WO1995003062A1 - Procedes et compositions de prevention du rejet immun de greffons organiques pleins - Google Patents

Procedes et compositions de prevention du rejet immun de greffons organiques pleins Download PDF

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WO1995003062A1
WO1995003062A1 PCT/US1994/008378 US9408378W WO9503062A1 WO 1995003062 A1 WO1995003062 A1 WO 1995003062A1 US 9408378 W US9408378 W US 9408378W WO 9503062 A1 WO9503062 A1 WO 9503062A1
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cells
recipient
stem cells
organ
solid
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PCT/US1994/008378
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English (en)
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Ronald J. Berenson
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Cellpro, Incorporated
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Priority to AU74044/94A priority Critical patent/AU7404494A/en
Publication of WO1995003062A1 publication Critical patent/WO1995003062A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins

Definitions

  • the present invention relates generally to solid organ transplantation and, more specifically, to methods and compositions suitable for inducing donor antigen- specific immunological tolerance in recipients of solid organ grafts.
  • kidney transplantation The science of transplantation of human organs has advanced to the point where more than 10,000 solid organ transplants, including heart, kidney, liver, and lung transplants, are performed in the U.S. annually.
  • solid organ transplants including heart, kidney, liver, and lung transplants, are performed in the U.S. annually.
  • the vast majority of these are cadaveric transplants, although some transplants, such as kidney transplants, are from living, usually related, donors.
  • Solid organ transplantation can be a highly cost-effective method for treating various diseases. For example, for end-stage renal disease kidney transplantation may be performed at approximately one-third of the cost of dialysis over the life of the patient (New Engl. J. Med. 77: 1243, 1991).
  • mixed chimerism a state in which there is a stable mixture of host and donor hematopoietic elements
  • the challenge then, as now, is to produce a durable chimeric state without eliciting graft versus host disease (GV ⁇ D) or precipitating acute organ rejection.
  • GV ⁇ D graft versus host disease
  • the preferred method of achieving a mixed chimeric state is to induce myelosuppression in the host by sublethal or total lymphoid irradiation, or by treatment with anti-lymphocyte serum (ALS), followed by transfusion of unfractionated donor bone marrow.
  • ALS anti-lymphocyte serum
  • mice In mice, this cell appears to be a small mononuclear cell which is la-, Thy-1-, and FcR+ (Gozzo et al., J. Immunol. 729: 1584, 1982; De Fazio et al., J. Immunol. 735:3035, 1985; De Fazio et al., Transplant Proc. 79:547, 1987).
  • the relevant cell type appears to be DR-, CD3-, CD2+, and CD 16+ (Thomas et al., Transplant. Proc. 23: 1 1, 1991). Starzle et al.
  • the present invention provides methods and compositions for preventing immune rejection of solid-organ grafts by tolerizing a recipient for solid- organ transplantation.
  • methods of tolerizing a recipient for solid-organ transplantation comprising the steps of (a) harvesting cells from an anticipated donor of a solid-organ, the cells including hematopoietic stem cells, (b) enriching hematopoietic stem cells from the harvested cells, and (c) infusing the enriched stem cells into a recipient, such that a state of tolerance to a transplanted solid-organ from the donor is induced in the recipient.
  • methods of tolerizing a recipient for solid-organ transplantation comprising the steps of (a) harvesting cells from an anticipated donor of a solid-organ, the cells including hematopoietic stem cells, (b) enriching hematopoietic stem cells from the harvested cells, (c) conditioning a recipient for sc ' ! -organ transplantation, and (d) infusing the enriched stem cells into the conditionec ⁇ ipient, such that a state o: tolerance to a transplanted solid-organ from the donor is induced in the recipient.
  • methods for transplanting solid organs comprising the steps of (a) harvesting cells from an anticipated donor of a solid-organ, the cells including hematopoietic stem cells, (b) enriching hematopoietic stem cells from the harvested cells, (c) infusing the enriched stem cells into an anticipated solid-organ graft recipient, such that a state of tolerance to a solid-organ from the donor is induced in the recipient, and (d) transplanting an organ from the donor into the recipient.
  • methods for transplanting solid organs comprising the steps of (a) harvesting cells from an anticipated donor of a solid-organ, the cells including hematopoietic stem cells, (b) enriching hematopoietic stem cells from the harvested cells, (c) transplanting an organ from the donor into the recipient, and (d) infusing the enriched stem cells into the recipient, such that a state of tolerance to a solid-organ from the donor is induced in the recipient.
  • methods for transplanting solid organs comprising the steps of (a) harvesting cells from an anticipated donor of a solid-organ, the cells including hematopoietic stem cells, (b) enriching hematopoietic stem cells from the harvested cells, (c) conditioning a recipient for solid-organ transplantation, and (d) infusing the enriched stem cells into the conditioned recipient, such that a state of tolerance to a solid-organ from the donor is induced in the recipient, and (e) transplanting an organ from the donor into the recipient.
  • methods for transplanting solid organs comprising the steps of (a) harvesting cells from an anticipated donor of a solid-organ, the cells including hematopoietic stem cells, (b) enriching hematopoietic stem cells from the harvested cells, (c) conditioning a recipient for solid-organ transplantation, (d) transplanting an organ from the donor into the conditioned recipient, and (e) infusing the enriched stem cells into the conditioned recipient, such that a state of tolerance to a solid-organ from the donor is induced in the recipient.
