WO2004002425A2 - Methodes permettant d'ameliorer l'acceptation d'une greffe par depletion des cellules souches hematopoietiques - Google Patents

Methodes permettant d'ameliorer l'acceptation d'une greffe par depletion des cellules souches hematopoietiques Download PDF

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WO2004002425A2
WO2004002425A2 PCT/US2003/020520 US0320520W WO2004002425A2 WO 2004002425 A2 WO2004002425 A2 WO 2004002425A2 US 0320520 W US0320520 W US 0320520W WO 2004002425 A2 WO2004002425 A2 WO 2004002425A2
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antibody
stem cells
hematopoietic stem
cells
therapeutic composition
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PCT/US2003/020520
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WO2004002425A3 (fr
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Julian D. Down
Mary E. White-Scharf
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Bio Transplant, Inc.
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Priority to AU2003245752A priority Critical patent/AU2003245752A1/en
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Publication of WO2004002425A3 publication Critical patent/WO2004002425A3/fr

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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates generally to a method for promoting acceptance in a recipient of a graft in the absence of myeloablative therapies.
  • the method includes the step of using antibodies to deplete and/or inactivate hematopoietic stem cells (HSC), preferably primitive hematopoietic stem cells (PHSC), and most preferably to selectively deplete and/or inactive at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells and subsequently administering bone marrow cells (BMC) or mobilized peripheral hematopoietic stem cells (MPHSC), preferably from a donor.
  • HSC hematopoietic stem cells
  • PHSC primitive hematopoietic stem cells
  • the mammalian hematopoietic system includes a heterogeneous array of cells ranging from large numbers of differentiated cells with defined function, such as B cells and T cells, to rare pluripotent stem cells with extensive developmental and proliferative potential. Methods for distinguishing stem cell lineage and developmental potential have been imprecise and have used phenotypic and functional characteristics.
  • the defining feature of a hematopoietic stem cell (HSC) that has been found to be useful is the ability of HSC to repopulate the hematopoietic system of a recipient after transplantation, particularly after whole body irradiation treatment. HSCin bone marrow have been found to be able to continuously regenerate all blood and immune cell lineages. HSC are also demonstrable in the yolk sac and later in the fetal liver.
  • Bone marrow cell (BMC) transplantation procedures have been utilized in replacement therapies both for treatment of hematologic disorders, such as hematologic cancers, and for modulating the immune system of recipients prior to solid organ transplants.
  • Bone marrow cells and mobilized peripheral stem cells (MPSC) provide sources of primitive hematopoietic stem cells as well as HSC.
  • MPSC mobilized peripheral stem cells
  • Both donor bone marrow cells and donor grafts bear major histocompatibility (MHC) markers as well as other cell surface antigens. Such markers permit an organism to determine self from non-self. When a patient's immune system is intact, non-self recognition can lead to rejection.
  • MHC major histocompatibility
  • Myeloablative conditioning regimes including high doses of total body irradiation (TBI) are often used in BMC transplantation in conjunction with treatments designed to prevent immunological rejection (e.g., cyclophosphamide).
  • TBI total body irradiation
  • Such conditioning is used for procuring engraftment of transplanted allogeneic donor BMC stem cells while eliminating or reducing GvHD in the recipient.
  • these treatments can have undesired side effects, such as toxicity (e.g. nephrotoxicity, hyperlipidemia, bone marrow suppression) and the complications of immunodeficiency (for example, infection and malignancy) on the recipient.
  • toxicity e.g. nephrotoxicity, hyperlipidemia, bone marrow suppression
  • immunodeficiency for example, infection and malignancy
  • non-myeloablative conditioning regimes comprise the use of reduced radiation together with immunosuppressive agents such as cytotoxic drugs (e.g., cyclophosphamide and/or fludarabine) and/or T cell depleting antibodies (e.g., anti-thymocyte globulin, CAMPATH, OKT3 and MEDI-507) to reduce the recipient's ability to reject the BMC.
  • immunosuppressive agents such as cytotoxic drugs (e.g., cyclophosphamide and/or fludarabine) and/or T cell depleting antibodies (e.g., anti-thymocyte globulin, CAMPATH, OKT3 and MEDI-507) to reduce the recipient's ability to reject the BMC.
  • cytotoxic drugs e.g., cyclophosphamide and/or fludarabine
  • T cell depleting antibodies e.g., anti-thymocyte globulin, CAMPATH, OKT3 and MEDI
  • DLI Donor lymphocyte infusion
  • GvHD graft-versus-host disease
  • the present invention relates to a method of promoting tolerance in a recipient to a graft in the absence of myeloablative conditioning.
  • an antibody such as a monoclonal antibody that depletes and/or inactivates hematopoietic stem cells (HSC), preferably primitive hematopoietic stem cells (PHSC), and most preferably to selectively deplete and/or inactive at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells is administered to the recipient prior to graft transplantation.
  • HSC hematopoietic stem cells
  • PHSC primitive hematopoietic stem cells
  • PHSC can be identified by the ability to form colonies that survive for at least four weeks and preferably beyond in a cobblestone area forming cell (CAFC) assay
  • CAFC cobblestone area forming cell
  • specific monoclonal antibodies are produced for use in the present invention, in particular in rats using human primitive hematopoietic stem cell membranes.
  • such an anti-PHSC antibody is an anti-c-kit (or anti- CD117) antibody, which is administered to the prospective recipient prior to introduction of donor BMC or MPHSC.
  • the antibody is a monoclonal antibody (MAb) and is chosen from the group comprising an anti-CD135 MAb, an anti-VEGFR2 (KDR) MAb, anti-CD133 (AC133) MAb, an anti-TIE MAb, an anti-TEK MAb, an anti-C1qRp MAb and an anti-CD117 MAb or combinations thereof.
  • MAb monoclonal antibody
  • Antibodies useful in practicing the invention are not limited to those capable of depleting or inactivating hematopoietic stem cells expressing the CD34 antigen but may include those capable of depleting or inactivating CD34 " cells as well.
  • the invention also relates to a method of facilitating engraftment of donor mammalian hematopoietic pluripotent stem cells in a mammalian recipient using an antibody capable of depleting or inactivating PHSC of the prospective recipient of donor hematopoietic stem cells.
  • Useful antibodies comprise monoclonal, recombinant, and humanized antibodies capable of depleting and/or inactivating hematopoietic stem cells (HSC), preferably primitive hematopoietic stem cells (PHSC), and most preferably capable of selectively depleting and/or inactiving at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells.
  • HSC hematopoietic stem cells
  • PHSC primitive hematopoietic stem cells
  • Such antibodies are administered to the recipient (or prospective graft recipient) prior to donor stem cell transplantation thereby reducing or eliminating the level of cytotoxic drugs or radiation administered or the requirement for donor T cells in the non-myeloablative protocols that are currently used as standard therapy.
  • TBI total body irradiation
  • It is one object of the present invention to facilitate use of a less toxic conditioning regimen for establishing mixed hematopoietic cell chimerism (a) in the treatment of malignant and non-malignant diseases, particularly of the blood; (b) in the promotion of immunological acceptance for cellular, tissue, and/or solid organ transplantation; (c) to prevent or reduce graft-versus-host disease (GvHD); (d) to provide a platform for administering donor-leukocyte infusions (DLI); (e) in the treatment of enzyme deficiency diseases; and (f) in the treatment of autoimmune diseases.
  • a less toxic conditioning regimen for establishing mixed hematopoietic cell chimerism (a) in the treatment of malignant and non-malignant diseases, particularly of the blood; (b) in the promotion of immunological acceptance for cellular, tissue, and/or solid organ transplantation; (c) to prevent or reduce graft-versus-host disease (GvHD); (d) to provide a platform for administering donor-leukocyte in
  • the present invention also relates to a process for inducing acceptance in a recipient mammal of a first species to a graft from a donor mammal of a second species by introducing, such as by intravenous or intraperitoneal injection into the recipient mammal, at least one antibody that specifically binds to and depletes and/or inactivates hematopoietic stem cells (HSC), preferably primitive hematopoietic stem cells (PHSC), and most preferably to selectively deplete and/or inactive at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells of the recipient; and, preferably subsequently, introducing hematopoietic stem cells of the donor into the recipient mammal, and further, preferably implanting a graft from the donor or from a syngeneic donor in the recipient.
  • HSC hematopoietic
  • the recipient mammal can be, by way of example, a human.
  • the donor mammal can be, by way of example, a human, a non-human primate, a swine, e.g., a miniature swine, or a sheep.
  • the swine is from a highly inbred herd (Sachs et al., USSN 09/378,684, the disclosure of which is hereby incorporated by reference) such that different members of the herd can be donors of BMC and graft(s) to a single recipient.
  • the present invention contemplates processes wherein multiple donor hematopoietic stem cell administrations are provided to a recipient.
  • multiple administrations of donor stem cells are provided prior to the implantation of a graft. Two, three, four, five, or more administrations can be provided.
  • a subsequent administration of donor hematopoietic stem cells is provided: at least two days, one week, one month, or six months after the previous administration of stem cells.
  • the methods of the invention contemplate inactivating natural killer cells, preferably graft reactive or xenoreactive, preferably swine reactive, NK cells, of the recipient mammal, and preferably by administering to the recipient mammal at least one antibody capable of binding to and depleting or inactivating natural killer cells of the recipient mammal, especially wherein the administration of antibodies, or other treatment to inactivate natural killer cells, occurs prior to introducing hematopoietic stem cells to the recipient mammal or prior to implanting the graft in the recipient.
  • natural killer cells preferably graft reactive or xenoreactive, preferably swine reactive, NK cells
  • FIGURE 1 shows staining intensity profiles for biotinylated pSCF and ACK45 and 3C1 antibodies on P815 cells with or without precoating with either ACK2 or 2B8.
  • FIGURE 2 is the timeline protocol for in vivo treatment with 2B8 MAbs in mice.
  • FIGURE 3 shows effect of 2B8 antibody or Busulfex treatment of B6 mice on the number of bone marrow CFU-Cs (left panel-individual mice) and CAFCs
  • FIGURE 4 shows effect of 2B8 antibody or Busulfex treatment of BALB/c mice on the number of bone marrow CFU-Cs (left panel-individual mice) and CAFCs (right panel - pooled for each treatment group with 95% confidence limits).
  • FIGURE 5 is the timeline protocol for in vivo treatment with 2B8 MAb in mice.
  • FIGURE 6 shows the number of lineage negative (Lin-) bone marrow cells expressing Sca-1 following treatment with anti-CD117 MAb (2 x 0.05 or 0.125 mg 2B8 at -4 and -2 days or -9 and -7 days).
  • FIGURE 7 shows the effect of 2B8 antibody treatment of BALB/c mice at two different doses and times on the number of bone marrow CFU-Cs (left panel-individual mice) and CAFCs (right panel - pooled for each treatment group with 95% confidence limits).
  • FIGURE 8 is the timeline protocol for in vivo treatment with 3C1 MAb in mice.
  • FIGURE 9 shows the number of lineage negative (Lin-) bone marrow cells expressing either Sca-1 or c-kit (ACK45) following treatment with anti- CD117 MAb (2 x 0.125 mg 3C1 at -9 and -7 days).
