WO2000066714A2 - Toxine de type shiga utilisee en tant qu'agent d'enrichissement et de purge - Google Patents

Toxine de type shiga utilisee en tant qu'agent d'enrichissement et de purge Download PDF

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WO2000066714A2
WO2000066714A2 PCT/US2000/011927 US0011927W WO0066714A2 WO 2000066714 A2 WO2000066714 A2 WO 2000066714A2 US 0011927 W US0011927 W US 0011927W WO 0066714 A2 WO0066714 A2 WO 0066714A2
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cells
shiga
toxin
hematopoietic progenitor
progenitor cells
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WO2000066714A3 (fr
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Linda M. Pilarski
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The Governors Of The University Of Alberta
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0093—Purging against cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the hematopoietic system involves both red blood cells and a heterogeneous population of white blood cells which include lymphocytes, monocytes, polymorphonuclear cells, stem cells, hematopoietic progenitor cells and platelet generating cells.
  • White blood cells also include the cells which comprise the adaptive immune system, the cells which are responsible for innate immunity and the cells that mediate inflammatory processes.
  • White blood cells are found in the primary lymphoid organs, the bone marrow and the thymus, and the peripheral lymphoid organs including the spleen, lymph nodes, Peyer's patches, and mucosal surfaces. In contrast to the primary lymphoid organs and the spleen which are linked only by the circulatory system, the peripheral lymphoid organs are linked by both the blood and the lymphatic system.
  • Both red and white blood cells are generated by hematopoietic stem cells.
  • Multipotential and committed stem cell progenitors are primarily located in the bone marrow. However, during the clinical process known as mobilization, stem cells are stimulated to leave the bone marrow and enter the blood where they can be harvested for use in autologous or allogeneic hematopoietic transplantation.
  • the earliest stem cell is a multipotential cell that is able to renew itself as well as give rise to more committed progenitors and, ultimately, all other cells of the hematopoietic system.
  • hematopoietic progenitor cells are localized in the bone marrow.
  • Hematopoietic transplantation was initially performed with bone marrow harvested from a cancer patient at a time of minimal disease when hopefully the number of malignant cells in the bone marrow was at its lowest level, or was derived from a normal volunteer whose blood type had been matched to that of the recipient to minimize graft versus host disease (a serious complication in which the grafted cells react strongly against the cells of the recipient).
  • cancer patients are treated with drugs and growth factors which cause the hematopoietic progenitor cells to both increase in number and migrate from the bone marrow to the blood.
  • the mobilized hematopoietic progenitor cells are harvested by leukapheresis and stored by freezing until required for transplant. Overall, mobilized blood transplants engraft quicker than bone marrow harvests, and are conventionally thought to include fewer malignant cells. However, this last assumption may be problematic.
  • a variety of methods have been developed to purge malignant cells from populations that include hematopoietic progenitor cells, including purification by positive selection of CD34 hematopoietic progenitor cells, negative selection to remove non- hematopoietic progenitor cells, and in vitro expansion of hematopoietic progenitor cells.
  • Hematopoietic progenitor cells are also used as a source of progenitors to autologous dendritic cells, which can then be used for vaccination strategies to treat cancer or other diseases.
  • Dendritic cells are "professional" antigen presenting cells. For example, antigens that may not normally stimulate an effective immune response, (e.g., tumor antigens) become considerably more imunogenic when they are "fed” to dendritic cells which then present them to the immune system.
  • tumor cells and dendritic cells may be fused such that tumor antigens become an intrinsic part of the dendritic cell makeup. These cells can then be used to promote an anti-tumor response or to create a tumor vaccine.
  • the invention pertains to a method for enriching hematopoietic progenitor cells, by administering to the cells a shiga-like toxin (SLT).
  • SLT shiga-like toxin
  • the cells can be from, for example, unfractionated mobilized blood, or, preferably, from a preliminarily enriched sample of hematopoietic progenitor cells. After the administration of SLT, the hematopoietic progenitor cells are enriched.
  • the invention also relates to a method for the ex vivo purging of shiga-like toxin sensitive cells and enrichment of hematopoietic progenitor cells.
  • the method involves contacting the cells with a shiga-like toxin and allowing sufficient time for enrichment of the hematopoietic progenitor cells and purging of the shiga-like toxin sensitive cells.
  • the cells are obtained from a human suffering from cancer, for example.
  • the invention also pertains to methods to enrich hematopoietic progenitor cells for ex vivo expansion of hematopoietic progenitor cells prior to other procedures.
