematopoietic cells themselves, without a subsequent graft, may also be used to treat genetic or acquired deficiencies.
  • the CD34-positive cells could also be genetically engineered prior to intrathymic implant to produce proteins, chemicals or specific enhancers or suppressors of cell function. Because stem cells may produce progeny, a single application/injection/treatment may be sufficient for the patient's lifetime.
  • CD34-positive hematopoietic cells substantially depleted of mature T and B lymphocytes, reduces the risk of graft versus host disease in the recipient individual while providing the ability to modify the cellular constituency of the hematopoietic system of the recipient over a prolonged period.
  • the CD34 antigen is present on substantially all hematopoietic precursor cells, but is substantially absent from more mature hematopoietic cells.
  • CD34- positive hematopoietic cells include those cells which express the CD34 antigen, among other surface antigens, and include totipotent stem cells as well as committed progenitor cells. The level of expression of the CD34 antigen will vary from one cell type to another.
  • a cell is operationally defined as CD34-positive if it expresses sufficient CD34 antigen to be detected by a given method of assay, e.g., by flow microfluorimetry using a fluorescence-activated cell sorter (FACS) , by immunofluorescence or immunoperoxidase staining using a fluorescence or light microscope, by radioimmunoassay, or by immunoaffinity chromatography, among numerous other methods which will be readily apparent to one skilled in the art. See, for example, Lansdorp and Thomas, in Bone Marrow Processing and Purging. A.P. Gee (ed.), Boca Raton: CRC Press (1991) pg. 351.
  • FACS fluorescence-activated cell sorter
  • the CD34-positive hematopoietic cells may be obtained from a variety of blood products of the intended donor.
  • the present invention provides methods for inducing tolerance to the tissue or organ of the donor in the intended recipient, it obviates the necessity of employing as a donor an individual who matches or closely matches the recipient's histocompatibility type.
  • allogeneic and xenogeneic transplants are made possible by the present invention without employing myeloablation.
  • immunosuppressive regimens can be used which are less toxic to the transplant recipient than those regimens employed in the absence of tolerance to antigens on the donor's CD34-positive cells.
  • baboons As a source of xenografts for humans, baboons have been used in xenograft transplants over the past 20 years with surprising success and, therefore, are considered phylogenetically quite similar to humans, allowing the present invention to be extended to xenograft applications.
  • Other species may also serve as graft donors, such as swine which can serve as a source of, e.g., skin and other tissues for engraftment onto humans.
  • Sources of CD34-positive cells include bone marrow, peripheral blood, umbilical cord blood, fetal liver, and spleen of the intended tissue donor.
  • Bone marrow is a particularly rich source of precursor cells (1-2% of marrow) , but alternate sources may be preferable because of the discomfort associated with bone marrow aspiration.
  • Bone marrow is typically aspirated from the iliac crest, but may be obtained from other sites (such as the sternum or vertebral bodies) if necessitated by prior or concurrent disease or therapy. In the case of cadaveric donors, vertebral bodies are a convenient source of large quantities of CD34-positive hematopoietic cells.
  • peripheral blood contains fewer precursor cells (typically ⁇ 1% of peripheral blood mononuclear cells) , it is generally easier to obtain than bone marrow.
  • the number of precursor cells circulating in peripheral blood can be increased by prior exposure of the donor to certain growth factors, such as, for example, G-CSF or SCF (KL) , and/or certain drugs.
  • G-CSF G-CSF
  • SCF SCF
  • certain drugs such as, for example, antibody to VLA-4, given intravenously, results in release of CD34-positive hematopoietic cells from marrow stores into peripheral blood.
  • administering can facilitate expeditious recovery of CD34-positive cells from peripheral blood.
  • blood may be obtained by venipuncture or by one or more aphereses on a blood separator.
  • a bone marrow or peripheral blood specimen or apheresis product into precursor and mature cells, (such as CD34-positive and CD34-negative populations)
  • Methods for the preparation of buffy coats and mononuclear cell fractions are well-known in the art (e.g., Kumar and Lykke, Pathology 16:53 (1984)). Separation of precursor cells from more mature cells can be accomplished by any of a variety of methods known to those skilled in the art, including immunoaffinity chromatography (Basch et al., J. Immunol.
  • CD34-negative fractions is rarely complete.