  • the invention subsequent to the step of enriching the stem cells and prior to the step of infusing the enriched stem cells into a conditioned recipient, further comprises the steps of (a) freezing the enriched stem cells, and (b) thawing the frozen stem cells.
  • the recipient may be conditioned by administration of an immunosuppressive agent selected from the group consisting of an anti-lymphocyte serum, anti-thymocyte globulin, anti-lymphocyte globulin, cyclosporine A, FK506, azathioprine, and cyclophosphamide.
  • an immunosuppressive agent selected from the group consisting of an anti-lymphocyte serum, anti-thymocyte globulin, anti-lymphocyte globulin, cyclosporine A, FK506, azathioprine, and cyclophosphamide.
  • the recipient may be conditioned by administration of a corticosteroid or CD3 monoclonal antibody, or by irradiation.
  • the solid-organ is selected from the group consisting of kidney, lung, liver, heart, pancreas and skin and multi-visceral, and the harvested cells are selected from the group consisting of bone marrow, mobilized peripheral blood, fetal liver, and cord blood.
  • the hematopoietic stem cells may be enriched from the harvested cells by immunoselection.
  • the invention further comprises, subsequent to the step of enriching the stem cells, expanding the number of stem cells in vitro.
  • the hematopoietic stem cells are CD34 + cells.
  • CD34 + cells As will be readiiy understood by one of ordinary skill in the art given the disclosure provided herein, homologues of CD34 + cells exist in animals other than humans, and thus are preferably utilized within other embodiments of the invention ⁇ e.g., for xenograft transplantations).
  • the present invention provides methods and compositions (also referred to as “medicaments”) for preventing immune rejection of solid-organ grafts by tolerizing a recipient for solid-organ transplantation.
  • methods and compositions also referred to as “medicaments” for preventing immune rejection of solid-organ grafts by tolerizing a recipient for solid-organ transplantation.
  • advantages which this invention provides are a reduction in the duration of, or elimination of, chronic immunosuppressive therapy, and in addition, improved graft survival.
  • methods for tolerizing a recipient for solid-organ transplantation comprising the steps of (a) harvesting cells from an anticipated donor of a solid-organ, the cells including hematopoietic stem cells, (b) enriching hematopoietic stem cells from the harvested cells, and (c) infusing the enriched stem cells into a recipient, such that a state of tolerance to a transplanted solid-organ from the donor is induced in the recipient.
  • a state of tolerance to a transplanted solid organ-donor has been induced if tolerance to donor antigens has been induced in the recipient.
  • hematopoietic stem cells are harvested from an anticipated donor of a solid-organ.
  • hematopoietic stem cells are defined as those cells which are capable of both replacing themselves and of giving rise to all of the cells of the hematopoietic lineages, including myeloid, erythroid, lymphoid and megakaryocytic. They may also be defined by their antigenic makeup.
  • human hematopoietic stem cells are known to express the CD34 antigen, among other antigens, at the cell surface and to lack lineage-specific antigens, such as B lymphocyte markers, T lymphocyte markers, etc..
  • the CD34 antigen is present on human hematopoietic stem cells and progenitor cells, but is absent from mature hematopoietic cells (reviewed in Knapp, W et al., eds., Leucocyte Typing IV, Oxford: Oxford UP, pp. 816-830, 1989; and Sutherland and Keating, J. Hematotherapy 7: 1 15-129, 1992). That this marker is not completely specific for stem cells (as opposed to progenitor cells) is believed to be inconsequential in the method of the instant invention, provided that the marker chosen does not identify mature hematopoietic cells.
  • antigens which may be useful for identifying and selecting hematopoietic stem cells include various growth factor receptors, such as the receptor for stem cell factor (SCF).
  • SCF stem cell factor
  • Hematopoietic stem cells may be harvested from any of a variety of tissues, including for example, fetal liver and spleen, umbilical cord blood, bone marrow of living or cadaveric organ donors, and peripheral blood of living donors.
  • Bone marrow is a particularly rich source of stem cells (typically 1-2% of marrow mononuclear cells are CD34 + ), and is typically obtained by aspiration (under general anesthesia, if from a living donor) from the iliac crest, but may be obtained from other sites (such as the sternum, ribs, and vertebral bodies) if necessary.
  • marrow is obtained at the same time as the organ to be transplanted.
  • Hematopoietic stem cells may also be obtained from peripheral blood.
  • peripheral blood contains fewer stem cells (typically ⁇ 1% of peripheral blood mononuclear cells are CD34 + ) than bone marrow, it is generally easier to obtain from a living donor.
  • the number of stem cells circulating in peripheral blood may be increased in the donor by prior exposure of the donor to certain growth factors, such as G-CSF, GM-CSF, IL-1 or SCF; to certain drugs, such as 5-fluorouracil; and/or to certain antibodies, such as anti- VLA4.
  • Peripheral blood collected from donors who have been pre-treated in this manner to increase the number of circulating CD34 + cells are said to have been "mobilized.”
  • blood may be collected by venipuncture or by one or more aphereses.
  • an anticoagulant is added to the blood, such as acid-citrate-dextrose (ACD), ethylenediamine tetraacetic acid (EDTA), heparin, or citrate-phosphate-dextrose-adenine (CPDA).