  • FIGURE 10 shows the effect of 3C1 antibody treatment of BALB/c mice at a dose of 2 x 0.125 mg on the number of bone marrow CFU-Cs (left panel- individual mice) and CAFCs (right panel - pooled for each treatment group with 95% confidence limits).
  • FIGURE 11 shows the effect of 2B8 and rabbit complement (C) treatment as compared to untreated and isotype control plus C and 2B8 alone on the frequency of bone marrow CFU-Cs (left panel) and CAFCs (right panel with 95% confidence limits).
  • FIGURE 12 shows the staining of Mo7e cells with the anti-human CD117 MAbs Nu c-kit and 104D2 and with bio-pSCF following precoating of the cells with SR-1.
  • FIGURE 13 illustrates how two groups of anti-human can be defined by their ability to recognize different epitopes on the c-kit molecule and to either allow binding or prevent binding of the c-kit ligand (SCF).
  • SCF c-kit ligand
  • FIGURE 14 shows the expression of c-kit in SP positive and SP negative cells of human bone marrow as determined from staining either with the 104D2 or O.N. 183 anti-CD117 MAbs.
  • FIGURE 15 shows the effect of different anti-human CD117 MAbs and rabbit complement (C) treatment, with or without addition of rat lgG2b anti- mouse lgG1 , on lysis of Mo7e cells.
  • FIGURE 16 shows the effect of human complement (Hu C) vs. rabbit complement (R C) on Mo7e cell lysis using the SR-1 anti-c-kit antibody. Cells were incubated overnight with either 10% human serum or rabbit complement.
  • FIGURE 17 shows the effect of SR-1 and rabbit complement (C) treatment of human bone marrow as compared to untreated and isotype control plus C and SR-1 alone on the frequency of CFU-Cs (left panel) and CAFCs (right panel with 95% confidence limits).
  • FIGURE 18 shows the effect of SR-1, O.N. 181 , O.N. 182 and O.N. 183 with and without rabbit complement on the frequency of colony formation following treatment of human bone marrow.
  • Antibody as used herein includes fragments and derivatives of antibodies Antibody includes, but is not intended to be limited to, monoclonal and recombinant antibodies. They may be mouse, rat, porcine, bovine, ovine or human. Anti-human primitive hematopoietic stem cell (hPHSC) antibodies are chosen for their ability to inactivate or deplete human PHSC. An antibody “derivative” as used herein means a chimeric or humanized antibody, single chain antibody, bispecific antibody or other such antibody which binds to the same epitope as its parent.
  • hPHSC hematopoietic stem cell
  • an antibody “fragment” as used herein means a portion of an antibody, by way of example such portions of antibodies shall include but not be limited to CDR, Fab, or such other portions, which bind to the same epitope or any portion thereof as recognized by the chosen anti- HSC antibody of the present invention.
  • CAFC Cobblestone Area-Forming Cell
  • the agent is capable of inhibiting the number of CAFC seen at four weeks and preferably beyond 5, 7, 10 weeks, by at least 50%, preferably 60%, 70%, 80%, 90%, 93%, 95%, most preferably by 98%, as compared to the untreated control group, e.g. if a cell is lysed, it is considered to have been inactivated and/or depleted.
  • Discordant species combination refers to two species in which hyperacute rejection occurs when an organ, tissue or cell 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 concordant species. Concordant species combinations do not exhibit hyperacute rejection.
  • Donor leukocyte cells typically are leukocytes obtained from one member of a species, a donor, for infusion into a recipient, frequently for the purpose of introducing immunologically reactive cells into the recipient capable of depleting T-cells, especially mature T-cells and/or malignant cells in the recipient.
  • DLI can be cells derived from the recipient that have been genetically engineered to perform the same function as those from a donor.
  • “Graft”, as used herein, refers to a body part, organ, tissue, or cells.
  • Organs such as liver, kidney, heart or lung; body parts, such as bone or skeletal matrix; tissue, such as skin, intestines, endocrine glands; and cells such as pancreatic cells and hematopoietic progenitor stem cells of various types, are all examples of potential grafts.
  • Help reducing agent is an agent, such as for example an immunosuppressive drug, which results in the reduction of cytokine release.
  • help reducing agents are cyclosporine, FK- 506, and rapamycin.
  • a help reducing agent must be administered in sufficient dose to give the level of inhibition of cytokine release that will result in tolerance.
  • the help reducing agent should be administered in the absence of treatments that promote cytokine, e.g., IL-2, release.
  • Putative 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 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 acceptance of the graft for at least about three months or longer) is indicative that the putative agent is, at the dosage tested, a help reducing agent.
  • 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 that is administered during the reduction of help.
  • the desired level of help 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.
  • Hematopoietic space refers to a condition created in the bone marrow which promotes engraftment of administered stem cells, such as donor BMC or mobilized peripheral stem cells (MPSC).
  • stem cells such as donor BMC or mobilized peripheral stem cells (MPSC).
  • MPSC mobilized peripheral stem cells
  • Hematopoietic space-creating irradiation refers to irradiation directed to the hematopoietic tissue, i.e., to tissue in which stem cells are found, e.g., the bone marrow. It is of sufficient intensity to kill or inactivate a substantial number of hematopoietic cells. It is often given as whole body irradiation.
  • Hematopoietic stem cell refers to undifferentiated cells that serve as precursors for multiple cell lineages, including myeloid and lymphoid, and that are demonstrable in bone marrow, mobilized peripheral blood, yolk sac and fetal liver. HSC are pluripotent. Frequently, HSC express the CD34 marker. When CD34 negative, HSC typically express c-kit.
  • Immunosuppressive agent capable of inactivating thymic or lymph node T cells is an agent, e.g., a chemical agent, such as for example an antibody or 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, rapamycin and antigen-presenting cell inhibitors (e.g., co-stimulatory blockade inhibitors).
  • An agent should be administered in a sufficient dose to result in significant inactivation of thymic or lymph node T cells which are not inactivated by administration of an 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.
  • 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.
  • histocompatibility refers to the similarities between different individuals of surface antigens on white blood cells and other tissues and organ. The level of histocompatibility describes how well matched or how antigenically similar a recipient is to a donor.
  • the major histocompatibility antigens are the human leucocyte antigens (HLA) known as HLA-A, HLA-B, and HLA-DR. Each person has two sets of these antigens, one set inherited from each parent. The best kind of match is an identical match wherein all six HLA antigens are the same between recipient and donor. Donors and recipients considered mismatched at one antigen are considered a "5 of 6" match, and so forth.
  • Allogeneic refers to the situation wherein most or all of the antigens are matched such as would be the case if one twin acted as a donor for an identical twin, or if a brother and sister acted as donor and recipient. When the donor and the recipient are of different species, especially discordant species, this is referred to as "xenogeneic.”
  • Miniature swine refers to a wholly or partially inbred miniature swine (See for example USSN 09/378,684 filed 8/20/99, the disclosure of which is hereby incorporated by reference in its entirety).
  • Mated chimerism refers to a situation wherein donor HSC co-exist with recipient HSC. This may be determined empirically such as by obtaining a bone marrow sample from the recipient after HSC administration and finding donor stem cells engrafted among the recipient cells. At least 1%, preferably at least 10% and more preferably at least 30, 50 or 75 % of the recipient's engrafted bone marrow comprises donor stem cells. Methods for induction of mixed chimerism are known in the art (Sykes, US Pat No. 6,006,752).
  • Purified preparations of donor hematopoietic stem cells or preparations including PHSC can be used in the methods of the present invention.
  • the desired hematopoietic stem cells can be separated out of a complex preparation such as from plasma after mobilization of the hematopoietic cells.
  • the complex preparation itself can be administered to a recipient.
  • Hematopoietic stem cells including PHSC can be obtained from fetal, neonatal, immature, or mature animals. Stem cells derived from the cord blood of the recipient or the donor can be used. See U.S. Pat. No. 5,192,553 and 5,004,681 , the disclosure of each of which is hereby incorporated by reference.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).
  • PHSC Primary hematopoietic stem cell
  • a cell such as for example, a bone marrow cell, a fetal liver cell, a spleen cell, a fetal cord blood cell, a muscle stem cell, or a skin stem cell, which is pluripotent capable of developing into all myeloid and lymphoid lineages, and that by virtue of being able to self-renew, can provide long-term hematopoietic reconstitution (LTRC).
  • LTRC long-term hematopoietic reconstitution
  • PHSC cells are distinguishable from progenitor hematopoietic stem cells (ProHSC) in that ProHSC: 1) confer 30 day radioprotection or short-term reconstituting cell (STRC) activity when transplanted in vivo; 2) give rise to CFU-s after 12 days when transplanted into lethally irradiated mice; and 3) show little or no proliferative response to single hematopoietic growth factors (HGFs) but proliferate maximally to multiple HGF combinations that always include steel factor (SLF).
  • ProHSC progenitor hematopoietic stem cells
  • the primitive hematopoietic stem cell is distinguishable from its "committed hematopoietic progenitor cell" (CHPC) descendants.
  • CHPC human hematopoietic progenitor cell
  • Committed progenitor cells generate short-term and transiently repopulating mature cells of blood and lymphoid tissues.
  • the two cell populations can be distinguished from each other experimentally, for example, by performing a Cobblestone- Forming Area Cell Assay (Ploemacher et al. Blood. 78:2527-33 (1991)).
  • the extent of long-term stem cell engraftment is determined by the frequency of cobblestone-area forming cells (CAFC) that appear after 4 to 5 weeks in stromal cell cultures when using human cells or 1-3 weeks when using murine cells.
  • CAFC cobblestone-area forming cells
  • primitive hematopoietic stem cells can be distinguished from committed hematopoietic progenitor cell populations because the CHPC appear earlier, over
  • “Promote acceptance of a graft”, as used herein, refers to lengthened functional ability of a donor graft in a recipient animal after graft transplantation, especially after the withdrawal or in the absence of continuous immunosuppressant administration. A donor graft is typically rejected within minutes of its introduction into a recipient in the absence of additional treatment of an immunocompetant recipient.
  • Selective or “selectively” or “selected”, as used herein refers to a particular population of hematopoietic stem cells capable of survival for at least four weeks or longer in a CAFC assay that are distinguishable by cell surface molecules or patterns thereof from mature blood cells. Subsets of the population may be distinguishable in CAFC assay also based upon the how much longer than 4-5 weeks the members are able to proliferate in CAFC assay.
  • “Short course of a help reducing agent”, as used herein, means a transitory non-chronic course of treatment.
  • the treatment should begin before or at about the time of transplantation of the graft.
  • the treatment can begin before or at about the time of the recipient's first exposure to donor antigens.
  • the treatment lasts for a time which is approximately equal to or less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • the duration of the treatment can be extended to a time approximately equal to or less than two, three, four, five, or ten times, the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • the duration will usually be at least equal to the time required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • about 12 days of treatment is sufficient.
  • CsA cyclosporin A
  • Other experiments in monkeys show that IL-2 administered on day 8, 9, or 10 of CsA treatment will result in rejection of the transplanted tissue.
  • 8, 9, or 10 days of treatment are probably insufficient in pigs.