  • the method involves treatment of, for example, an unfractionated or pre-enriched hematopoietic progenitor cell population with a shiga-like toxin.
  • the shiga-like toxin treated cells are maintained in culture for a sufficient length of time (e.g., about 5-10 days or longer) to permit in vitro expansion of the shiga-like toxin enriched hematopoietic progenitor cells.
  • the method may also involve, for example, direct reinfusion of the cells, cryopreservation of the cells, or manipulation of the cells to alter their properties prior to reinfusion.
  • the invention also pertains to a method of strengthening the hematological system of a mammal.
  • the method includes treating hematopoietic progenitor cells with a shiga-like toxin, allowing the hematopoietic progenitor cells to become enriched, and administering the hematopoietic progenitor cells to the mammal.
  • the mammal has undergone chemotherapy or radiation therapy or is suffering from an acquired or congenital immune deficiency.
  • the invention pertains to a method for treating a mammal suffering from a hematological disorder, for example, a genetic disorder or a cancer.
  • the method includes removing hematopoietic progenitor cells from the mammal, treating the cells with a shiga-like toxin, allowing sufficient time for the purging of the shiga-like toxin sensitive cells and the enrichment of the hematopoietic progenitor cells, and transplanting the cells back into the mammal.
  • the mammal is a human.
  • the cells may be transfected with genes prior to, concurrent with, or subsequent to treatment with the shiga-like toxin.
  • the invention also relates to a method for purging of malignant cells by shiga- like toxin for those cancers where CD77 receptors or binding of shiga-like toxins by the malignant cells, or by subpopulations of malignant cells, are not detectable.
  • the method includes removing a sample of cells from a mammal, advantageously, from the bone marrow or blood. The cells are then treated with an effective amount of shiga-like toxin cultured for a sufficient length of time and, advantageously, administered to the mammal.
  • the invention also pertains to methods for the generation of dendritic cells.
  • the method involves treating a culture of hematopoietic progenitor cells with a sufficient amount of a Shiga-like toxin as an enriching step, followed by culturing the cells for an appropriate period of time to generate dendritic cells.
  • the dendritic cells could subsequently be used to present tumor or other antigens, or may be fused to the tumor cell making tumor antigens an intrinsic component of the dendritic cell surface.
  • the initial culture of cells in pre-enriched for hematopoietic progenitor cells.
  • the present invention also relates to a preparation containing hematopoietic progenitor cells.
  • the hematopoietic progenitor cells in the preparation have been previously treated with a shiga-like toxin and, advantageously, purged substantially of shiga-like toxin sensitive cells.
  • the cells may be taken from, for example, the blood or bone marrow of a mammal, e.g., a human.
  • the invention pertains to a method of enriching hematopoietic progenitor cells by administering to the cells a shiga-like toxin.
  • the treated cells are subsequently cultured for a sufficient amount of time such that the hematopoietic progenitor cells are enriched.
  • the cells may be pre-enriched for hematopoietic progenitor cells prior to the administration of the shiga-like toxin.
  • the shiga-like toxin may, advantageously, be administered to the culture at a concentration, for example, of about 5-10 ⁇ g/ml.
  • the term “enriching,” “enrichment,” or “enriched” includes any increase in the overall numbers or proportion of a particular cell type in a given cell population. Preferably, both the absolute number and the proportion of the cell type of interest in the culture is increased. For example, the number of hematopoietic progenitor cells may be increased, for example, about 50% or greater, 100% or greater, 250% or greater, or, in a particularly preferred embodiment, the number of hematopoietic progenitor cells may be increased 500%.
  • pre-enriched includes any culture which comprises a certain type of cell of interest in a greater proportion than it is generally found in an unfractioned sample taken from the same source.
  • the term also includes taking cells from sites where toxin has been locally injected to stimulate, increase, or enrich cells of interest in vivo.
  • the cells enriched in vivo may include mesenchymal stem cells.
  • the culture may be pre-enriched for hematopoietic progenitor cells.
  • Pre- enriched cultures may comprise about, for example, 5% or greater, 10% or greater, 20% or greater, 30% or greater , 40% or more hematopoietic progenitor cells than in the initial unfractioned culture.
  • Methods of pre-enriching cultures for hematopoietic progenitor cells are known in the art (e.g., Bertonlini et al. Haematologica (1998) 83(9):824-48).