  • ACD acid-citrate-dextrose
  • EDTA ethylenediamine tetraacetic acid
  • CPDA citrate-phosphate-dextrose-adenine
  • Peripheral blood may be obtained at the same time as the organ to be transplanted or it may be obtained at a later time, for example, sufficiently in advance of infusion to allow for processing but not so far in advance as to necessitate cryopreservation.
  • a buffy coat or mononuclear cell (MNC) fraction from the bone marrow or peripheral blood specimen in order to reduce the number of contaminating red cells in the specimen.
  • MNC mononuclear cell
  • a peripheral blood or bone marrow specimen, or buffy coat or mononuclear cell fraction thereof may also be enriched for hematopoietic stem cells by a method such as counterflow centrifugal elutriation or immunoselection. Briefly, in counterflow centrifugal elutriation, cells are fractionated according to size, density, or a combination of both, using a rotor especially designed for this purpose (such as the JE- 10X from Beckman Instruments, Palo Alto, CA).
  • fractions collected when the rotor is stopped but medium is still flowing is generally regarded as the progenitor (s ⁇ ell) -rich fraction (Noga et al., in Bone Marrow Purging and Processing, NY: Alan k ⁇ Jss, p. 345, 1990).
  • Immunoselection is generally preferred over centrifugal elutriation because both the yield and purity of stem cells is superior, and because sterility can be more easily maintained. Immunoselection may be accomplished by negative immunoselection, positive immunoselection, or a combination of the two. Briefly, "positive" selection refers to 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) or receptor;- s expressed at the cell surface).
  • “Negative” selection refers to the exclusion or depletion of cells by some means, usually immunological, on the basis of t ir lack of expression of a specific characteristic or set of characteristics (again, usually a surface antigen(s) or receptor(s)).
  • hematopoietic stem cells are positively immunoselected on the basis of their expression of the CD34 marker utilizing a polyclonal or monoclonal anti-CD34 antibody, or fragment thereof.
  • a variety of anti- CD34 antibodies have been described in the literature, many of which are commercially available. These include 12 8 (Andrews et al., Blood 67:842, 1986), My 10 (Civin et al., J. Immunol.
  • hematopoietic stem cells may be negatively selected on the basis of their lack of expression of lineage-defining antigens.
  • a cocktail of monoclonal or polyclonal antibodies, or fragments thereof, which includes antibodies to myeloid, erythrocytic, lymphoid, and megakaryocytic lineages can be used to bind CD34" cells.
  • lineage-defining antigens antibodies to which may be useful for negative selection of CD34 cells are CD15 and CD33 (myeloid markers); CD19, CD20, CD4, CD8, CD 10, and CD45 (lymphoid markers); glycophorin (erythroid marker), etc.
  • Immunoselection of hematopoietic stem cells may be accomplished by any of a variety of means known to those skilled in the art, including immunoaffinity chromatography, fluorescence activated cell sorting, panning (Wysocki and Sato, Proc. Nail. Acad. Sci. (USA) 75:2844, 1978), magnetic activated cell sorting (Miltenyi et al., Cytomefry 77:231, 1990), and cytolysis.
  • immunoselection of a heterogeneous population of cells such as in a bone marrow aspirate or a peripheral blood specimen or apheresis product, to yield target and non-target fractions is rarely complete.
  • a given target cell population ⁇ e.g., CD34 + cells
  • the ratio of target to non-target cells in the enriched fraction is at least 10X the ratio of target to non-target cells in the starting population.
  • the ratio of target to non-target cells in the enriched fraction will be 30-100X the ratio of target to non-target cells in the starting population.
  • the resultant labeled cells are then flowed, without static incubation, through a column containing immobilized avidin, such that the labeled cells are substantially bound to the immobilized avidin.
  • the labeled cells can be recovered from the column, if desired, in a subsequent step, by agitating the solid phase such that the cells are released in substantially viable condition.
  • This capture efficiency, or yield, is particularly important when one is attempting to select a rare population of cells, such as hematopoietic stem cells, from among more abundant cell types present in a heterogeneous mixture, such as a blood or bone marrow specimen. Yet a further advantage resides in the ability to recover substantially all of the cells which are bound to the solid phase in a condition suitable for transplantation. Cells are said to be suitable for transplantation if they are substantially viable and substantially biologically active. In addition, it is preferable that the cells be free of surface antibody, especially mouse antibody, which may induce a human anti-mouse antibody (HAMA) response in transplant host.
  • HAMA human anti-mouse antibody
  • Immunoselection may be performed utilizing devices such as those described in co-pending patent applications U.S.S.N. 08/005,891, entitled “Improved Apparatus and Method for Cell Separation,” and U.S.S.N. 07/599,796, entitled “An Apparatus and Method for Separating Particles Using a Pliable Vessel,” both of which are herein incorporated by reference Briefly, t ,?