  • a dose of 10 mg/kg CsA 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 5 mg/kg prevents rejection (by long-term immune suppression) but does not result in tolerance.
  • “Short course of a immunosuppressive agent”, as used herein, 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 of 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.
  • Hematopoietic stromal tissue may be obtained from a variety of hematopoietic tissues to include, but is not meant to be limited to, fetal or neonatal thymus or liver tissue or a combination thereof or bone marrow or spleen.
  • “Therapeutically effective amount” as used herein means the amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, promoting acceptance of a graft or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a therapeutically effective amount can be determined through the use of a Cobblestone Area Forming Cell Assay or by observing primitive hematopoietic stem cell depletion after administration of a therapeutic antibody composition.
  • Thymic or lymph node 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, such as for example, an ATG preparation.
  • Thymic or lymph node thymocytes refers to thymocytes which are resistant to inactivation by traditional methods of thymocyte inactivation, e.g., inactivation by a single intravenous administration of an ATG preparation.
  • Thymic irradiation refers to a treatment in which at least half, and preferably at least 75, 90, or 95% of the administered irradiation is targeted to the thymus.
  • Whole body irradiation even if the thymus is irradiated in the process of delivering the whole body irradiation, is not considered thymic irradiation.
  • Thimic space as used herein, is a state created by a treatment that facilitates the migration to and/or development in the recipient thymus of donor hematopoietic cells of a type which can delete or inactivate recipient thymocytes that recognize donor antigens. It is believed that the effect is mediated by elimination of recipient cells in the thymus.
  • Tolerance refers to an inhibition of a graft recipient's immune response that would otherwise occur, e.g., in response to the introduction of a non-self MHC antigen into the recipient. Tolerance can involve humoral, cellular, or both humoral and cellular responses. Tolerance, as used herein, 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. Tolerance, as used herein, refers to a donor antigen-specific inhibition of the immune system as opposed to the broad-spectrum inhibition of the immune system seen with immunosuppressants.
  • CD117 expression in the adult is known to be limited to committed lymphohematopoietic progenitors and primitive hematopoietic stem cells (Kodama et al. J Exp Med. 176:351-361 (1992); Katayama et al. Blood 82:2353-2360 (1993); Matsuzaki et al. J Exp Med. 178:1283-1292 (1993)), mature mast cells, intestinal interstitial cells of Cajal, spermatogonial stem cells and oocytes (Katayama et al.
  • c-kit tyrosine kinase receptor for stem cell factor (SCF). Its expression has been shown to be more limited and more consistent on pluripotent hematopoietic stem cells, myeloid progenitor populations, and lymphoid progenitor populations than CD45 or CD34 (Sato et al. Blood, 94:2548-2554 (1999)).
  • Radioisotope( 131 l)-labeled anti-CD45 MAb (a pan leukocyte and mature bllod cell marker) is capable of providing for subsequent engraftment of donor stem cells in syngeneic as well as allogeneic recipients in mice (Matthews et al, Blood, 93:737-745 (1999)), and is the basis for a Phase I clinical trial in patients using 131 l-labeled anti-CD45 combined with 120 mg/kg cyclophosphamide (CyP) and 12 Gy, a myeloablative dose of total body irradiation (TBI) (Matthews et al, Blood, 94:1237-1247 (1999)).
  • TBI total body irradiation
  • Utilization of the present invention allows reduction or avoidance of total body irradiation and reduced toxicity is possible. Also.use of antibodies against markers like CD45 is less advantageous because they would deplete a much wider number of cell types, including mature blood cells, thereby leading to excessive immunosuppression and/or toxicity
  • the inventors have discovered that to achieve long-term donor cell re- population of the recipient hematopoietic system while facilitating donor hematopoietic stem cell engraftment, it is advantageous to deplete hematopoietic stem cells from a prospective graft recipient prior to performing a bone marrow transplant.
  • HSC hematopoietic stem cells
  • PHSC primitive hematopoietic stem cells
  • Busulfan (1 ,4-butanediol dimethylsulfonate) has been shown to be more effective than other cytotoxic drugs at depleting primitive hematopoietic stem cells and its use has been shown to facilitate long-term donor cell re- population (Down and Ploemacher. Exp Hematol. 21:913-921 (1993); Down et al. BrJ Cancer 70: 611-616 (1994); Westerhof et al. Cancer Res. 60: 5470- 5478 (2000) . Further studies (data not shown) have demonstrated that Busulfex® (Orphan Medical, Inc.; Minnetonka, MN) depletes cells bearing the c-kit marker.
  • Treatments or agents that lead to myeloablation may not necessarily deplete primitive hematopoietic stem cells.
  • chemotherapeutic agents that result in acute myelosuppression without depleting primitive hematopoietic stem cells include cyclophosphamide, melphalan, 5-fluoruracil and thiotepa (Down and Ploemacher. Exp Hematol 21 : 913-921 (1993); Down et al. Br J Cancer. 70:611-616 (1994); Down et al. Bone Marrow Transplant. 21:327-330 (1998)).
  • treatments or agents that lead to depletion of primitive hematopoietic stem cells may not necessarily lead to myeloablation.
  • the present invention provides improved methods for inducing mixed chimerism and tolerance in a recipient through antibody-mediated removal of HSC, preferably PHSC, and most preferably through selective depletion and/or inactivation of at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells prior to the introduction of donor hematopoietic stem cells such as bone marrow cells or mobilized peripheral stem cells.
  • HSC preferably PHSC
  • the invention provides a method for inducing mixed chimerism and tolerance in a recipient by depletion and/or inactivation of the recipient's HSC, preferably PHSC, and most preferably through selective depletion and/or inactivation of at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells and depletion of the recipient's T-cells, prior to administration of donor hematopoietic stem cells.
  • the present invention provides a method for inducing mixed chimerism and tolerance by depletion of donor and recipient T cells and depletion and/or inactivation of recipient HSC, preferably PHSC, and most preferably through selective depletion and/or inactivation of at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells.
  • the present invention finds use in the treatment of malignant and non- malignant diseases, the induction of immunological acceptance for cellular and/or solid organ transplantation, in the prevention or reduction of graft-versus- host disease (GvHD), for providing a platform for administering donor-leukocyte infusions (DLI), and in the treatment of enzyme deficiency diseases and autoimmune diseases.
  • GvHD graft-versus- host disease
  • DLI donor-leukocyte infusions
  • enzyme deficiency diseases and autoimmune diseases e.g., enzyme deficiency diseases and autoimmune diseases.
  • Suitable combinations with short-term immune modulating agents e.g., T-cell-depleting antibodies
  • the present invention provides methods for improving graft acceptance while reducing GvHD.
  • the present invention provides improved methods for treating hematological disorders.
  • the methods include the steps of non-myeloablative conditioning of the recipient by reducing or eliminating both the recipient's T cell population and the recipient's HSC, preferably PHSC, and most preferably through selective depletion and/or inactivation of at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells and infusing donor hematopoietic stem cells such as bone marrow cells or mobilized peripheral stem cells into the recipient.
  • a short course of help reducing agent may be administered to the recipient; an organ, tissue or cell graft may be provided; and/or a donor lymphocyte infusion may be administered.
  • hematopoietic stem cells which are pluripotent, are isolated and utilized to raise an antibody or antibodies specific for primitive hematopoietic stem cells.
  • Cell lines producing an antibody are isolated and specific monoclonal antibodies are collected using methods known to those of skill in the art such as for example those described in Harlow, E., et al., (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)). see example.
  • Each specific antibody is tested for its ability to deplete and/or inactivate hematopoietic stem cells, preferably primitive hematopoietic stem cells, and most preferably selected primitive hematopoietic stem cells from bone marrow utilizing a CAFC assay.
  • Those antibodies capable of depleting and/or inactivating at least about 50%, at least about 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, 93%, 95% and most preferably at least about 98% of the pluripotent hematopoietic stem cells from a bone marrow preparation are selected for use.
  • cocktails of monoclonal antibodies are utilized and/or polyclonal antibodies are used.
  • the chosen antibody or antibodies are administered to a recipient in need thereof for inducing mixed chimerism and tolerance.
  • the methods of the present invention allow utilization of the graft- versus-leukemia (GvL) effects for immunotherapy of blood borne malignancies and engraftment-promoting effects of donor T cells to facilitate formation of mixed chimerism and tolerance in the recipient while minimizing GvHD, especially in HLA-mismatched pairs and in xenogeneic transplantation procedures. Additionally, immunosuppressive therapy can be reduced or substantially eliminated.
  • the methods of the present invention are also useful for exploiting the graft-versus-host reaction obtained when donor hematopoietic cells are administered to a recipient.
  • the present invention provides a method of promoting graft acceptance in a recipient by depleting and/or inactivating the recipient's HSC, preferably PHSC, and most preferably through selective depletion and/or inactivation of at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while avoiding the use of immunosuppressive regimens by administering to the recipient an antibody, such as a monoclonal antibody, that depletes and/or inactivates pluripotent primitive hematopoietic stem cells preferentially and selectively while not significantly reducing mature blood cell numbers.
  • an antibody such as a monoclonal antibody
  • a useful depleting and/or inactivating antibody is identified by CAFC assay in which cytokines from the same species as the species from which the hematpoietic stem cells were harvested are provided.
  • the antibody e.g. monoclonal antibody, is capable of eliminating at least about 50%, at least about 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, 93%, 95% and most preferably at least about 98% of the hematopoietic stem cells that survive for at least four weeks or longer under the culture conditions provided by the CAFC assay.
  • such antibody is an anti-c-kit (or anti- CD117) antibody.
  • the antibodies useful in practicing the invention are not limited to those capable of depleting or inactivating cells with the CD117 antigen but may include those capable of depleting or inactivating CD117 negative (CD117 " ) cells as well.
  • the method includes the additional step of administering to the recipient an agent, such as for example an antibody or combination of antibodies, capable of depleting cells expressing CD4 and CD8 prior to the administration of donor hematopoietic cells.
  • the antibody is a monoclonal antibody (MAb) and is chosen from the group comprising an anti-CD135 MAb, an anti-VEGFR2 (KDR) MAb, anti-CD133 (AC133) MAb, an anti-TIE MAb, an anti-TEK MAb, an anti-C1qRp MAb and an anti-CD117 MAb or combinations thereof.
  • MAb monoclonal antibody
  • Antibodies useful in practicing the invention are not limited to those capable of depleting or inactivating hematopoietic stem cells expressing the CD34 antigen but may include those capable of depleting or inactivating CD34 " cells as well.
  • C1qRp is the human homologue of the mouse stem cell antigen AA4.1 (Danet et al., Proc Natl Acad Sci U S. A. 99(16): 10441 -5 (2002)).
  • the present invention also relates to methods for engrafting donor mammalian hematopoietic stem cells, preferably primitive hematopoietic stem cells, and most preferably selected primitive hematopoietic stem cells in a mammalian recipient which uses anti-hematopoietic-stem-cell antibodies, preferably PHSC antibodies, and most preferably selective PHSC antibodies.
  • an anti-c-kit antibody which is capable of depleting or inactivating sufficient numbers of hematopoietic stem cells (HSC), preferably primitive hematopoietic stem cells (PHSC), and most preferably at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells prior to donor stem cell transplantation is used.