  • separation and substantial purification or enrichment of CD34-positive hematopoietic cells is considered to have been accomplished if the target fraction is comprised of at least about 10% CD34-positive cells, typically at least about 50%, more typically at least about 70% CD34-positive hematopoietic cells, preferably at least about 90%, more preferably about 95%, and even more preferably about 98 to 99% or more CD34-positive hematopoietic cells.
  • the CD34-positive hematopoietic cells may be positively selected or negatively selected.
  • positive selection is meant the capture of cells by some means, usually immunological, on the basis of their expression of a specific characteristic or set of characteristics (usually an antigen(s) expressed at the cell surface) .
  • CD34-positive cells can be positively selected by any of the above methods (except cytolysis, which would result in destruction of the desired cells) on the basis of their expression of the CD34 antigen utilizing an anti-CD34 antibody, such as the monoclonal antibodies 12.8, My-10, and 8G12 (commercially available from Becton Dickinson Co. , Mountain View, CA) , or Q-Bend 10 (commercially available from Biosystems Ltd. , Waterbeach, Cambridge, England) .
  • CD34-positive cells usually involves the use of one or more antibodies or fragments thereof, in some cases selection may involve the use of lectins or other types of receptors or ligands expressed on the cell surface.
  • antigens, receptors and ligands which may be useful, alone or in combination with other markers, for separating CD34-positive cells from CD34-negative cells are transferrin, the transferrin receptor, soybean agglutinin, c-kit ligand, c-kit receptor, HLA-DR, CD33, etc.
  • Negative selection means the exclusion or depletion of cells by some means, usually immunological, on the basis of their lack of expression of a specific characteristic or set of characteristics (again, usually a surface antigen) .
  • CD34-positive cells can be negatively selected by any of the above methods on the basis of their lack of expression of lineage-defining antigens, such as CD19 (for B lymphocytes) , CD3 (for T lymphocytes) , CD56 (for NK cells) , etc. , utilizing antibodies to the above-mentioned and other lineage-defining antigens.
  • CD34-positive hematopoietic cells By using a cocktail or mixture of monoclonal antibodies directed to red cell, platelet, granulocyte, lymphocyte and/or tumor cell antigens, it is possible to leave behind a population of cells which is highly enriched for CD34-positive cells.
  • Numerous monoclonal and polyclonal antibodies suitable for this purpose are known in the art (see, e.g., Leukocyte Typing IV, Knopp et al. (eds.), Oxford UP, 1989) and are commercially available from a wide variety of sources (for example, Becton Dickinson Co., Mountain View, CA; Coulter Immunology, Hialeah, FL; Ortho Diagnostics, Raritan, NJ, etc.).
  • the CD34-positive hematopoietic cells can also be separated from mature cells by a combination of negative and positive selection techniques.
  • the separated CD34-positive hematopoietic cells may be used immediately suspended in an isotonic solution, stored frozen in a DMSO medium or other suitable freezing medium and thawed at a later date for use, and/or inoculated into a suitable vessel containing a culture medium comprising a conditioned medium and nutritive medium, optionally supplemented with a source of growth factors and, optionally, human or other animal plasma or serum. If cultured, the resultant cell suspension may be cultured under conditions and for a time sufficient to increase the number of hematopoietic precursor cells relative to the number of such cells present initially in the blood product.
  • the cells may then be separated by any of a variety of methods, such as centrifugation or filtration, from the medium in which they have been cultured, and may be washed one or more times with fresh medium or buffer.
  • the cells may be re-separated into CD34-positive and -negative fractions, prior to resuspension to a desired concentration in a medium or buffer suitable for injection.
  • a cell separator device is also provided for separating target cells from non-target cells, one embodiment being the CEPRATE SCTM cell separation system described in Berenson et al. fAdv. Bone Marrow Purging & Processings. N.Y.: Wiley-Liss, 1992, pg. 449).
  • the donor CD3 -positive hematopoietic cells can then be introduced into the thymus of the transplant recipient.
  • the thymus provides the CD34- positive cells with an extended half-life, and the implanted cells are treated as "self" by the host, thereby resulting in donor-specific tolerance to the graft.
  • Thoracic organ transplantation provides a convenient opportunity to implant donor marrow components directly into the thymus, which is readily accessible during the cardiac or lung transplant operation. In the absence of thoracic organ transplantation, a small left anterior thoracotomy is performed in the second intercostal space which provides excellent access to the thymus.