  • a cell separator including a column assembly for separating target cells from a sample fluid, the column assembly including a column, a sample fluid supply bag and a fluid collection bag wherein the column is provided for receiving the sample fluid from the sample fluid supply bag and for separating the target cells from the sample fluid and retaining the target cells, and wherein the fluid collection bag is provided for receiving the target cells after being released from the column, said cell separator comprising an agitation means for agitating the contents of the column to assist in releasing the sample cells retained in the column, the agitation means being responsive to a drive signal for varying the amount of agitation of the contents of the column to vary the rate at which the sample cells are released, column sensor means for providing a column signal indicative of the optical density of fluid flowing out of the column and into the fluid collection bag, a column valve means responsive to a column valve control signal for selectively enabling the fluid coming out of the column to flow into the fluid collection bag, and a data processor means for controlling the operation of the cell separator, the data
  • One embodiment of this invention is the CEPRATE SC cell separation system which is manufactured by CellPro (Bothell, WA).
  • the '796 application describes a vessel having an inlet through which a mixture of target and non-target cells may be introduced and an outlet through which the fluid may exit, at least a portion of which is pliable, and a bed of binding material disposed within the vessel, the binding material attracting the target cells such that they become bound thereto and being porous enough to allow the non-target cells to pass therethrough. Deformation of the pliable portion of the vessel causes relative movement in the binding material, thereby creating the necessary degree of agitation to cause the target cells to become dislodged from the binding material.
  • One embodiment of this invention is the CEPRATE LC laboratory column, which is commercially available from CellPro.
  • this may be accomplished by inoculating the enriched cells into a suitable vessel, containing a nutritive medium supplemented with a source of growth factors and, optionally, human or other animal plasma or serum, and incubating the resultant culture in a humidified atmosphere containing approximately 5% CO2 for a period of time sufficient to yield the desired increase in cell number.
  • a particularly preferred method of expanding hematopoietic stem cells is described in co-pending patent application U.S.S.N.
  • enriched hematopoietic stem cells are inoculated into a culture vessel containing a culture medium comprising a nutritive medium and a source of growth factors and cultured under agitative conditions sufficient to maintain the cells substantially in suspension and for a time sufficient to increase the number of said cells.
  • a culture medium comprising a nutritive medium and a source of growth factors
  • enriched hematopoietic stem cells are stored frozen.
  • CD34 + cells are suspended in a physiologically acceptable medium containing human plasma, or another source of human or animal protein, and a cryoprotective agent, such as DMSO, and frozen at a controlled rate to a temperature between approximately -80°C and -196°C.
  • Cryopreserved stem cells are preferably thawed immediately prior to transfusion. It is usually desirable to thaw the cells as rapidly as possible, for example, by placing the vessel in which the cells were frozen in a 37°C water bath.
  • the cells may be pelleted by centrifugation, the DMF° - containing freezing medium withdrawn and discarded, and the cells resuspended in a physiologically acceptable medium.
  • the cells may also be washed one or more times, if desired, in a suitable buffer or medium. Since the amount of DMSO used in freezing the cells is small and since some cell loss is inevitable during centrifugation and washing, it is generally preferred to transfuse the cells immediately after thawing, without performing any additional manipulations.
  • enriched stem cells are infused into a recipient which has previously been conditioned for solid- organ transplantation.
  • methods include immunosuppressive regimens such as administration of anti-lymphocyte serum (ALS), anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG), cyclosporine A, FK506, azathioprine, corticosteroids (for example, prednisone), cyclophosphamide, and/or CD3 monoclonal antibody, total lymphoid irradiation, partial lymphoid irradiation, or some combination of the above (for example, ALS, cyclosporin, azathioprine and prednisone are commonly co-administered).
  • the terms ALS, ATG, and ALG are used interchangeably.
  • the recipient is conditioned according to the following regimen: ALS (20 mg/kg body weight intravenously from the first through the seventh to fourteenth day post-transplant) / cyclosporine (3 to 7 mg/kg daily) / prednisone (0J25 mg/kg daily).
  • ALS is discontinued between days 7 and 14 post-transplant, unless an acute rejection episode occurs, in which case it is resumed until the episode resolves.
  • Prednisone is tapered, typically by 5 mg per week, beginning about week 12 post-transplant.
  • Cyclosporine is generally continued at least until the organ transplant host exhibits mixed chimerism, typically 5 to 8 weeks post- transplant, but may be continued longer. Ideally, the cyclosporine dose is tapered gradually once mixed chimerism has been attained in the host; in some cases, it may even be possible to withdraw the host from cyclosporine altogether.
  • the enriched stem cells are then infused into a recipient. This may occur either before, coincident with or subsequent to, organ transplantation.
  • the stem cell composition is comprised of at least about 30% CD34+ cells, preferably at least about 50% CD34 + cells, and most preferably at least about 60% CD34 + cells.
  • Cell viability of the composition should preferably be at least about 80% and more preferably at least about 90% viable.
  • the enriched stem cell composition may be administered to the recipient intravenously via a needle and syringe or, via a standard blood administration set.
  • a single stem cell may be sufficient for engraftment and may lead to durable chimerism in the host, it is generally preferable to give a larger dose of stem cells in order to maximize the likelihood of engraftment.
  • the enriched cells are provided to the recipient at a concentration of between about 0.3 x 10 ⁇ and 10 x 10 ⁇ CD34 + cells/kg, more often between about 1 x 10 6 and 3 x 10 6 CD34 + cells/kg, and usually at least about 2 x 10 6 CD34 + cells/kg.
  • the cells can be administered in any convenient volume, but it is usually preferred to utilize the smallest volume which is practicable.