  • the anti-hematopoietic-stem-cell antibody is administered at a dose and using a schedule capable of reducing or eliminating the level of cytotoxic drugs or radiation administered to a recipient while facilitating induction of tolerance and mixed chimerism and promoting acceptance of a graft.
  • the present invention also relates to a method for inducing tolerance in a recipient mammal of a first species to a graft from a donor mammal of a second species.
  • the method includes: introducing into the recipient mammal such as by intravenous injection or by intraperitoneal injection or intramuscularly, etc., an antibody that binds to and depletes hematopoietic stem cells (HSC), preferably primitive hematopoietic stem cells (PHSC), and most preferably to selectively deplete and/or inactive at least 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 93%, at least 95%, at least 98% of the PHSC of the recipient while maintaining mature blood cells ; and, preferably thereafter, introducing hematopoietic stem cells of the donor into the recipient mammal.
  • HSC hematopoietic stem cells
  • PHSC primitive hematopoietic stem cells
  • the mature T-cells of the recipient are also depleted prior to donor stem cell infusion (for example, BMC or PMSC) while not depleting donor T cells.
  • donor stem cell infusion for example, BMC or PMSC
  • mixed chimerism has been induced prior to the transplant or infusion.
  • antibody depletion of the hematopoietic stem cells especially primitive hematopoietic stem cells, most preferably selected PHSC of the recipient is believed to facilitate the long-term engraftment of the donor hematopoietic stem cells.
  • the donor hematopoietic stem cells are believed to prepare the recipient for subsequent engraftment by inducing tolerance at both the T and B cell levels.
  • the method for inducing tolerance in a recipient mammal of a first species to a graft from a donor mammal of a second species involves the following steps: (1) Administering an agent that depletes and/or inactivates HSC, preferably PHSC, and most preferably at least about 50%, at least about 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, 93%, 95 % and most preferably at least about 98% of the primitive hematopoietic stem cells that are capable of surviving for at least four weeks or more under the culture conditions provided by the CAFC assay.
  • the PHSC antibody in a pharmacologically acceptable vehicle and selectively depletes or inactivates the PHSCs of the recipient.
  • donor hematopoietic cells Administering a sufficient number of donor hematopoietic cells to the recipient such that donor stem cells engraft and give rise to mixed chimerism.
  • the donor hematopoietic cells are administered following one or more of the treatments disclosed herein, e.g., those described below;
  • immune suppressive antibodies or drugs such as for example, administering an inhibitor of cell proliferation, e.g., deoxyspergualin (DSG), or an anti-metabolite, e.g. brequinar, or an anti-T cell antibody, e.g., one or both of an anti-CD4 or anti-CD8 antibody.
  • an inhibitor of cell proliferation e.g., deoxyspergualin (DSG)
  • an anti-metabolite e.g. brequinar
  • an anti-T cell antibody e.g., one or both of an anti-CD4 or anti-CD8 antibody.
  • treatments other than whole body irradiation
  • thymic space in the recipient, such as for example, 1) by irradiating the thymus of the recipient, e.g., by administering between 1 and 10, more preferably between 3 and 7, e.g., 7 Gy, of thymic irradiation and/or 2) by administering anti-T cell antibodies to the recipient in a sufficient dose to inactivate recipient thymocytes.
  • thymic space treatments for the creation of thymic space include: the administration of steroids, corticosteroids, brequinar, co-stimulatory blockade or an immune suppressant chemical or drug, such as for example rapamycin, cyclosporine (available as Sandimmune®, Novartis Pharmaceutical Corp., East Hanover, NJ), or FK506 (available from Fijisawa Healthcare, Inc.).
  • Treatment to create thymic space should be administered, or at least begun, prior to the administration of donor hematopoietic stem cells.
  • An effective treatment should deplete single positive thymocytes to an extent that engraftment and the formation of mixed chimerism is optimized in the absence of the creation of hematopoietic space, e.g., hematopoietic space created by whole body irradiation.
  • the recipient's single positive thymocytes are depleted by at least 20, 40, 60, or 80%. Treatments that result in between 10 and 90% depletion are preferred.
  • the length of the treatment will vary with dosage and the effectiveness of the agent but will generally be less than 60, 30, or 15 days.
  • the treatment should last at least 7, and more preferably 10, or 14 days in length.
  • an immunosuppressive chemical or drug e.g., cyclosporine
  • the treatment e.g., the administration of cyclosporine
  • the agent should be on a daily basis or as needed to maintain a level of the agent that allows the desired level of depletion in the recipient.
  • a particularly preferred treatment is the administration of an immunosuppressive chemical, e.g., cyclosporine, for more than 7 and less than 30 days.
  • a useful regimen in rodents is 20 mg/kg/day cyclosporine for 14 days ending on the third day before administration of stem cells.
  • the dose and/or timing of administration of the agent should not affect the concentration of donor stem cells administered.
  • an anti-CD4, anti-CD8 antibody composition is administered to the recipient, the composition should be substantially cleared prior to administration of the donor cells so as not to deplete donor T-cells.
  • mixed chimerism is induced in the recipient and the state of mixed chimerism is formed in the absence of the induction of hematopoietic space, e.g., in the absence of hematopoietic space created by space creating irradiation, e.g., whole body irradiation.
  • the number of donor hematopoietic cells administered to the recipient can be increased by either increasing the number of stem cells provided in a particular administration or by providing repeated administrations of donor stem cells.
  • Repe ⁇ ted stem cell administration can promote engraftment, mixed chimerism, and long-term acceptance in graft recipients.
  • the invention also includes methods in which multiple hematopoietic stem cell administrations are provided to a recipient. Multiple administrations can substantially reduce or eliminate the need for hematopoietic space-creating irradiation. Administrations can be given prior to, at the time of, or after graft implantation. In preferred embodiments multiple administrations of stem cells are provided prior to the implantation of a graft. Two, three, four, five, or more administrations can be provided. The period between administrations of hematopoietic stem cells can be varied.
  • a subsequent administration of hematopoietic stem cell is provided: at least two days, one week, one month, or six months after the previous administration of stem cells; when the recipient begins to show signs of host lymphocyte response to donor antigen; when the level of chimerism decreases; when the level of chimerism falls below a predetermined value; when the level of chimerism reaches or falls below a level where staining with a monoclonal antibody specific for a donor PBMC antigen is equal to or falls below staining with an isotype control which does not bind to PBMC , e.g. when the donor specific monoclonal stains less than 1-2% of the cells; or generally, as is needed to maintain a level of mixed chimerism sufficient to maintain tolerance to donor antigen.
  • Post graft administration of hematopoietic stem cells can be provided: at least two days, one week, one month, or six months after the previous administration of stem cells; at least two days, one week, one month, six months, or at any time in the life span of the recipient after the implantation of the graft; when the recipient begins to show signs of rejection, e.g., as evidenced by a decline in function of the grafted organ, by a change in the host-donor-specific antibody response, or by a change in the host-lymphocyte response to donor antigen; when the level of chimerism decreases; when the level of chimerism falls below a predetermined value; wh2n the level of chimerism reaches or falls below a level where staining with a monoclonal antibody specific for a donor PBMC antigen is equal to or falls below staining with an isotype
  • one or more of the administrations can include a number of donor hematopoietic cells that is at least 25% to 200% as great as, the number of bone marrow hematopoietic cells found in an adult of the recipient species.
  • Preferred embodiments include use of cytokine mobilization of hematopoietic progenitor cells and leukapheresis.
  • the method includes inactivating natural killer cells, preferably graft reactive NK cells, of the recipient mammal.
  • natural killer cells preferably graft reactive NK cells
  • This can be accomplished, e.g., by introducing into the recipient mammal an antibody capable of binding to natural killer cells.
  • the administration of antibodies, or other treatment to inactivate natural killer cells can be given prior to introducing the hematopoietic stem cells into the recipient mammal or prior to implanting the graft in the recipient.
  • This antibody can be the same or different from an antibody used to inactivate T cells.
  • the method includes inactivating T cells, preferably graft reactive T cells, e.g., by introducing into the recipient mammal an antibody capable of binding to T cells of the recipient mammal, either prior to introducing the hematopoietic stem cells into the recipient or prior to implanting the graft.
  • This antibody can be the same or different from an antibody used to inactivate natural killer cells.
  • anti-NK antibody is anti-human thymocyte polyclonal anti-serum (ATG).
  • AGT anti-human thymocyte polyclonal anti-serum
  • a second anti-mature T cell antibody can be administered as well, which lyses T cells as well as NK cells. Lysing T cells is advantageous for both bone marrow and graft survival.
  • Anti-T cell antibodies are present, along with anti-NK antibodies, in anti-thymocyte anti-serum. Repeated doses of antibodies, e.g., anti-NK or anti-T cell antibodies, may be preferable.
  • the recipient does not receive treatments that stimulate the release of a cytokine by mature T cells.
  • the recipient should not receive a substance, e.g., a steroid drug, e.g., prednisone (17,21-dihydroxypregna- 1,4-diene-3,11, 20-trione), at a dosage or concentration which stimulates the release of a cytokine by mature T cells in the recipient.
  • a substance e.g., a steroid drug, e.g., prednisone (17,21-dihydroxypregna- 1,4-diene-3,11, 20-trione
  • the recipient is free of such treatment from the time stem cells are first administered until the graft is implanted or until mixed chimerism and tolerance is established.
  • the method includes the administration of an agent such as a drug or other chemical agent, which induces tolerance to unmatched class I and/or minor antigens on the graft which is introduced into the recipient.
  • an agent such as a drug or other chemical agent, which induces tolerance to unmatched class I and/or minor antigens on the graft which is introduced into the recipient.
  • a short course of help reducing treatment such as a short course of high-dose cyclosporine, preferably is administered at the time the graft is introduced into the recipient.
  • the duration of the short course of help reducing treatment is approximately equal to or is less than the period required for mature T cells of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen; in more preferred embodiments, the duration is approximately equal to or is less than two, three, four, five, or ten times, the period required for a mature T cell of the recipient species to initiate rejection of an antigen after first being stimulated by the antigen.
  • Thymic or lymph node thymocytes or T cells might otherwise inhibit the engraftment or survival of the administered donor cells.
  • Such inactivation can be accomplished by one or more of: 1) irradiating the thymus of the recipient mammal with a dose of radiation sufficient to inactivate thymocytes, e.g., 1-10 Gy, more preferably between 3 and 7, e.g., about 3.5 or 7 Gy of thymic irradiation; 2) administering one or repeated doses of an anti-T cell or anti- thymocyte antibody; or 3) administering to the recipient a short course of an immunosuppressant chemical or drug, as is described herein. Inactivation of thymocytes or T cells most preferably is performed prior to donor hematopoietic stem cell or graft transplantation.
  • the method includes diminishing or inhibiting thymocyte or T cell activity, preferably the activity of thymic or lymph node T cells by administering to the recipient a short course of an immunosuppressive agent, e.g., a chemical or drug, e.g., cyclosporine, sufficient to inactivate thymocytes or T cells, preferably thymic or lymph node T cells.