  • the purified CD34-positive donor cells is injected in different portions of at least one lobe and typically both lobes of the thymus.
  • the thoracotomy incision is closed and a chest radiograph can be used to ensure full left lung inflation.
  • Advances in videoscopic, thoracoscopy, mediastinoscopy and CT-guided or ultrasound guides percutaneous procedures make intrathymic delivery of cells possible without thoracic surgery. Since injection of cells is accomplished by simple inoculation through a needle tip, unusual/sophisticated instrumentation is not necessary.
  • percutaneous injection can be readily accomplished.
  • In utero injection using ultrasound or fetoscopy is also possible for use of the present methods in fetuses.
  • selective catheterization of thymic veins (via the innominate vein) or arteries allows fluoroscopically- guided injection of cells into the thymus in the radiology suite.
  • CD34-positive hematopoietic cells may be administered via the peripheral blood circulation to the thymus, including the use of agents which target said cells to the thymus.
  • the tolerizing CD34-positive hematopoietic cells from the donor can be implanted in the thymus of the transplant recipient prior to, at the time of, or shortly following graft transplantation.
  • the CD34-positive cells are typically implanted from one to two weeks prior to graft transplant, more often about two to four weeks prior to transplant, or in some cases from four to eight weeks or more prior to transplant.
  • the recipient can be assayed for the development of hematopoietic microchimerism such that timing of implantation can be coordinated to achieve levels of tolerance.
  • the level of tolerance can be assessed in the recipient indirectly by determining the level of microchimerism in the blood using markers specific for the donor cells as measured by polymerase chain reaction, etc. , as set forth in more detail below.
  • probes can be used to demonstrate peripheral hematopoietic microchimerism in blood samples from the transplant recipient.
  • PCR analysis and oligonucleotide probes are generally described in, e.g., Erlich et al., Arch. Pathol. Lab. Med. 117: 482-485 (1993); Gaur et al., J. Mol. Evol. 9:599-609 (1992); and Nuovo, PCR In Si tu Hybridization. Protocols and Applications.
  • Probes specific to individual donors are used not only to identify donor cells in recipient peripheral blood, but also to detect donor cells in recipient lymphoid tissue.
  • a microchimerism typically of at least about 0.01% cells derived from the donor is sufficient to alter immune status in recipients; levels of from about 0.01% to 1.0%, up to 5% or higher are sufficient to establish tolerance to the graft in the recipient.
  • Other methods for measuring tolerance include acceptance of small patches of donor skin grafts on the recipient skin.
  • a diminished mixed lymphocyte reaction (MLR) of the recipient's lymphocytes to those of the donor may also be indicative of a level of tolerance.
  • MLR mixed lymphocyte reaction
  • the CD34-positive hematopoietic cells can also be implanted into the thymus at approximately the same time as the graft transplant. This will often occur when the CD34-positive cells are not available in advance, e.g., as with a cadaveric donor.
  • the graft is a heart, heart-lung or lung transplant the thymus can be conveniently implanted with the CD34-positive cells during the same surgical procedure.
  • Donor CD34-positive hematopoietic cells can also be administered to the graft recipient post-transplant, either to induce tolerance or to maintain a level of tolerance that was previously established. To induce tolerance the cells are implanted as soon after the transplant as possible, typically within about 1-14 days of the transplant procedure. Percutaneous inoculation of CD34- positive donor cells into the thymus under CT guidance, or videoscopic introduction of the stem cells, permits intrathymic implantation of cells at a variety of timepoints. Maintenance of a desired level of tolerance may require periodic intrathymic implants of CD34-positive hematopoietic donor cells in the transplant recipient.
  • the substantially pure preparation of CD34-positive cells are administered into the thymus in an amount sufficient to induce tolerance in the recipient to the graft being transplanted. This is referred to herein as a "tolerizing" dose.
  • a tolerizing dose can range from about 1 x 10 5 CD34- positive stem cells up to about 3 x 10 8 cells or more, but more typically from 5 x 10 5 to 1 x 10 8 to induce hematopoietic microchimerism, tolerance and subsequently prolongation of graft survival.