  • hematopoietic progenitor cells includes stem cells and other cells which differentiate through hematopoiesis to become red and white blood cells. Stem cells are pluripotent and able to differentiate along a number of pathways, thereby generating, for example, erythrocytes, granulocytes, monocytes, mast cells, lymphocytes, and megakaryocytes. Stem cells are few in number, normally occurring with a frequency of about one stem cell per 10 4 bone marrow cells. However, in mobilized blood samples hematopoietic progenitor cells comprise, for example, 1 -7% of the mononuclear cell population. In this application, the terms “mesenchymal progenitor cells” and “mesenchymal stem cells are used interchangeably and include the broadest art recognized definition of each..
  • hematopoietic progenitor cells also includes committed progenitor cells which give rise to a specific hematopoietic lineage of cells, for example the lymphoid cells, the macrophage/monocytic cells, the polymorphonuclear cells and the megakaryocytes.
  • stem cell is usually reserved for multipotential cells that are also able to self-renew (i.e. give rise to more multipotential stem cells).
  • the term also includes mesenchymal progenitor cells. The study of stem cells has been hampered by their low frequency and the difficulty of maintaining them in tissue culture. As a result, little is known about the regulation of their proliferation and differentiation.
  • stem cells are maintained at homeostatic levels throughout adult life; however, when there is an increased demand for hematopoiesis, stem cells display an enormous proliferation capacity. This can be demonstrated in mice whose hematopoietic systems have been completely destroyed by a lethal dose of x-rays (950 rads). Such irradiated mice will die within 10 days unless they are infused with normal bone marrow cells from a syngeneic (genetically identical) mouse.
  • hematopoiesis Early in hematopoiesis, a pluripotent stem cell differentiates along one of two pathways giving rise to either a lymphoid stem cell or a myeloid stem cell. The types and amounts of growth factors present in the microenviroment in which a particular stem cell resides controls its differentiation. Lymphoid and myeloid stem cells differentiate into progenitor cells, which have lost the capacity for self-renewal and are committed to a given cell lineage. The lymphoid stem cell generates T and B progenitor lymphocytes. The myeloid stem cell generates progenitor cells for red blood cells (erythrocytes), the various white blood cells (neutrophils, easinophils, basophils, monocytes, mast cells), and platelets. The term "hematopoietic progenitor cell” includes pluripotent stem cells, lymphoid stem cells, myeloid stem cells, mesenchymal progenitor cells and other primitive progenitor cells.
  • the cell surface marker CD34 is usually used to identify populations of hematopoietic progenitor cells.
  • CD34 is expressed on other cell types and often occurs on malignant hematopoietic cells, when used in conjunction with other surface markers and physical parameters, it is considered to be specific for most hematopoietic progenitor cells.
  • Hematopoietic progenitor cells can be identified clinically through CD34 together with a low intensity of the leukocyte marker CD45 and low/medium forward and side light scattering properties (as measured by flow cytometry). However, certain hematopoietic progenitor cells lack CD34 and are identified by other techniques known in the art.
  • shiga-like toxin includes cytotoxins similar in structure and function to shiga toxin as well as shiga toxins.
  • the term includes verotoxins which, based upon structural similarity to shiga toxins by sequencing of relevant genes, are often referred to as shiga-like toxins (SLTs).
  • SLTs include SLT-1 (verotoxinl), SLTII (verotoxin 2), and SLTIII.
  • Variants of SLTII include verotoxin 2c, verotoxin 2e, SLTII, vtx2ha; SLTIIvh, vtx2hb, SLTIIc, SLTIIvp, etc.
  • the term also encompasses the presently unknown SLTs or variants thereof that may be discovered in the future, since their characterization as an SLT or variant thereof will be readily determinable by persons skilled in the art.
  • Shiga-like toxins are involved in the etiology of the hemolytic uremic syndrome (Karmali, M.A. et al. (1985) J. Infect. Dis. 151 :775), and haemorrhagic colitis (Riley, L.W. et al (1983) N Engl. J. Med. 308:681).
  • cell cytotoxicity is mediated via the binding of the B subunit of the holotoxin to the receptor glycolipid, globotriaosylceramide (Gb 3 ) of sensitive cells (Lingwood, CA et al (1993) Advances in Lipid Research Academic Press. 25 : 189-211 ).
  • shiga-like toxin toxicity has also been observed for cells which have no detectable binding with SLT- IB (VT- 1B) nor Gb expression
  • shiga-like toxins may also affect cells through the use of other modes of entry.