  • an immunosuppressive agent e.g., a chemical or drug, e.g., cyclosporine
  • the duration of the short course of immunosuppressive agent is: approximately equal to 30 days; approximately equal to or less than 8-12 days, preferably about 10 days; approximately equal to or less than two, three, four, five, or ten times the 8-12 or 10 day period.
  • the short course can begin: before or at about the of time the treatment to induce tolerance is begun, e.g., at about the time stem cells are introduced into the recipient; on the day the treatment to induce tolerance is begun, e.g., on the day stem cells are introduced into the recipient; within 1, 2, 4, 6, 8, 10, or 30 days before or after the treatment to induce tolerance is begun, e.g., within 1 , 2, 4, 6, 8, 10, or 30 days before or after stem cells are introduced into the recipient.
  • the short course of an immunosuppressive can be administered in conjunction with an anti-T cell antibody.
  • T cells e.g., thymic or lymph node T cells
  • ATG antibody intravenous administrations of ATG antibody, or similar, preparations.
  • inventions include (optionally): the step of, prior to hematopoietic stem cell transplantation, creating hematopoietic space, e.g., by irradiating the recipient mammal with low-dose, e.g., less than 4, preferably less than 3, more preferably less than 2 or 1 Gy, whole body irradiation to deplete or partially deplete the bone marrow of the recipient. As is discussed herein this treatment may be reduced or entirely eliminated.
  • low-dose e.g., less than 4, preferably less than 3, more preferably less than 2 or 1 Gy
  • the method includes the step of introducing into the recipient a donor graft such as for example a heart, pancreas, liver, or kidney in addition to administration of hematopoietic cells.
  • a donor graft such as for example a heart, pancreas, liver, or kidney in addition to administration of hematopoietic cells. This process can be used in conjunction with any of the other methods disclosed herein.
  • the methods of the present invention also provide for the treatment of patients having a hematological disorder such as for example a hematological malignancy e.g., leukemia or relapsed multiple myeloma.
  • a hematological malignancy e.g., leukemia or relapsed multiple myeloma.
  • the methods of the present invention also provide for treatment of a patient, especially a human patient, having non-neoplastic disorders such as for example sickle cell anemia or thalassemia.
  • the present invention provides a method for treating a hematological disorder in a patient in need thereof comprising: 1) administering a myeloreductive non-myeloablative treatment (for example such as described in US Patent Nos. 5,876,708 and 6,006,752, the disclosures of which are incorporated herein by reference) in an amount that mixed chimerism is induced in the recipient; 2) depleting the recipient of primitive hematopoietic stem cells by administering the antibody of the present invention in a therapeutic composition; and 3) introducing into the recipient hematopoietic cells from an allogeneic donor to form chimeric bone marrow in the recipient.
  • a myeloreductive non-myeloablative treatment for example such as described in US Patent Nos. 5,876,708 and 6,006,752, the disclosures of which are incorporated herein by reference
  • each of the recited steps is a separate discrete administration or agent.
  • the donor stem cells are provided as allogeneic bone marrow, mobilized peripheral blood cells, or cord blood cells.
  • the donor stem cells in some instances, can be expanded ex vivo for transplantation.
  • the donor and the recipient can be the same species (allogeneic) or each can be of a different species (xenogeneic) e.g non-human primate, swine.
  • the donor is both a source of the hematopoietic cells (e.g BMC or MPSC) and of the graft (e.g. organ, tissue or cells e.g. leukocytes).
  • the donor is a mammal such as for example an inbred miniature swine (US Patent Applic. No. 09/378,684 filed 8/20/99, the disclosure of which is incorporated herein by reference).
  • the donor hematopoietic stem cells and the graft can be from the same mammal or can be from a different mammal of the same species as the HSC donor which are MHC matched or highly inbred, eg. the first donor can be from the same herd as the second donor wherein the herd members are inbred at MHC loci or locus.
  • donor hematopoietic stem cell administration is given after the agent utilized to deplete or inactivate recipient T-cells has been substantially cleared from the circulatory system.
  • both administered anti-T-cell antibodies and anti-PHSC antibodies have been cleared from the circulatory system of the recipient.
  • the myeloreductive treatment includes treating the recipient, prior to introduction of the donor stem cells, with a cytoreductive agent selected from one or more of alkylating agents (e.g., nitrogen mustards [such as mechloretamine], cyclophosphamide, elphalan and chlorambucil), alkyl sulphonates (e.g., busulphan), nitrosoureas (e.g., carmustine, lomustine, semustine and streptozocine), triazenes (e.g., dacarbazine), antimetabolites (e.g., folic acid analogs such as methotrexate), pyrimidine analogs (e.g.
  • alkylating agents e.g., nitrogen mustards [such as mechloretamine], cyclophosphamide, elphalan and chlorambucil
  • alkyl sulphonates e.g., busulphan
  • nitrosoureas
  • fluorouracil and cytarabine purine analogs (e.g., fludarabine, idarubicin, cytosine arabinoside, mercaptopurine and thioguanine), vinca alkaloids (e.g., vinblastine, vincristine and vendesine), epipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., actinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitomycin), dibromomannitol, deoxyspergualine (DSG), dimethyl myleran and thiotepa.
  • purine analogs e.g., fludarabine, idarubicin, cytosine arabinoside, mercaptopurine and thioguanine
  • vinca alkaloids e.g., vinblastine, vincristine and vendesine
  • Preferred myeloreductive non-myeloablative agents are alkylating agents, e.g., cyclophosphamide, or fludarabine or similar substances, however, hematopoietic space creating antibodies or drugs, e.g., inhibitors of cell proliferation, e.g., DSG, or an anti-metabolite, e.g. brequinar, or an anti-T cell antibody, e.g., one or both of an anti-CD4 or anti-CD8 antibody can be used as a myeloreductive non-myeloablative agent.
  • alkylating agents e.g., cyclophosphamide, or fludarabine or similar substances
  • hematopoietic space creating antibodies or drugs e.g., inhibitors of cell proliferation, e.g., DSG, or an anti-metabolite, e.g. brequinar, or an anti-T cell antibody, e.g., one or both of an anti
  • Antibodies suitable for inactivating T cells include anti-T cell antibodies (including humanized versions thereof) for example an ATG preparation, OKT3, BTI-322® (US Patent No. 5,730,979 the disclosure of which is hereby incorporated by reference).
  • Antibodies suitable for depleting and/or inactivating hematopoietic stem cells in the recipient include antibodies directed against isolated progenitor cells (for example antibody A3C6E2, exemplified in US Patent No. 5,808,002) and/or antibodies directed against primitive hematopoietic cells and/or antibodies directed against one or more known markers of primitive hematopoietic cells such as for example: CD117, (for example antibody SR-1 exemplified in US Patent Nos.
  • antibodies for depleting and/or inactivating endothelial cells which produce factors such as for example granulocyte-, granulocyte-macrophage-, and macrophage colony-stimulating factors, which stimulate and support primitive hematopoietic stem cells may be administered either alone or in combination with the above mentioned antibodies.
  • Such antibodies can be combined in a pharmaceutically acceptable carrier to form a therapeutic composition and administered to a patient in need thereof at a therapeutically effective dose.
  • Antibodies to hematopoietic stem cells can for example be administered intravenously or intraperitoneally or intramuscularly.
  • the characteristics of the carrier will depend on the route of administration.
  • Such a composition may also contain (in addition to at least one anti-HSC antibody and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • the immunosuppressant regimen includes treatment with one or more of a macrolide immunosuppressant, azathioprine, steroids (e.g., prednisone, methyl prednisolone), or sub-lethal nonmyleoablative irradiation of lymphocyte-containing tissue.
  • a macrolide immunosuppressant e.g., azathioprine, steroids (e.g., prednisone, methyl prednisolone), or sub-lethal nonmyleoablative irradiation of lymphocyte-containing tissue.
  • Treatments which deplete or inactivate recipient T cells may do so by directly or indirectly depleting, inactivating, or reducing recipient T cell activity.
  • Indirect methods of altering T cell activity include reducing the number of cells capable of forming T cells such as for example primitive hematopoietic stem cells, and/or reducing levels of factors, such as for example cytokines, which support and maintain T cells and/or their precursors.
  • Treatments which inhibit recipient primitive hematopoietic stem cells capable to maturing into recipient T cells should be administered prior to donor stem cell administration.
  • T cell activity suppressors can be administered at any time in the course of the method but should not be administered in such a manner that donor T cell engraftment will be substantially affected when they are administered.
  • treatments which selectively deplete or inactivate recipient T cells are provided both before and after the administration of donor hematopoietic stem cells. Treatment prior to the administration of donor hematopoietic stem cells is believed most desirable in that it will condition the recipient for the receipt of the donor hematopoietic stem cells.
  • treatments to inhibit T cell activity e.g., anti-T-cell antibodies, cyclosporine or antibodies capable of depleting and/or inactivating recipient hematopoietic stem cells (PHSC) preferably PHSC, and most preferably selected PHSC
  • PHSC hematopoietic stem cells
  • an anti-HSC treatment agent for example the administration of antibodies, will be given to the patient about 1, 2, 3, 4, or 5 days prior to stem cell transplantation depending upon the rate of clearance of the agent from the recipient's circulation and upon the level of depletion desired.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 93%, at least 95%, at least 98% depletion and/or inactivation of PHSC is achieved. It may be desirable to repeat pre- stem cell administrations every until the patient shows excess antibodies in the serum and about 80, 90, or 99% depletion of peripheral T cells and then to perform the donor HSC transplantation. Treatments can also be administered one, two, three, or more times after donor hematopoietic stem cell transplantation. Typically, a post-stem cell transplant treatment will be given about 1 , 2, 3, 4, or 5 days after bone marrow transplantation.
  • two or more T-cell inhibiting modalities or treatments can be combined.
  • an antibody e.g., an anti-T-cell antibody, an agent capable of selectively depleting and/or inactivating HSC, for example an antibody of the present invention alone or in combination with a chemical agent such as Bulsulfex®, an immunosuppressant e.g., cyclosporine, and thymic irradiation, are all administered to the recipient.
  • the immunosuppressant can be administered once, or more than once, but the administrations should be short term and not chronic or long term administration. In general, this will mean the treatment is administered for not more than 30, 45, 60, 90, or 120 days, and in many treatments this means administration on 1 , 2, 3, 4, 5, or fewer days.
  • Cyclosporine and similar agents will generally be administered for not more than 30, 45, 60, 90, or 120 days.
  • PHSC depleting agents such as antibodies will generally be administered for 1 , 2, 3, 4, 5, or fewer days prior to donor stem cell infusion.
  • donor leukocytes are administered to a patient after achievement of mixed chimerism has been confirmed. While not wishing to be bound by theory, the donor leukocyte infusion (DLI) is believed to provide additional GVL activity-donor leukocytes are believed to further reduce the number of cancer cells in the patient. The need for or appropriateness of donor leukocyte administration can be evidenced by incomplete tumor regression. Donor leukocyte administration should be delayed for at least 10, 20, 30, 35 or 60 days after the administration of any myeloreductive, non-myeloablative treatment. Initial trials showed a delay of about 35 days to be suitable.