  • Dosing is of logistical importance because it determines whether a sufficient number of donor CD34-positive hematopoietic cells could be procured from one or two vertebral bodies, ribs, sternum, or iliac crest, all of which might be available early in an organ donor operation.
  • the equivalent of 2 vertebral bodies of marrow can be processed within 6-10 hours, assuring simultaneous implantation of donor stem cells during the subsequent organ implant operation, if desired.
  • a requirement for larger doses of stem cells necessitates complete vertebral body marrow procurement following organ harvest and thus may require up to 12-24 hours for processing.
  • a cardiac transplant can proceed and the donor marrow fractions are delivered to the recipient after the cardiac transplant.
  • the dose of cells required for engraftment determines whether a single donor can supply enough stem cells for multiple organ transplant recipients. Up to 14 x 10 8 marrow cells can potentially be recovered from a cadaver donor, with CD34-positive cells more prevalent (at 4.6%) in marrow obtained from vertebral bodies.
  • CD34-positive hematopoietic cells of the present invention can be employed without immunosuppressive therapies or in conjunction with such therapies, if desired.
  • Adjunct immunosuppressive therapy may also be employed, i.e., chemical (e.g., cyclosporin A, steroids, and/or azathioprine) anti-lymphocyte globulin or serum, or radiological yeloablation, and may be at levels less than those which would be typically employed in the absence of intrathymic implantation of CD34-positive hematopoietic stem cells.
  • therapies ca»n also accompany the present methods to facilitate tolerance and/or acceptance of the graft, such as, e.g., 0KT3, FKS06, other immunosuppressive drugs, and therapies to reduce humorally mediated rejection such as photophoresis, plasmapheresis, soluble complement fractions, cobra venom factor, genetic manipulation of donor organ endothelium or the complement activation cascade, methods for downregulating VCAM-1 or E- selectin expression or cytokine production and the like.
  • humorally mediated rejection such as photophoresis, plasmapheresis, soluble complement fractions, cobra venom factor, genetic manipulation of donor organ endothelium or the complement activation cascade, methods for downregulating VCAM-1 or E- selectin expression or cytokine production and the like.
  • donor CD34- positive hematopoietic cells intended for intrathymic implant can be genetically engineered to encode a gene product of interest.
  • Methods for retroviral transduction of stem cells are described in, e.g., Emery et al., Blood 81:2460-2465 (1993) , which is incorporated herein by reference.
  • This Example describes the intrathymic inoculation of CD34-positive marrow fractions for the induction of hematopoietic microchimerism and prolongation of skin allograft survival.
  • Initial studies were designed to assess the production of hematopoietic microchimerism by direct introduction into the thymus of a CD34-positive hematopoietic cell-rich donor marrow fraction defined by an anti-CD34 antibody, 12.8.
  • CD34-positive hematopoietic cells were isolated from marrow specimens obtained from adult male donor baboons and enriched to approximately greater than 98% pure according to the procedure described in Andrews et al. , Blood 80:1693-1701 (1992), incorporated herein by reference.
  • Peripheral blood obtained from each recipient was examined for the presence of male donor cells, detected by PCR amplification of a Y chromosome-specific (male-specific) determinant as described in Reits a and Harrison, Cytogenet. Cell Genet. 64:213-216 (1993), incorporated herein by reference. This assessment was made more than twice on each blood and marrow specimen, several months apart, to confirm the reproducibility of PCR results.
  • Two animals were treated with standard triple drug immunosuppression, consisting of cyclosporin A (sufficient to maintain whole blood HPLC levels at 300-500 ng/dl) , dexamethasone (0.5/mg/kg/day) , and azathioprine, 3 mg/kg/day.
  • the two control animals also with CD34-positive hematopoietic cell injections, received no immunosuppression.
  • the two immunosuppressed recipients developed peripheral blood microchimerism within two weeks of donor cell injection.
  • microchimerism as evidenced by PCR amplification of the male-specific determinant persisted for at least 16 months from the time of donor cell implantation (Table 1) .
  • microchimerism developed even earlier, at three days following donor cell implantation in one animal, at one week in the other.
  • Microchimerism persisted for at least 6 months in one animal, and 9 months in the other.
  • CD2+PBL 26-28 0/2 1/1 [4*10*] 4/5[l*10*] 1/2[7*10 3 ]
  • This Example demonstrates that intrathymic CD34- positive hematopoietic cell implantation and/or development of microchimerism prolongs survival of grafts from the cell donor.