  • the toxins inhibit protein synthesis via the A subunit, an N-glycanase which removes a specific adenine base in the 28S RNA of the 60S RNA ribosomal subunit.
  • the verotoxin A subunit may be one of the most potent inhibitor of protein synthesis yet described, being effective at a concentration of about 8 pM.
  • Gb 3 is referred to as the CD77 antigen in the hematopoietic system and shows a restricted pattern of expression limited to a subset of activated B-cells in the germinal (follicular) center (Murray, L.J. et al., supra).
  • CD77 expression is prevalent in certain hematological cancers of B cells, such as Burkitt's lymphoma represented by the available cell line, Daudi.
  • Daudi Burkitt's lymphoma represented by the available cell line, Daudi.
  • the sensitivity of Daudi cells to SLTs has previously been established (U.S. patent Serial No. 5,801,145).
  • the language "shiga-like toxin sensitive" includes any effect of a shiga-like toxin interacting with or contacting a cell or biological system.
  • shiga-like toxin sensitive cells explicitly includes all cells that are sensitive to the toxin whether or not they express CD77 (Gb 3 ) or exhibit detectable binding of SLT- IB. Normally, but not exclusively, shiga-like toxins bind to Gb 3 , present on colonic and kidney endolethial cells, which permits the internalization of the toxin and leads to cell death.
  • Shiga-like toxin- 1 has been shown to be an effective purging agent for malignant B and/or plasma cells from patients with non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia (B-CLL) and multiple myeloma.
  • B-CLL B-chronic lymphocytic leukemia
  • multiple myeloma Although many cells, e.g., malignant B cells, bind and internalize SLT-1 via binding to the CD77 receptor, this mechanism is not universal. For example, some cells, which lack detectable binding of the SLT- IB subunit to CD77, are killed by treatment with SLT-1.
  • Shiga-like toxins represent ideal purging agents for many reasons. For example, they are cytotoxic throughout the cell-cycle and differ in cell-cycle dependence patterns from that of conventional chemotherapeutic drugs. They possess an impressive ability to eliminate clonogenic tumor cells, but show no toxicity against normal bone marrow progenitors. Shiga-like toxins are very soluble in aqueous media and can be easily removed prior to reinfusion. They also possess a theoretical lack of cross-resistance with prior in vivo drug regimens because of their distinct mode of action.
  • the invention pertains to a method of strengthening the hematological system of a mammal.
  • the method includes treating hematopoietic progenitor cells with a shiga-like toxin, allowing the hematopoietic progenitor cells to become enriched, and administering the hematopoietic progenitor cells to the mammal.
  • the cell sample may be pre-enriched before the treatment with the shiga-like toxin.
  • the cells may be transfected with a gene and, advantageously, be used to treat a mammal suffering from a genetic hematological disorder. The transfection may occur prior to, or after treatment of the cells with shiga-like toxin.
  • mammal includes, for example, rodents, cows, sheep, horses, dogs, cats, and primates.
  • the preferred mammal is a human.
  • the mammal may have an acquired or congenital immune deficiency.
  • the mammal may have under gone chemotherapy or radiation therapy.
  • the mammal may be suffering, recovering from, or undergoing treatment for cancer, e.g., breast, ovarian, brain, testicle, lung, prostate, colon, leukemia, bowel, or small cell lung cancer.
  • cancer e.g., breast, ovarian, brain, testicle, lung, prostate, colon, leukemia, bowel, or small cell lung cancer.
  • aggressive chemotherapy techniques may be developed which may be able to treat certain cancers, e.g., bowel or prostate, which are presently unresponsive to current techniques.
  • the mammal is suffering from a chemotherapy- sensitive disease susceptible to high dose chemotherapy regimens followed by stem cell rescue, including, for example, leukemia, lymphomas, multiple myeloma, breast cancer, testicular cancer, and small cell lung cancer.
  • the mammal may be suffering from a genetic disorder, such as sickle cell anemia, acquired or congenital aplastic anemia, thalessimia, or a severe immunodeficiency disorder.
  • the mammal may be suffering from an acquired immune deficiency disorder, e.g., AIDS.
  • hematological system includes all red and white blood cells and all tissues known to one skilled in the art to be associated with the blood, white blood cells and immune system. This includes, for example, the blood, lymph, all primary and secondary lymphoid organs, including the thymus, bone marrow, spleen, lymph nodes, Peyer's patches, mucosa, gut and skin.