  • DLI donor leukocyte infusion
  • the donor leukocyte infusion is delayed to avoid introduction of relatively large numbers of donor immune cells into the patient during the period when induced pro-inflammatory conditions exist. Delay allows the DLI recipient to recover from conditioning and to be less susceptible to GVHD as a result of the DLI, especially when mismatched donor tissue is used. While not wishing to be bound by theory, it is thought that the donor leukocyte infusion converts the mixed chimeric state of the recipient to one which is fully chimeric while limiting the graft cell mediated immune attack to the hematopoietic compartment, thereby minimizing GVHD and maximizing GVL effects.
  • the method includes creating thymic space in the recipient utilizing an agent other than thymic irradiation.
  • thymic space can be created by irradiating the thymus of the recipient, for example, by administering between 100 and 1 ,000, more preferably between 300 and 700, e.g., 700 rads, of thymic irradiation.
  • thymic space is created by administering anti-T cell antibodies in sufficient dose to inactivate thymocytes.
  • Other agents for the creation of thymic space include: the administration of brequinar, or an immune suppressant chemical or drug such as for example rapamycin, cyclosporine, or FK506.
  • an effective agent or combination of agents should deplete single positive thymocytes to an extent that engraftment and the formation of mixed chimerism is optimized.
  • the recipient's single positive thymocytes are depleted by at least 20, 40, 60, or 80%. Treatments which result in between 10 and 90% depletion are preferred.
  • the recipient does not receive additional treatments which stimulate the release of a cytokine by mature T cells.
  • the recipient should not receive a substance, e.g., a steroid drug, e.g., Prednisone (17, 21-dihydroxypregna-1 , 4diene-3, 11 , 20-trione), at a dosage or concentration which stimulates the release of a cytokine by mature T cells in the recipient.
  • a substance e.g., a steroid drug, e.g., Prednisone (17, 21-dihydroxypregna-1 , 4diene-3, 11 , 20-trione
  • the subject is free of such treatment from the time stem cells are first administered until mixed chimerism is established and/or donor leukocytes are administered.
  • an agent for example 15-deoxyspergualin, mycophenolate mofetil (MMF), brequinar sodium, or a similar agent, which inhibits the production, levels, or activity of anti-donor antibodies in the recipient may be utilized, especially in cases of increasing mismatch such as for example xenogeneic transplantation.
  • MMF mycophenolate mofetil
  • brequinar sodium or a similar agent, which inhibits the production, levels, or activity of anti-donor antibodies in the recipient may be utilized, especially in cases of increasing mismatch such as for example xenogeneic transplantation.
  • the method includes: inhibiting natural killer cells of the recipient preferably prior to introducing donor tissue into the subject, for example by introducing into the recipient a drug such as for example deoxyspergualin (DSG) (Bristol-Myers- Sqibbs), an antibody such as anti-lgM or ATG which is capable of binding to and depleting or inactivating natural killer cells of the subject or for example by depleting natural antibodies from the blood of the recipient.
  • DSG deoxyspergualin
  • an antibody such as anti-lgM or ATG which is capable of binding to and depleting or inactivating natural killer cells of the subject or for example by depleting natural antibodies from the blood of the recipient.
  • Depletion from the blood can also be achieved, by way of example, by contacting the recipient's blood with an epitope which absorbs preformed anti-donor antibody.
  • the epitope can be coupled to an insoluble substrate and provided for example as an affinity column.
  • an alpha (1-3) galactose linkage epitope-affinity matrix e.g., matrix bound linear B type VI carbohydrate
  • an alpha (1-3) galactose linkage epitope-affinity matrix e.g., matrix bound linear B type VI carbohydrate
  • natural antibody depletion can be achieved by hemoperfusing an organ, e.g., a liver or a kidney, obtained from a mammal of the donor species.
  • an organ hemoperfusion antibodies in the blood bind to antigens on the cell surfaces of the organ and are thus removed from the blood.
  • DSG or similar drugs
  • anti-lgM antibodies and hemoperfusion
  • the methods of the present invention can also include the management of GVHD responses post-transplantation by administration of immunosuppressants, or by use of engineered stem cells which give rise to small molecule ablatable T cells or other hematopoietic cells. See, for example, U.S. Pat. No. 5,834,266.
  • the invention disclosed herein permits improved hematopoietic stem cell (HSC) engraftment in patients without the need to administer life threatening levels of ablative therapy, greatly improving the survival and cure rates of numerous hematopoietic diseases that do not require high-dose ablation which currently rely on the transplantation of HSC.
  • HSC hematopoietic stem cell
  • diseases and conditions treatable by the methods of the present invention include: congenital B- and T- lymphocyte disorders, such as predominantly antibody defects, X-linked agammaglobulinemia, common variable immunodeficiency, immunodeficiency with thymoma, selective IgA deficiency, X-linked immunodeficiency with hyper- IgM antibody deficiency with normal immunoglobulins, subclass deficiency, poor response to polysaccharide antigens, or X-linked lymphoproliferative syndrome; or a combined immunodeficiency-primary defect in cellular immunity, such as severe combined immunodeficiency, autosomal recessive and X-linked, adenosine deaminase deficiency, defective expression of histocompatibility antigens, deficiency of T cell receptors, Omen's syndrome, cellular immunodeficiency with immunoglobulins (Nezelofs syndrome), purine nucleoside phosphorylase
  • disorders of phagocytic function such as disorders of production and consumption, abnormal production, Kostmann's syndrome, Schwachman's syndrome, cyclic neutropenia, Primary B- and T-lymphocyte disorders, X-linked hyper-lgM, X-linked agammaglobulinemia, ataxia telangiectasia, cartilage-hair hypoplasia, IgA deficiency; disorders of migration and chemotaxis, general defects in leukocyte mobility, non-specific disorders, such as Kartogener's syndrome, lazy leukocyte syndrome, hyper-lgE syndrome, Chediak-Higaschi syndrome; or disorders of intracellular killing, such as chronic granulomatous disease, myeloperoxidase deficiency, gluthathione reductase and peroxidase deficiency, glucose-o- phosphate dehydrogenase deficiency; or a deficiency of leukocyte function antigen 1 (LFA-1).
  • LFA-1 le
  • the invention also finds utility in bone marrow transplantation for such hematologic disorders as marrow aplasia, Fanconi's aplasia, Diamond-Blackfan syndrome, hemoglobinopathies, ⁇ -thalassemia major, sickle cell anemia, neutrophil disorders, congenital neutropenia, chronic granulomatous disease, Chediak-Higashi syndrome, Osteopetrosis; immune deficiency disorders, such as severe combined immunodeficiency disease, ADA-deficient SCID, reticular dysgenesis, bare lymphocyte syndrome, PNP deficiency, LFA-1 deficiency, ataxis telangiectasia, or Wiskoff-Aldrich syndrome; metabolic disorders, such as mucopolysaccharidoses, Hurler's syndrome, Hunter's syndrome, Sanfilippo's syndrome, leukodystrophies, metachromatic leukodystrophy, adrenoleucodystrophy, sphingolipidoses, Neimann-P
  • Isolating of the desired antibody from ascitic fluid by immunoaffinity chromatography takes advantage of the allotypic difference existing between the immunoglobulins of the rat receiving the producing hybridoma and the MAb secreted by the latter (Bazin, H., Cormont F. and DeClercq, L.. J.
  • Wistar, Sprague-Dawley, Lewis and Louvain rats are among the rat varieties suitable for use as the source of antibody-producing cells for fusion.
  • the immunogen can be derived from any hematopoietic stem cell or any primitive hematopoietic cell line, such as for example the cell line produced by the method described in US Pat. No 6,280,718, the disclosure of which is hereby incorporated by reference. Human origin of these cells is preferred.
  • human bone marrow cells which can be phenotypically and functionally demonstrated to be primitive hematopoietic stem cells will be used to obtain a protein preparation, such as a membrane protein preparation, for use as the antigen.
  • CD117 would be obtained from the human acute megakaryocytic cell line designated Mo7e that is known to express CD117 (US Patent Nos.
  • Hybridomas expressing a single monoclonal antibody will be isolated.
  • the MAb collected will be used as the test antibody.
  • the human bone marrow cells will be resuspended at 10 6 cells/ml in
  • Iscove's Modified Dulbecco's Medium (IMDM; JHR Biosciences Inc; Kansas) supplemented with 10% fetal bovine serum (FBS), gentamycin, 50 ⁇ M ⁇ - mercaptoethanol and 0, 1 , 5 and 10 ⁇ g/ml of an isotype control antibody or the test antibody.
  • FBS fetal bovine serum
  • a CAFC assay will be performed as above as described by Breems ,et al. utilizing human cytokines and a time point for colony formation of about four weeks. Hydridomas producing antibodies resulting in at least a 50% depletion of colonies as compared to controls will be further characterized for use.
  • the human bone cell suspensions will be processed as follows: i. incubate at 37°C for 18 hours with or without addition of 200 ng/ml human stem cell factor (hSCF); ii. incubate at 4°C for 1 hour to coat cells with the antibody that is isolated as described above, wash a.id resuspend in media containing rabbit complement and then incubate at 37°C for a further hour; iii. incubate at 4°C for 1 hour to coat cells with the antibody, washe, co- culture with human peripheral blood monocytes and then incubate at 37°C for 18 hours.
  • hSCF human stem cell factor
  • the cells will be plated in methyl cellulose cultures (see below) to provide a rapid screening for the initial dose range for efficacy and provide some insight as to whether the antibodies alone are inhibitory or whether they require complement or antibody dependent cellular cytotoxicity (ADCC) for complete action.
  • ADCC antibody dependent cellular cytotoxicity
  • Methods are described for treating patients using the therapeutic composition(s) of the present invention to include treating patients with blood cancers, such as non-Hodgkin's lymphomas.
  • blood cancers such as non-Hodgkin's lymphomas.
  • CsA cyclosporine
  • HLA-identical allogeneic or autologous bone marrow transplantation has led to durable remissions in only 0-23% of patients.
  • animal studies have shown that MHC-disparate bone marrow transplants can mediate anti-tumor effects that greatly exceed those achieved with MHC-matched BMT.
  • the potential of HLVmismatched bone marrow transplantation as immunotherapy for hematologic malignancies has not yet been exploited, largely due to the high incidence of intractable GVHD and the potentially lethal failure of marrow engraftment associated with standard ablative conditioning regimens.
  • the protocol disclosed herein is optimized to further reduce toxicities by using an antibody of the present invention in addition to an anti-T cell antibody and Tl, thereby eliminating the need for immunosuppressive agents such as cyclophosphamide and myeloablative whole body irradiation.
  • immunosuppressive agents such as cyclophosphamide and myeloablative whole body irradiation.
  • patients will undergo non- myeloablative conditioning therapy followed by allogeneic mobilized peripheral hematopoietic stem cell (MPHSC) transplant.
  • Eligibility criteria will include chemotherapy-refractory hematologic malignancy, ECOG performance status of 2 or less, age of 65 years or less, and adequate organ function.
  • a less than three of six HLA antigen-mismatched related donor may be required.
  • Patients and donors will be typed using standard serological techniques for HLA-A and B, and SSOP-or SSP-based analyses for HLA-DR.