  • Skin grafts were used as a test of donor-specific tolerance because skin grafts express non-MHC antigens which are not tolerized by hematopoietic cells and thus are a more difficult model of allograft acceptance.
  • Example I two juvenile female baboons were injected intrathymically with lxlO 6 CD34-positive marrow cells from an unrelated male donor baboon. Unlike Example I, CD34- positive cells were injected into both lobes of the recipient thymus. Because chimerism had developed earlier in the unimmunosuppressed animals in the previous study, neither of these two recipients was given immunosuppression. Using PCR amplification of the male-specific sequence, donor cells were detectable in the blood and marrow of both animals after transplantation, demonstrating hematopoietic chimerism.
  • the repeat third-party skin allografts were not rejected in an accelerated fashion.
  • This finding, as well as the survival of the first set of third-party allografts to 24 days, is indicative of a non-specific suppression of alloreactivity associated with microchimerism and/or intrathymic CD34-positive hematopoietic cell engraftment.
  • the two recipient baboons, who each served as third-party skin donors for the other may have inadvertently shared some key MHC antigens with the recipient. The latter possibility is unlikely since the recipient demonstrated full in vitro alloreactivity to this third-party donor on mixed lymphocyte culture.
  • the presence of alloantibody to donor antigens introduced in the thymus was determined using a two-color immunofluorescence technique and analyzed on a flow cytometer. Antibody in recipient serum binding to donor cells but not autologous cells or cells from the other recipient was interpreted as antibody to donor alloantigens. As shown in Table 2, in recipients of allogeneic CD34-positive hematopoietic cells and skin grafts, both recipients developed antibody to the cell donor. Results from a serum sample drawn after skin grafting are also shown. The animals identified as RS and RU are the CD34-positive cell recipients. Animal 90221 is the cell donor.
  • HLA class I MAb Positive Positive Positive
  • This Example describes the use of the present invention in primate xenograft transplantation by demonstrating that intrathymic injection of CD34+ fractions of human donor marrow can induce hematopoietic microchimerism in baboon recipients without immunosuppression.
  • a human donor- to-baboon recipient protocol was used as a model for baboon- to-human concordant xenograft organ (liver, kidney, heart, etc.) transplants.
  • the two recipient baboons for the xenografts were both juvenile females (10 to 12 weeks of age) , as in the allograft Examples.
  • Recipient serum was drawn pre-transplant to assess the presence of preformed anti-human antibodies.
  • 6.5 x 10 5 human CD34-positive donor hematopoietic cells were injected into 4 sites in both lobes of the recipient thymus, using a left thoracotomy approach.
  • One recipient was treated with standard triple drug immunosuppression (cyclosporin A, azathioprine, and steroids) ; the other recipient animal was not immunosuppressed.
  • Peripheral blood samples were taken at intervals to assess the development of hematopoietic microchimerism, as assayed by PCR amplification of human Y chromosome determinants. Serum was taken to assess the development of xenoantibodies.
  • skin grafts were placed on both recipients from both the human xenograft marrow donor and from an unrelated baboon allograft skin donor. Grafts were biopsied at 10, 18, and 28 days for PCR analysis to assess the presence of donor DNA.
  • xenoantibodies were not present in these juvenile baboons prior to CD34-positive hematopoietic cell transplantation.
  • the animal receiving human CD34-positive hematopoietic cells without immunosuppression (SD) developed antibody to cells from the human following hematopoietic cell implantation.
  • the animal that received human CD34-positive hematopoietic cells with triple therapy immunosuppression (SC) did not develop antibody to donor cells after stem cell infusion but did make antibody following challenge with a skin graft from the stem cell donor.
  • the host response to the human cell donor xenograft skin was quite different than to the skin allograft from the unrelated baboon.
  • the human xenograft stem cell donor

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Abstract

La prise de greffes chez des receveurs est favorisée par implantation de cellules hématopoiétiques CD34-positives, provenant du donneur du greffon, dans le thymus du receveur. Ces procédés favorisent la prise aussi bien d'allogreffes que d'hétérogreffes. L'invention concerne également un procédé permettant l'apport d'un produit génique désiré chez un mammifère hôte par implantation dans le thymus dudit hôte de cellules parentes hématopoiétiques qui codent le produit génique désiré. Les cellules hématopoiétiques CD34-positives peuvent être produites par génie génétique pour donner le produit génique désiré chez le receveur.