  • that volume is typically about 5 is and is usually between about 3 and 10 mis. Since the volume is small compared to the volume of unfractionated marrow (200-1000 mis), infusion into the host can take place relatively rapidly, for example, in a matter of minutes.
  • solid-organ grafts may be readily transplanted utilizing the methods and compositions described herein.
  • Representative examples of solid-organ grafts include kidney, lung, liver, heart, pancreas and skin grafts, as well as multi-organ grafts, such as heart and lung, kidney and pancreas, or kidney and liver grafts, and multi-visceral grafts comprised of all intra-abdominal organs.
  • Grafts may be allografts or xenografts.
  • An allograft is defined as a graft from one member of a species (the donor) to another member of the same species (the recipient or host), wherein the donor and recipient may or may not be matched at the major histocompatibility complex (MHC).
  • a xenograft is defined as a graft from a member of one species to a member of a different species. Typically, xenografting is done between phylogenetically related animals, for example, from one mammal, such as a pig or monkey, to another mammal, such as a human.
  • a graft may be primary, meaning that it is the first graft of a given organ type for that recipient, or secondary, meaning that the recipient has received an earlier graft of the same organ type (which was rejected or which ceased to function effectively).
  • organ transplantation may be accomplished either prior to, or subsequent to infusion of the enriched hematopoietic cells.
  • Methods for the preservation and transplantation of organs may vary depending upon the organ to be transplanted and the source of the organ. Such procedures are readily known to those of skill in the art ⁇ see e.g., Richard Simmons et al., in Principles of Surgery, Schwartz et al., eds., N.Y. : McGraw Hill, pp. 349-442, 1974; Starzle, Experience in Renal Transplantation, Philadelphia: L.B. Sanders, 1964; Starzle, Experience in Hepatic Transplantation, Philadelphia: L.B.
  • a kidney may be harvested from a heart-beating donor, and preserved by cold pulsatile perfusion on a Waters MOX 100 machine using a perfusate solution such as Belzer's.
  • the ischemic interval is typically about 18-25 hours.
  • the induction of tolerance in a solid organ graft recipient may be assessed in a number of ways, including both / ' // vitro and in vivo methods.
  • tolerance can be assessed by long-term acceptance of the grafted organ without concomitant, exogenously supplied immunosuppression.
  • tolerance can be assessed by one-way mixed lymphocyte cultures (MLC) and by cell-mediated lympholysis (CML) activity.
  • MLC mixed lymphocyte cultures
  • CML cell-mediated lympholysis
  • a tolerized recipient is expected to exhibit reduced or absent proliferation to donor lymphoid cells in one-way MLC, while continuing to exhibit proliferation in response to third-party lymphoid cells.
  • a tolerized recipient is expected to exhibit absent or reduced cytolysis of donor cells in a CML assay, while maintaining cytolysis of third-party cells.
  • Stimulating cells are mononuclear cells obtained from the spleen of the organ donor and prepared as described above; because the donor is typically cadaveric, the stimulating cells will usually be stored frozen in a suitable medium containing a cryoprotectant and thawed immediately prior to use. If the donor happens to be living, peripheral blood mononuclear cells can be used instead of spleen cells and the requirement for frozen storage may be obviated. Stimulating cells are irradiated with 2500 rads prior to use in MLC; this prevents them from proliferating in culture (hence the term one-way MLC).
  • Typical microliters of stimulating cells are added to each well containing responding cells and the microtiter trays are incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide. After approximately 5 days culture, 1 uCi of tritiated thymidine is added to each well containing cells. The cultures are harvested 18-20 hours later and counted in a scintillation counter.
  • unrelated third party cells and irradiated host cells are also used to control for non-specific proliferation. If tolerance has been established in the host, host cells will not proliferate, as judged by their incorporation of tritiated thymidine, in response to the donor cells to any greater extent than they proliferate in response to autologous or unrelated third party cells.
  • CML assays are also well-known to those skilled in the art and are described, for example, in Mishell and Shiigi (ibid.), p. 128. Briefly, responding cells and stimulating cells are prepared as described above and co-cultured at 4 X 10 ⁇ cells each in approximately 2 ml of a suitable medium, such as RPMI/10% fetal calf serum (FCS), for 5 days at 37°C. At this time, the cells are harvested, counted, and resuspended at various effector : target ratios with - > lCr-labeled donor cells as targets. After 3-5 hours, the supernatants are harvested and the specific target cell lysis is quantified relative to appropriate controls. If the host has been tolerized to the donor, there should be no significant chromium release above background.
  • a suitable medium such as RPMI/10% fetal calf serum (FCS)
  • hematopoietic stem celL are harvested from the bone • ⁇ of an organ donor.
  • the stem cells are partially enriched and reinfused, bet 1 days 3 and 5 post-transplant, into an organ graft recipient who has been condi > for transplantation by one or more methods selected from the group consisting o_ total lymphoid irradiation (TLI), cyclosporine therapy, CD3 monoclonal antibody therapy, and anti-lymphocyte serum (ALS) therapy, such that a state of mixed chimerism is attained in the organ recipient.
  • TLI total lymphoid irradiation
  • cyclosporine therapy CD3 monoclonal antibody therapy
  • ALS anti-lymphocyte serum
  • the organ recipient may be gradually withdrawn from immunosuppressive therapy.
  • hematopoietic stem cells are, by definition, capable of self-renewal as well as differentiation into all of the various hematopoietic lineages.