  • TC such as for example anti-CD117 in a pharmacologically acceptable carrier
  • Day -7 1 mg/kg
  • the second and subsequent doses should be 1.0 mg/kg, administered over 2 hours, without steroid pre-medication.
  • Diphenhydramine and acetominophen may be administered prior to the second and subsequent doses, however.
  • one or more additional doses can be administered as long as excess antibody has been cleared from the circulation by the time of donor MPHSC infusion.
  • the first dose of anti-T cell antibody should be on day -7 (0.1 mg/kg) followed on day -6 by a dose of 0.5 mg/kg.
  • the initial dose should be preceded 1-4 hours by the administration of intravenous methylprednisolone sodium succinate (8 mg/kg up to a maximum dose of 500 mg), along with diphenhydramine(50 mg p.o.), and acetominophen, (650 mg, p.o.).
  • the therapeutic composition and anti-T cell monoclonal antibodies may be administered concurrently or in succession.
  • 700 cGy of thymic irradiation can be administered in a single dose on Day -1 ,
  • a field size of approximately 8 cm wide and 10 cm in longitudinal dimension should be used, with the midpoint of the upper edge of the field at the sternal notch.
  • the dose should be calculated at a depth of approximately 6 cm, guided by results of a lateral chest roentgenogram for the approximate location of the thymus.
  • a 10 MV x-ray machine at maximum dose rate should be used. No pre-medication for nausea should be required.
  • the details of field sizes, dose calculation, energy, beam spoiling, and dose rate are recorded on the coronary flow reserve (CRF).
  • thymic irradiation can be eliminated by increasing the dose of donor mononuclear cells transplanted and/or by administering an antigen-presenting cell inhibitory agent such as a co-stimulatory blocker, e.g. one or more agents that inhibit the CD40-CD40 ligand interaction and/or the
  • Cyclosporine either Neoral ® or Sandimmune ® or equivalent should be administered orally starting on Day-1.
  • the initial dose should be 6 mg/kg given twice with doses administeredapproximately 12 hours apart, followed by a single dose of 4 mg/kg given late in the evening on the day of transplantation (Day 0).
  • the dose should be 4 mg/kg given twice a day, and adjusted to provide a trough whole blood concentration of 400-500 ng/mL, as measured by a monoclonal antibody-based assay, or the equivalent if a different assay or serum rather than whole blood are used, to determine trough cyclosporine concentrations.
  • Cyclosporine should be continued for 35 days, then tapered over 7 days and discontinued on Day 42. If the patient shows signs of GvHD, cyclosporine coverage may be extended.
  • CP is used for cytoreduction of the malignancy, it may be given intravenously, 50 mg/kg/d, (with dosing based on actual or ideal body weight, whichever is less) on days -6 through -3.
  • Dexamethasone may be used at a dose of 20 mg/d prior to each dose of CP.
  • Hematopoietic stem cell donors will undergo treatment with recombinant G-CSF (Filgrastim; Amgen Corp; Thousand Oaks, CA) 5-10 micrograms/kg/day subcutaneously for 4-7 days to mobilize hematopoietic progenitor cells into the peripheral blood where they may be collected by a 3- 4 blood volume (15-18 I) leukapheresis using standard techniques. Cytokine mobilization should be monitored, and the day of leukapheresis may be determined by the CD34 + cell count and total white blood cell count in peripheral blood. The leukapheresis product may then be further processed to enrich for the hematopoietic stem cells, e.g.
  • the cells may be cryopreserved until needed for infusion into the recipient at the time of transplantation.
  • the total WBC count of the donor should not exceed 70 x 10 3 /mm 3 . If this level is reached, the dose of Filgrastim should be reduced and the WBC count followed daily until it falls below this value. Every effort should be made to collect an adequate cell product from these patients, and preferably a high-dose (>5 x 10 6 CD34 + cells/kg).
  • donors may be leukapheresed and aliquots of the product can be cryopreserved for potential administration of donor leukocyte infusions to increase levels of chimerism and enhance the donor vs. tumor therapeutic effect.
  • unmobilized bone marrow may be used in place of mobilized peripheral blood (MPHSC).
  • Donor bone marrow may be procured under anesthesia by standard techniques. A target number of 3 x 10 8 /kg nucleated cells may be sought.
  • plasma may be removed from donor marrow prior to transplantation.
  • red blood cells are depleted from the donor marrow using a CS-3000 Blood Cell Separator (Baxter-Fenwal, Round Lake, IL).
  • Donor Lymphocyte Infusion can be administered to qualified patients, preferably on day 35, and subsequently as deemed potentially beneficial.
  • DLI is used as an anti-tumor immunotherapy and can be administered to patients who do not exhibit symptoms of GvHD. It is a preferred treatment for those patients demonstrating mixed chimerism ( ⁇ 90% donor cells) and for those patients testing positive for disease.
  • DLI can be administered as a single dose of 1 x 10 7 T cells/kg in donor peripheral blood or it may be given as a series of graded doses starting as low as 5 x 10 6 T cells/kg and increasing up to 5 x 10 7 T cells/kg.
  • Patients with confirmed GvHD should not be given DLI. For those patients who become full chimeras after the initial transplant and who do not have GvHD, they may be administered DLI at the discretion of the physician; however, advisement is that the potential benefits may be outweighed by the risks of developing GvHD.
  • FCM Flow cytometry
  • VNTR variable number of tandem repeats
  • STR short tandem repeat
  • 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 a match or any degree of mismatch at MHC loci or other loci which influence graft rejection.
  • class I and class II MHC loci the donor and recipient can be: matched at class I and mismatched at class II; mismatched at class I and matched at class II; mismatched at class I and mismatched at class II; matched at class I, matched at class II.
  • mismatched at MHC class I means mismatched for one or more MHC class I loci, e.g., in the case of humans, mismatched at one or more of HLA-A, HLA-B, or HLA-C.
  • 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.
  • 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.
  • a neoplastic disorder particularly a disorder which is resistant to normal modes of therapy, e.g., chemotherapy or radiation therapy.
  • the methods of the invention are also particularly useful in replacing tissue or organ with a patient who is believed to be, based on past experience or current expectations, unreliable with respect to the careful self- administration of chronic immunosuppressive regimens which would otherwise be required following the transplantation of a mismatched mammalian organ or tissue.
  • 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 kidney, liver, heart, lung, thymus, pancreas e.g.
  • islet cells, 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.
  • Blockers of the CD40 ligand-CD40 or CD28-B7 interactions can be administered repeatedly.
  • blockers can be administered one, two, three or more times prior to donor bone marrow transplantation.
  • a pre-bone marrow transplantation dose is 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.
  • a blocker can also be administered one, two, three, or more times after donor bone marrow transplantation.
  • a post-bone marrow transplant treatment is 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.
  • a blocker is administered at least once (and preferably two, three, or more times) prior to donor bone marrow transplantation and at lease once (and preferably two, three, or more times) after donor bone marrow transplantation.
  • any of the methods disclosed herein that comprise administering hematopoietic stem cells can further include multiple administrations of stem cells, such as a first and a second administration of stem cells are provided prior to the implantation of a graft; a first administration of donor stem cells is provided at the time of implantation of the graft.
  • a first administration of donor stem cells is provided prior to or at the time of implantation of a graft and a second administration of stem cells is provided subsequent to the implantation of a graft.
  • the period between administrations of hematopoietic stem cells can be varied.
  • a subsequent administration of hematopoietic stem cell is provided: at least two days, one week, one month, or six months after the previous administration of stem cells; at least two days, one week, one month, or six months after graft implantation.
  • the methods of the invention may further include the step of administering a second or subsequent dose of hematopoietic stem cells: when the recipient begins to show signs of rejection, e.g., as evidenced by a decline in function of the grafted organ, by a change in the recipient anti-donor antibody response, or by a change in the recipient lymphocyte response to donor antigen; when the level of chimerism decreases; when the level of chimerism falls below a predetermined value; when the level of chimerism reaches or falls below a level where staining with a monoclonal antibody specific for a donor MPHSC antigen is equal to or falls below staining with an isotype control which does not bind to MPHSC, e.g.
  • ACK2 purified in-house from hybridoma supematent
  • ACK4 Chip Laboratories Ltd., Hornby, Ontario, Canada
  • ACK45 BD Pharmingen
  • 2B8 e-Bioscience, San Diego, USA
  • 3C1 Cedarlane Laboratories Ltd.
  • the mouse mastocytoma cell line P815 obtained from ATCC No. TIB-64) was used to assess whether pre-coating the cells with a particular antibody has the ability to prevent binding of the biotinylated porcine stem cell factor (pSCF, BioTransplant Inc.) or other antibodies.
  • pSCF biotinylated porcine stem cell factor
  • antibodies were categorized in terms of their recognition of different epitopes on the c-kit molecule.
  • ACK2 and ACK45 appeared to share the same or a similar epitope at the ligand binding site as both ACK2 and ACK45 prevented staining with pSCF and ACK2 competed effectively with ACK45.
  • the antibodies 2B8, 3C1 and ACK2 did not interfere with SCF binding.
  • 2B8 and ACK2 appeared to recognize the same epitope as 2B8 coating prevented ACK2 staining.
  • 3C1 recognized a rather distinct epitope as it did not compete with any of the antibodies tested.
  • Pre-coating with ACK2 did produce lower staining with 3C1 to indicate some steric hindrance and suggesting that the epitope may be relatively close to the ligand binding site.
  • Results Figure 1 shows staining intensity profiles for biotinylated pSCF and
  • mice Two anti-c-kit MAbs from clones 2B8 and 3C1 that recognized a distinctly different epitope on the c-kit molecule and did not interfere with the binding of the c-kit ligand (see Table 3.1) were used for in vivo treatment of mice.
  • This treatment was compared with a non-myeloablative dose of busulfan (Busulfex ® , Orphan Medical, Inc., Minnetonka, MN) that is known to provide for maintained mixed chimerism in both syngeneic and allogeneic BMT recipients (Adams et al. 2001 ; Andersson et al. 2003).
  • This antibody was given according to the following doses and timing with respect to marrow harvest or BMT: 1. Saline alone.
  • Figure 2 is the schematic representation of the experimental protocol of Example 4.
  • Cells were plated in 35 mm Nalge/NUNC suspension dishes in 1ml complete methyl cellulose medium containing rmSCF, rmlL-3, rhlL-6 and rh erythropoietin (MethoCult GF M3434, Stem Cell Technologies Vancouver, BC, Canada). Typically, 5,000-20,000 cells were plated in 2 dishes. Colonies containing at least 50 cells were counted at day 7 after plating and CFU-C numbers per femur estimated from CFU frequency and femoral cellularity.
  • CAFC Cobblestone-Area Forming Cell
  • the harvested BMCs were pooled for each experimental group and plated in MyeloCult M5300 (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 10 "6 M hydrocortisone in limiting dilution on confluent layers of the murine stromal cell line 721 C5 (obtained from Dr. Rob Ploemacher, Erasmus University, Rotterdam, The Netherlands) according to Ploemacher et al. (1989 and 1991).
  • the number of CAFCs per femur and the 95% confidence intervals were calculated using a GW basic computer program according to the method devised by Fazekas de St. Groth (1982).