  • the language “strengthening the hematological system” includes increasing in absolute number or percentage of any type of blood cell.
  • blood cells that may be increased include, for example, stem cells, hematopoietic progenitor cells, erythrocytes, granulocytes, monocytes, mast cells, lymphocytes, and megakaryocytes.
  • hematological disorder includes any disorder associated with the hematological system or any of its components.
  • hematological disorders include cancer (e.g., Hodgkin's or non-Hodgkin's lymphoma, leukemia, or, preferably, multiple myeloma), paraproteinemias, AIDS, and genetic disorders.
  • paraproteinemia includes any disorder which may involve abnormal proteins in the blood.
  • the proteins may be affiliated with white or red blood cells or not.
  • paraproteinemias include, for example, Waldenstrom's macroglobulinemia, primary and secondary amyloidosis, monoclonal gammoplathies (including, for example, those of IgM, IgD, IgG, IgA, and IgE subtypes).
  • genetic hematological disorder includes disorders which result from abnormal genes in blood cells.
  • genetic hematological disorders include sickle cell anemia, thalessimia, paroxysmal nocturnal hemoglobulinemia, congenital and acquired sideroblastic anemia, congenital or acquired aplastic anemia and severe immunodeficiency disorders, including both congenital and acquired forms of the disease.
  • the method involves shiga-like toxin exposure of hematopoietic progenitor cells to shiga-like toxin prior to the transfection step to permit in vitro enrichment.
  • Exposure to the transfecting genetic material may immediately follow after toxin treatment or, alternatively, may occur after a period in culture to permit toxin-mediated enrichment of hematopoietic progenitor cells before transfection.
  • hematopoietic progenitor cells may be enriched by treatment with shiga-like toxin prior to, or after, transfection with vectors encoding genes for drug resistance, such that after reinfusion, hematopoietic progenitor cells would resist, for example, alkylating agents, allowing more selectivity of the drug for the tumor (Reese et al., PNAS, (1996) 93(24): 14088-93; Niitsu, Y., Chem Biol. Interact. (1998) 111-112:325-32; Reese et al., Clin. Cane. Res. (1999) 5(l):163-9).
  • the culture conditions may be advantageously designed to amplify the expansion of hematopoietic progenitor cells in preference to those having a certain phenotype.
  • in vitro expansion of normal hematopoietic progenitor cells may be carried out in order to remove clones of cells which have the Philadelphia chromosome in chronic myeloid leukemia. The procedure would be facilitated by treatment with Shiga-like toxin to enrich normal progenitors as indicated in this embodiment, followed by a further more prolonged culture to preferentially expand the numbers of normal progenitors, as is carried out by those skilled in the art.
  • the hematopoietic progenitor cells include cells from the mammal.
  • the cells may be taken from the mammal's blood, umbilical cord, bone marrow, or mesenchymal cells. In some instances, the mammal may have previously or concurrently undergone chemotherapy or radiation therapy.
  • the invention relates to a method for the ex vivo purging of shiga-like toxin sensitive cells and enrichment of hematopoietic progenitor cells, advantageously, in a culture.
  • the cells are obtained from a human suffering from a hematological disorder.
  • the method involves contacting the cells with a shiga-like toxin e.g., verotoxin, and allowing sufficient time, e.g., about 5 to 10 days, for enrichment of the hematopoietic progenitor cells and purging of the shiga-like toxin sensitive cells.
  • the shiga-like toxin may be administered at a concentration of about 5-10 ⁇ g/ml.
  • the method may further comprise washing the cells to remove excess shiga-like toxin before being administered to a mammal.
  • the cells are autologous mobilized blood cells, pre- enriched by a preliminary selection method, from patients with B lineage malignancies, for example multiple myeloma or other paraproteinemias, non-Hodgkin's lymphoma or B-CLL.
  • B lineage malignancies for example multiple myeloma or other paraproteinemias, non-Hodgkin's lymphoma or B-CLL.
  • B-CLL non-Hodgkin's lymphoma
  • shiga-like toxin purging of primary cancer cells from patients with multiple myeloma, B-lymphoma or B-CLL advantageously occurs at a concentration of shiga-like toxin of, for example, about 5 ⁇ g/ml to about 10 ⁇ g/ml of shiga-like toxin.
  • the toxicity of shiga-like toxin may not readily detectable until 5-10 days of culture post-exposure to shiga-like toxin.