  • hematopoietic stem cells can mediate a durable state of mixed chimerism.
  • stem cells can mediate tolerance by any of a variety of mechanisms, including active T cell- mediated suppression, clonal delet n, and clonal anergy. This is important since the precise mechanism by which tolerance is induced and maintained is not known; different mechanisms may be important at different times (for example, immediately post- transplant and much later) and may be mediated by more than one cell type.
  • hematopoietic stem cells are harvested fron he bone marrow of an organ donor and enriched by means of immunoselection for CD34 + cells.
  • the resultant cells are cryopreserved by freezing at a controlled rate to a temperature between approximately -80 and -196°C in a freezing medium comprising a physiologically acceptable medium in combination with a penetrating cryoprotectant and a source of protein.
  • the cryopreserved, enriched CD34 + ceils are thawed and reinfused into an organ recipient who has been conditioned since transplant by administration of ALS and cyclosporine.
  • ALS therapy is discontinued. Cyclosporine administration is continued at least until such time as the recipient attains a state of mixed chimerism, after which the cyclosporine dose may be tapered gradually and possibly even withdrawn.
  • the present invention provides methods and compositions suitable for tolerizing a recipient for solid-organ transplantation. In most (but not all cases), this produces a state of mixed chimerism (a state in which there is a stable mixture of host and donor hematopoietic elements). Briefly, there are a number of methods by which one skilled in the art can determine whether a solid organ graft recipient has attained a state of mixed chimerism. Typically, this is accomplished by isolating hematopoietic cells from the peripheral circulation and subjecting them to analysis by immunophenotyping. Other methods include mini-satellite detection, as described in Harano et al. ⁇ Bone Marrow Transplant 72:221, 1993), karyotyping and determination of immunoglobulin allotypes.
  • a blood specimen is obtained from a putatively tolerized recipient by phlebotomy.
  • the blood is collected in an anticoagulant, such as EDTA, heparin, ACD, or CPDA.
  • the blood specimen is centrifuged and the buffy coat which forms at the interface of the red cell and plasma layers is harvested.
  • the buffy coat may be fractionated by density gradient centrifugation, for example, using Ficoll-Hypaque or Percoll, to yield a mononuclear cell (MNC) fraction.
  • the MNC fraction is washed in a suitable buffer or medium and the cells are resuspended to a desired density.
  • Aliquots of cells are stained with various directly or indirectly labeled antibodies capable of distinguishing between cells of donor origin and cells of recipient origin (such as antibodies to private MHC determinants), and the resultant stained cell suspension is interrogated, for example, using a fluorescence activated cell sorter (FACS) or other means.
  • FACS fluorescence activated cell sorter
  • the solid organ graft recipient is a mixed chimera, hematopoietic cells expressing donor antigens, as well as hematopoietic cells expressing host antigens should be detected.
  • the proportion of chimerism may vary from less than 1% donor cells to approximately 90% donor cells.
  • the relative proportions of donor and host cells may vary as a function of the time post-transfusion.
  • chimerism will be evident in the host within approximately 15-45 days post-transfusion, although the time course may vary widely among individuals. In some instances, it may be difficult to demonstrate chimerism in the peripheral circulation, but be possible to demonstrate it in other blood-forming organs, such as the spleen or the marrow. Accordingly, if chimerism cannot be demonstrated in one tissue at one time point, it may be desirable to re-assay the same tissue at a later time point(s) or to assay a different tissue(s) before drawing a conclusion. In general, it is believed that a level of chimerism as low as 1% in any recipient tissue may be sufficient to induce tolerance in the host to donor-derived antigens.
  • a solid organ graft recipient to donor antigens, such as are expressed by the graft, thereby reducing or eliminating the need for expensive and, in many cases, toxic immunosuppressive agents in the host.
  • toxicities associated with commonly used immunosuppressive agents include liver toxicity (cyclosporine, azathio, rine), renal toxicity (cyclosporine), leukopenia and thrombocytopenia (total lymphoid irradiation, azathioprine, cyclophosphamide, actinomycin), hypertension (prednisone), and growth retardation (prednisone, actinomycin, total lymphoid irradiation).
  • chronic immunosuppression such as is typically required to maintain graft acceptance
  • infections including viral (EBV, CMV, VZV, etc.), bacterial, and fungal (Aspergillus, Nocardia, Toxoplasma, Cryptococcus, etc.) infections, and malignancy in the host.
  • the methods and compositions of the instant invention enable organ transplantation between unrelated individuals, without the need for matching donor and recipient.
  • the instant invention provides multiple substantial advantages over other art-accepted methods of maintaining solid organ graft survival and function.

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Abstract

La présente invention concerne un procédé augmentant la tolérance d'un receveur d'organe plein. Le procédé consiste (a) à prélever des cellules du donneur prévu de l'organe plein, les cellules prélevées incluant des cellules souches hématopoïétiques, (b) à enrichir les cellules souches hématopoïétiques provenant des cellules prélevées, et (c) à transfuser au receveur les cellules souches enrichies de façon à induire chez le receveur l'état de tolérance du donneur vis-à-vis de l'organe plein transplanté.