  • Figure 5 is the schematic representation of the experimental protocol of Example 4. Results
  • 3C1 at doses of 0.125 mp per mouse at -9 and -7 days prior to bone marrow harvesting for estimation of bone marrow CFU-C and CAFC content, according to the method in Example 4.
  • Figure 8 is the schematic representation of the experimental protocol of
  • FIG. 11 shows that 2B8 plus complement had very little effect on CFU-Cs or CAFCs forming at day 7 while a progressive loss of CAFCs were seen with time in culture giving about 20% survival for the most primitive CAFC day 35 subset.
  • Table 8.1 lists how all of the13 different anti-CD117 antibodies can either block or allow staining by bio-pSCF.
  • the anti-murine CD117 MAbs 2B8 and 3C1 were also tested on Mo7e cells but failed to show cross-reactive staining ( Figure 12).
  • Figure 13 illustrates how two classes of antibodies interact with the c-kit antigen.
  • the SP cells in the marrow are a rare population defined by the ability to exclude the Hoechst 33342 dye from two fluorescence emission characteristics and is a phenotype commonly regarded to be associated with primitive stem cells in both mouse and human bone marrow (Goodell et al. 1996; Goodell et al. 1997). It is thus of interest to evaluate whether the anti-CD117 MAbs also stain these cells consistent with expression on the stem cell population.
  • Stocks of vertebral bone marrow have been obtained from The National Disease Research Interchange (Philadelphia, PA) and are stored in liquid nitrogen.
  • HBSS+ Hanks Balanced Salt Solution (HBSS, Gibco) containing 2% Fetal Calf Serum and 10 mM HEPES buffer Gibco)
  • Hoechst 33342 Bis-Benzimide, Sigma
  • the cells were then incubated for 120 minutes at 37°C and then resuspended in cold HBSS+. The cells were then maintained on ice to prohibit leakage of the Hoechst dye from the cells and stained with 104D2-PE anti- CD117 MAb or O.N.
  • the different anti-human CD117 MAbs were screened for their ability to lyse Mo7e cells via rabbit complement.
  • Mo7e cells an acute megakaryoblastic leukemia cell line (c-kit + ), were incubated at 1 x 10 7 cells/ml with either mouse isotype controls or the anti-c-kit antibodies [10 ⁇ g/ml] at 4° C to allow for antibody binding.
  • the cells were resuspended in RPMI+2% FBS supplemented with either 10% or 20% rabbit complement (Pelfreeze). The cells were treated with complement for 4 hours at 37° C to allow for lysis.
  • Selected anti-human CD117 MAbs were also assessed for their ability to deplete various hematopoietic cell subsets present in human bone marrow.
  • Stocks of vertebral bone marrow have been obtained from The National Disease Research Interchange (Philadelphia, PA) and are stored in liquid nitrogen.
  • the cells were thawed and resuspended at 10 6 cells/ml in IMDM supplemented with 10% FBS, gentamycin, 50 ⁇ M ⁇ -mercaptoethanol and 10 ⁇ g/ml of an isotype control antibody or the test anti-CD117 antibody.
  • the cell suspensions were incubated at 4°C for 1 hour to coat cells with the antibody, washed and resuspended in media containing 10% rabbit complement and incubated at 37°C for a further four hours.
  • the functional assays consisted of evaluating the growth potential of short-term repopulating progenitors in the CFU-C assay and short- and long- term repopulating subsets in the CAFC assay.
  • treated cells were plated in 1 ml complete methyl cellulose medium containing rhSCF, rhlL-3, rhGM-CSF, rh G-CSF, rhlL-6 and rh erythropoietin (MethoCult GF + M4435, Stem Cell Technologies Vancouver, BC, Canada) and the number of colonies with mono- (BFU-E and CFU-E), bi- (CFU-GM) and multipotential (CFU-GEMM or CFU-MIX) characteristics determined after 14 days of growth.
  • the CAFC assay was based on the method of Breems et al. (1994) whereby test marrow cells were overlaid over a series of dilutions on 721 C5 stromal cell layers in MyeloCult H5100 medium (Stem Cell Technologies Vancouver, BC, Canada) supplemented with 10 "6 M hydrocortisone, 10 ng/ml rhlL-3 and 20 ng/ml rhG-CSF and the frequency of cobblestones at 2, 4, 6 and 8 weeks in culture determined from limiting dilution analysis.
  • Figure 17A shows that there was little effect of treatment with the SR-1 antibody plus rabbit complement on any of the CFU subsets. While there was no significant effect of this antibody on the CAFC frequency at the earliest time point of 2 weeks, subsequent CAFC subsets were significantly depleted out to 10 weeks in long-term culture (Figure 17B).
  • Figure 18 shows that none of the antibodies, either alone or in conjunction with complement had any effect on colony formation.
  • Examples 3 to 11 involving a number of different anti-mouse and anti- human c-kit monoclonal antibodies demonstrate that an agent can be developed commercially for application in stem cell transplant conditioning therapy.
  • Initial studies on in vivo treatment of mice with antibodies that recognise distinctly different epitopes of the c-kit molecule has shown that inhibition of binding of the c-kit ligand is a not a prerequisite for these antibodies to deplete hematopoietic stem cells.
  • ACK2 marked depletion of the committed hematopoietic progenitor population was found, as shown in the CFU-C and the early-forming CAFC frequencies in the BALB/c mouse strain.
  • CFU-Cs normal human bone marrow progenitor population
  • a monoclonal antibody such as anti-c-kit (anti-CD117) can be specifically directed against hematopoietic cells of in both mouse and human bone marrow and therefore can be included in a BMT conditioning regimen and replace agents such as busulfan or radiation in allowing for permanent engraftment of transplanted stem cells and thereby greatly reducing the toxiciticies associated with existing regimens.
  • Breems DA Blokland EA, Mau S, Ploemacher RE. Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay.
  • Ploemacher RE van der Sluijs JP, Voerman JSA, Brons NHC. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 1989; 74:2755-63.
  • Ploemacher RE van der Sluijs JP, Van Beurden CAJ, Baert MRM, Chan PL. Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 1991; 10:2527-33.

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Abstract

La présente invention concerne des compositions thérapeutiques et des méthodes qui permettent de moduler le système immunitaire pour améliorer l'acceptation d'une greffe et pour traiter une malignité hématologique en l'absence de thérapies myéloablatives. La méthodologie décrite comprend une étape qui consiste à appauvrir et/ou à inactiver les cellules souches hématopoïétiques du destinataire au moyen d'une composition thérapeutique comprenant au moins un anticorps spécifique pour appauvrir et/ou inactiver les cellules souches hématopoïétiques tout en préservant les cellules sanguines matures, de préférence l'anticorps appauvrit sélectivement et/ou inactive les cellules souches hématopoïétiques primitives. La composition thérapeutique est de préférence administrée avant l'administration de moelle osseuse, de cellules souches hématopoïétiques périphériques mobilisées ou de leucocytes du donneur.
PCT/US2003/020520 2002-06-28 2003-06-27 Methodes permettant d'ameliorer l'acceptation d'une greffe par depletion des cellules souches hematopoietiques WO2004002425A2 (fr)

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WO2008067115A2 (fr) 2006-11-03 2008-06-05 The Board Of Trustees Of The Leland Stanford Junior University Immunodéplétion sélective de niche de cellules souches endogènes pour greffe
WO2015050959A1 (fr) * 2013-10-01 2015-04-09 Yale University Anticorps anti-kits et leurs méthodes d'utilisation
US9155708B2 (en) 2008-10-10 2015-10-13 Probelte Pharma, S.A. Orally administrable immunostimulant product for aquaculture
US9156912B2 (en) * 2008-12-12 2015-10-13 The University Of Tokyo Immunological reconstitution promoter or prophylactic agent for infections each of which maintains graft-versus-tumor effect
WO2016022166A1 (fr) * 2014-08-07 2016-02-11 Nitor Therapeutics Utilisation d'inhibiteur de pnp pour traiter une récidive de tumeur maligne après transplantation de cellules souches hématopoïétiques
US9334332B2 (en) 2012-07-25 2016-05-10 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies
US9540443B2 (en) 2011-01-26 2017-01-10 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies
AU2017204125B1 (en) * 2016-06-17 2017-10-26 Crispr Therapeutics Ag Compositions and methods for the depletion of cd117+ cells
WO2018116178A1 (fr) * 2016-12-21 2018-06-28 Novartis Ag Conjugués anticorps-médicament pour l'ablation de cellules souches hématopoïétiques
WO2018183613A1 (fr) * 2017-03-31 2018-10-04 The Children's Medical Center Corporation Conditionnement médié par anticorps avec immunosuppression pour permettre une greffe allogénique
US10239943B2 (en) 2014-05-23 2019-03-26 Celldex Therapeutics, Inc. Treatment of eosinophil or mast cell related disorders
US10280225B2 (en) 2015-04-06 2019-05-07 President And Fellows Of Harvard College Compositions and methods for non-myeloablative conditioning
EP3186395B1 (fr) 2014-08-26 2019-09-25 The Board of Trustees of the Leland Stanford Junior University Greffe de cellules souches avec combinaison d'un agent ciblant des cellules souches et modulation de la signalisation immunorégulatrice
WO2019244082A3 (fr) * 2018-06-20 2020-03-05 Novartis Ag Conjugués anticorps-médicament pour l'ablation de cellules souches hématopoïétiques
WO2020112870A1 (fr) 2018-11-28 2020-06-04 Forty Seven, Inc. Csph génétiquement modifiées résistantes au traitement ablatif
US10894831B2 (en) 2015-08-26 2021-01-19 The Board Of Trustees Of The Leland Stanford Junior University Enhanced depletion of targeted cells with CD47 blockade and an immune costimulatory agonist
RU2781444C2 (ru) * 2016-12-21 2022-10-12 Новартис Аг Конъюгаты антитела и лекарственного средства для разрушения гемопоэтических стволовых клеток
EP3958879A4 (fr) * 2019-04-25 2023-05-10 Actinium Pharmaceuticals, Inc. Compositions et procédés d'immunodéplétion pour le traitement des maladies hématologiques malignes et non malignes
WO2024097131A1 (fr) * 2022-11-04 2024-05-10 The Board Of Trustees Of The Leland Stanford Junior University Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées production de myéloïdes

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US10189907B2 (en) 2011-01-26 2019-01-29 Celldex Therapeutics, Inc. Polynucleotides encoding anti-KIT antibodies
US9605081B2 (en) 2012-07-25 2017-03-28 Celldex Therapeutics, Inc. Polynucleotides encoding anti-kit antibodies
US10781267B2 (en) 2012-07-25 2020-09-22 Celldex Therapeutics, Inc. Methods of treating by administering anti-kit antibodies
US9334332B2 (en) 2012-07-25 2016-05-10 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies
US10184007B2 (en) 2012-07-25 2019-01-22 Celldex Therapeutics, Inc. Methods of treating a kit-associated cancer by administering anti-kit antibodies
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US10774146B2 (en) 2014-05-23 2020-09-15 Celldex Therapeutics, Inc. Treatment of eosinophil or mast cell related disorders
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JP2020504112A (ja) * 2016-12-21 2020-02-06 ノバルティス アーゲー 造血幹細胞を除去するための抗体薬物結合体
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