PCT/US1995/014773 1994-11-10 1995-11-09 Implantation intrathymique de cellules souches WO1996014853A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU42362/96A AU4236296A (en) 1994-11-10 1995-11-09 Intrathymic stem cell implantation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33760394A 1994-11-10 1994-11-10
US08/337,603 1994-11-10

Publications (1)

Publication Number Publication Date
WO1996014853A1 true WO1996014853A1 (fr) 1996-05-23

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PCT/US1995/014773 WO1996014853A1 (fr) 1994-11-10 1995-11-09 Implantation intrathymique de cellules souches

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Country Link
AU (1) AU4236296A (fr)
WO (1) WO1996014853A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075291A2 (fr) * 1999-06-08 2000-12-14 Novartis Ag EXPRESSION FORCEE DE BCL-xL
WO2001079452A2 (fr) * 2000-04-17 2001-10-25 Novartis Ag Construction tissulaire composite et organe composite tissu thymique-moelle induisant une tolerance
WO2002031110A2 (fr) * 2000-10-13 2002-04-18 Monash University Therapie genique aux cellules souches hematopoietiques
WO2002030351A2 (fr) * 2000-10-13 2002-04-18 Monash University Ameliorations de l'acceptation d'un greffon par manipulation de la regeneration thymique
US20130216495A1 (en) * 2012-02-21 2013-08-22 Baxter Healthcare Sa Pharmaceutical composition comprising cd34+ cells
EP3384006A4 (fr) * 2015-12-04 2019-06-12 Fred Hutchinson Cancer Research Center Utilisations de populations étendues de cellules souches/progénitrices hématopoïétiques

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WO1995003062A1 (fr) * 1993-07-21 1995-02-02 Cellpro, Incorporated Procedes et compositions de prevention du rejet immun de greffons organiques pleins

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WO1995003062A1 (fr) * 1993-07-21 1995-02-02 Cellpro, Incorporated Procedes et compositions de prevention du rejet immun de greffons organiques pleins

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075291A3 (fr) * 1999-06-08 2001-06-28 Novartis Ag Expression forcee de bcl-xl
WO2000075291A2 (fr) * 1999-06-08 2000-12-14 Novartis Ag EXPRESSION FORCEE DE BCL-xL
EP1428872A2 (fr) * 2000-04-17 2004-06-16 Novartis AG Construction tissulaire composite et organe composite tissu thymique-moelle induisant une tolerance
WO2001079452A2 (fr) * 2000-04-17 2001-10-25 Novartis Ag Construction tissulaire composite et organe composite tissu thymique-moelle induisant une tolerance
WO2001079452A3 (fr) * 2000-04-17 2002-04-18 Novartis Ag Construction tissulaire composite et organe composite tissu thymique-moelle induisant une tolerance
EP1428872A3 (fr) * 2000-04-17 2004-12-01 Novartis AG Construction tissulaire composite et organe composite tissu thymique-moelle induisant une tolerance
WO2002031110A2 (fr) * 2000-10-13 2002-04-18 Monash University Therapie genique aux cellules souches hematopoietiques
WO2002030351A3 (fr) * 2000-10-13 2002-07-04 Richard Boyd Ameliorations de l'acceptation d'un greffon par manipulation de la regeneration thymique
WO2002031110A3 (fr) * 2000-10-13 2002-06-20 Richard Boyd Therapie genique aux cellules souches hematopoietiques
WO2002030351A2 (fr) * 2000-10-13 2002-04-18 Monash University Ameliorations de l'acceptation d'un greffon par manipulation de la regeneration thymique
US20130216495A1 (en) * 2012-02-21 2013-08-22 Baxter Healthcare Sa Pharmaceutical composition comprising cd34+ cells
EP3384006A4 (fr) * 2015-12-04 2019-06-12 Fred Hutchinson Cancer Research Center Utilisations de populations étendues de cellules souches/progénitrices hématopoïétiques
US10813949B2 (en) 2015-12-04 2020-10-27 Fred Hutchinson Cancer Research Center Uses of expanded populations of hematopoietic stem/progenitor cells

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