PCT/US1994/008378 1993-07-21 1994-07-21 Procedes et compositions de prevention du rejet immun de greffons organiques pleins WO1995003062A1 (fr)

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996014853A1 (fr) * 1994-11-10 1996-05-23 University Of Washington Implantation intrathymique de cellules souches
WO1998052582A2 (fr) * 1997-05-23 1998-11-26 Hadasit Medical Research Services And Development Ltd. Traitement tolerogenique non myeloablatif
WO1999025367A2 (fr) * 1997-11-14 1999-05-27 The General Hospital Corporation Traitement de troubles hematologiques
EP0955948A1 (fr) * 1995-10-26 1999-11-17 Paul P. Latta Induction d'une tolerance immunologique
WO2001011011A3 (fr) * 1999-08-05 2001-05-10 Leo T Furcht Cellules souches adultes toutes-puissantes et procede d'isolement
US6428782B1 (en) 1997-05-23 2002-08-06 Hadasit Medical Research Services And Development Ltd. Non-myeloablative tolerogenic treatment
US6447767B1 (en) 1997-05-23 2002-09-10 Hadasit Medical Research Services And Development Ltd. Non-myeloablative tolerogenic treatment
EP1367899A2 (fr) * 2001-02-14 2003-12-10 Leo T. Furcht Cellules souches adultes totipotentes, sources de ces cellules, procedes d'obtention et de maintien de ces dernieres, procedes de differentiation de ces cellules, procedes d'utilisation correspondants et cellules derivees des cellules susmentionnees
US7015037B1 (en) 1999-08-05 2006-03-21 Regents Of The University Of Minnesota Multiponent adult stem cells and methods for isolation
US8252280B1 (en) 1999-08-05 2012-08-28 Regents Of The University Of Minnesota MAPC generation of muscle
US8426200B2 (en) 2003-07-02 2013-04-23 Regents Of The University Of Minnesota Neuronal differentiation of stem cells
US8609412B2 (en) 1999-08-05 2013-12-17 Regents Of The University Of Minnesota Mapc generation of lung tissue
US9005964B2 (en) 2006-11-24 2015-04-14 Regents Of The University Of Minnesota Endodermal progenitor cells
EP3384006A4 (fr) * 2015-12-04 2019-06-12 Fred Hutchinson Cancer Research Center Utilisations de populations étendues de cellules souches/progénitrices hématopoïétiques
WO2019195657A1 (fr) * 2018-04-05 2019-10-10 Medeor Therapeutics, Inc. Compositions permettant d'établir un chimérisme mixte et leurs procédés de fabrication
WO2019195659A1 (fr) * 2018-04-05 2019-10-10 Medeor Therapeutics, Inc. Compositions cellulaires dérivées de donneurs d'organes antérieurs et leurs procédés de fabrication et des méthodes d'utilisation
US10638734B2 (en) 2004-01-05 2020-05-05 Abt Holding Company Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof
US11273179B2 (en) 2018-03-12 2022-03-15 Medeor Therapeutics, Inc. Methods for treating non-cancerous disorders using hematopoietic cells
US11435350B2 (en) 2018-09-18 2022-09-06 Medeor Therapeutics, Inc. Methods of analysis of blood from deceased donors
US11813376B2 (en) 2018-09-18 2023-11-14 Medeor Therapeutics, Inc. Cellular compositions derived from deceased donors to promote graft tolerance and manufacture and uses thereof

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WO1992018615A1 (fr) * 1991-04-09 1992-10-29 Indiana University Foundation Systeme et procede de maintien en vie de cellules hematopoïetiques
WO1993009234A2 (fr) * 1991-11-05 1993-05-13 The Board Of Trustees Of The Leland Stanford Junior University Cellules supresseurs et souches

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1992018615A1 (fr) * 1991-04-09 1992-10-29 Indiana University Foundation Systeme et procede de maintien en vie de cellules hematopoïetiques
WO1993009234A2 (fr) * 1991-11-05 1993-05-13 The Board Of Trustees Of The Leland Stanford Junior University Cellules supresseurs et souches

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WO1996014853A1 (fr) * 1994-11-10 1996-05-23 University Of Washington Implantation intrathymique de cellules souches
EP0955948A4 (fr) * 1995-10-26 1999-11-17
US7361333B2 (en) 1995-10-26 2008-04-22 Latta Paul P Prevention of diabetes through induction of immunological tolerance
US7361334B2 (en) 1995-10-26 2008-04-22 Latta Paul P Method of treatment of diabetes through induction of immunological tolerance
EP1586284A1 (fr) * 1995-10-26 2005-10-19 Paul P. Latta Induction d'une tolérance immunologique
EP0955948A1 (fr) * 1995-10-26 1999-11-17 Paul P. Latta Induction d'une tolerance immunologique
US6447767B1 (en) 1997-05-23 2002-09-10 Hadasit Medical Research Services And Development Ltd. Non-myeloablative tolerogenic treatment
US6428782B1 (en) 1997-05-23 2002-08-06 Hadasit Medical Research Services And Development Ltd. Non-myeloablative tolerogenic treatment
WO1998052582A3 (fr) * 1997-05-23 1999-02-25 Hadasit Med Res Service Traitement tolerogenique non myeloablatif
WO1998052582A2 (fr) * 1997-05-23 1998-11-26 Hadasit Medical Research Services And Development Ltd. Traitement tolerogenique non myeloablatif
US7892578B2 (en) 1997-11-14 2011-02-22 The General Hospital Corporation Treatment of hematologic disorders
WO1999025367A3 (fr) * 1997-11-14 1999-08-05 Gen Hospital Corp Traitement de troubles hematologiques
US6558662B2 (en) 1997-11-14 2003-05-06 The General Hospital Corporation Treatment of hematologic disorders
WO1999025367A2 (fr) * 1997-11-14 1999-05-27 The General Hospital Corporation Traitement de troubles hematologiques
US7408039B2 (en) 1997-11-14 2008-08-05 The General Hospital Corporation Kits for treatment of hematologic disorders
US7659118B2 (en) 1999-08-05 2010-02-09 Abt Holding Company Multipotent adult stem cells
US10226485B2 (en) 1999-08-05 2019-03-12 Abt Holding Company Multipotent adult stem cells and methods for isolation
US7015037B1 (en) 1999-08-05 2006-03-21 Regents Of The University Of Minnesota Multiponent adult stem cells and methods for isolation
US8609412B2 (en) 1999-08-05 2013-12-17 Regents Of The University Of Minnesota Mapc generation of lung tissue
US8252280B1 (en) 1999-08-05 2012-08-28 Regents Of The University Of Minnesota MAPC generation of muscle
EP2348104A1 (fr) * 1999-08-05 2011-07-27 Mcl Llc Cellules souches adultes multipotentes et procédés d'isolation
WO2001011011A3 (fr) * 1999-08-05 2001-05-10 Leo T Furcht Cellules souches adultes toutes-puissantes et procede d'isolement
EP1367899A2 (fr) * 2001-02-14 2003-12-10 Leo T. Furcht Cellules souches adultes totipotentes, sources de ces cellules, procedes d'obtention et de maintien de ces dernieres, procedes de differentiation de ces cellules, procedes d'utilisation correspondants et cellules derivees des cellules susmentionnees
EP1367899A4 (fr) * 2001-02-14 2004-07-28 Leo T Furcht Cellules souches adultes totipotentes, sources de ces cellules, procedes d'obtention et de maintien de ces dernieres, procedes de differentiation de ces cellules, procedes d'utilisation correspondants et cellules derivees des cellules susmentionnees
EP1491093A2 (fr) 2001-02-14 2004-12-29 Leo T. Furcht Céllules souches adultes totipotentes, sources de ces céllules, procédés d'obtention et de maintien de ces dernières, procédés de differentiation de ces céllules, procédés d'utilisation correspondants et céllules dérivées des céllules susmentionnées
EP1491093A3 (fr) * 2001-02-14 2005-05-04 Leo T. Furcht Cellules souches adultes totipotentes, sources de ces cellules, procedes d'obtention et de maintien de ces dernieres, procedes de differentiation de ces cellules, procedes d'utilisation correspondants et cellules derivees des cellules susmentionnees
US7838289B2 (en) 2001-02-14 2010-11-23 Abt Holding Company Assay utilizing multipotent adult stem cells
US8426200B2 (en) 2003-07-02 2013-04-23 Regents Of The University Of Minnesota Neuronal differentiation of stem cells
US10638734B2 (en) 2004-01-05 2020-05-05 Abt Holding Company Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof
US9005964B2 (en) 2006-11-24 2015-04-14 Regents Of The University Of Minnesota Endodermal progenitor cells
US10813949B2 (en) 2015-12-04 2020-10-27 Fred Hutchinson Cancer Research Center Uses of expanded populations of hematopoietic stem/progenitor cells
EP3384006A4 (fr) * 2015-12-04 2019-06-12 Fred Hutchinson Cancer Research Center Utilisations de populations étendues de cellules souches/progénitrices hématopoïétiques
US11273179B2 (en) 2018-03-12 2022-03-15 Medeor Therapeutics, Inc. Methods for treating non-cancerous disorders using hematopoietic cells
WO2019195657A1 (fr) * 2018-04-05 2019-10-10 Medeor Therapeutics, Inc. Compositions permettant d'établir un chimérisme mixte et leurs procédés de fabrication
US10842821B2 (en) 2018-04-05 2020-11-24 Medeor Therapeutics, Inc. Cellular compositions derived from prior organ donors and methods of manufacture and use thereof
US10881692B2 (en) 2018-04-05 2021-01-05 Medeor Therapeutics, Inc. Compositions for establishing mixed chimerism and methods of manufacture thereof
WO2019195659A1 (fr) * 2018-04-05 2019-10-10 Medeor Therapeutics, Inc. Compositions cellulaires dérivées de donneurs d'organes antérieurs et leurs procédés de fabrication et des méthodes d'utilisation
US11701392B2 (en) 2018-04-05 2023-07-18 Medeor Therapeutics, Inc. Compositions for establishing mixed chimerism and methods of manufacture thereof
US11819521B2 (en) 2018-04-05 2023-11-21 Medeor Therapeutics, Inc. Cellular compositions derived from prior organ donors and methods of manufacture and use thereof
US11435350B2 (en) 2018-09-18 2022-09-06 Medeor Therapeutics, Inc. Methods of analysis of blood from deceased donors
US11813376B2 (en) 2018-09-18 2023-11-14 Medeor Therapeutics, Inc. Cellular compositions derived from deceased donors to promote graft tolerance and manufacture and uses thereof

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