  • the invention also pertains to methods to enrich hematopoietic progenitor cells prior to procedures to cause ex-vivo expansion of hematopoietic progenitor cells.
  • the method involves treatment of, for example, an unfractionated or pre-enriched hematopoietic progenitor cell population with a shiga like toxin, (at a concentration of, for example, about 5-10 ⁇ g/ml).
  • the shiga like toxin treated cells are maintained in culture for a sufficient length of time (for example, about 5-10 days or longer) to permit in vitro expansion of the shiga-like toxin enriched hematopoietic progenitor cells.
  • the method may involve direct reinfusion or cryopreservation of the cells.
  • the cells are manipulated (e.g., genetically) to alter their properties prior to reinfusion.
  • the invention relates to a method of purging shiga-like toxin sensitive cells.
  • the shiga-like toxin sensitive cells do not express CD77.
  • the shiga-like toxin sensitive cells are freshly prepared and have not been frozen.
  • the invention also relates to a method for purging of malignant cells by shiga- like toxin for those cancers where CD77 receptors or binding of SLT- IB by malignant cells, or by subpopulations of malignant cells, are not detectable.
  • the malignant cells may bind to the whole SLT and/or SLT-1 A.
  • the methods include removing cells from a patient, advantageously, from the blood or bone marrow.
  • the cells are then treated with an effective amount of shiga-like toxin (e.g., about 5-10 ⁇ g/ml), cultured for an appropriate length of time, and, preferably, administered to the patient.
  • the methods may include washing the cells to remove unbound toxin followed by direct reinfusion, or cryopreservation followed by reinfusion into the patient.
  • the cells are freshly prepared and have not been frozen or otherwise permeabilized by artificial preservation techniques.
  • freshly prepared includes cells which have not been frozen, cryopreserved, or otherwise permeabilized by preservation techniques which may alter the cell's permeability, e.g., to shiga-like toxin.
  • binding of SLTB-FITC has been detected for cryopreserved myeloma plasma cells, (Lacasse et al. Blood 94(8) 2901-10 (1999)) when freshly isolated biopsy samples from similar patients were studied, no significant numbers of SLTB-binding plasma cells were detected.
  • the cells may be cultured for a short period of time (e.g., 1-2 days) to allow the initiation of toxicity.
  • the cells may be cryopreserved prior to reinfusion into the patient or reinfused directly.
  • the final stages of shiga-like toxin initiated cell death may occur within the reinfused patient.
  • the shiga-like toxin treated cells remain in culture for 5-10 days under conditions where hematopoietic progenitor cells survive or are enriched and allow the shiga-like toxin sensitive cells to be purged in culture.
  • the cells may be reinfused into the patient directly or after cryopreservation.
  • the cells may be removed from the bone marrow of a patient suffering from multiple myeloma, or from the blood of a patient with B cell chronic lymphocytic leukemia.
  • the shiga-like toxin sensitive cells being purged may or may not exhibit shiga-like toxin binding and may or may not express CD77.
  • the invention also pertains to methods for the generation of dendritic cells.
  • dendritic cells may be advantageously used in tumor vaccines or for other immunological purposes (DiNicola, M. et al., Cytokines Mol. Ther. (1998) 4(4):266- 273; Choi, D. et al., Clin Cancer Res. (1998) 4(11):2709-16; Soligo, D. et al. Br. J. Haematol. (1998) 101(2):352-63).
  • the method involves treating a culture of hematopoietic progenitor cells with a sufficient amount of a Shiga-like toxin (e.g., 5-10 ⁇ g/ml) and culturing the cells for an appropriate period of time to generate dendritic cells.
  • a Shiga-like toxin e.g., 5-10 ⁇ g/ml
  • dendritic cells may be generated by methods known to those skilled in the art.
  • the dendritic cells could subsequently be used to present tumor or other antigens, or may be fused to the tumor cell making tumor antigens an intrinsic component of the dendritic cell surface.
  • the initial culture of cells is pre-enriched for hematopoietic progenitor cells.
  • the antigens presented by the dendritic cells may be specific for, for example, breast, ovarian, testicular, prostate, lung, bowel, rectal, prostate, pancreatic, stomach, brain, or skin (e.g., melanoma) cancers.
  • Methods of using dendritic cells are known to those skilled in the art.
  • the term "dendritic cells” include Langerhans cells, interstitial dendritic cells, interdigitating dendritic cells, follicular dendritic cells and circulating dendritic cells. Langerhans cells are found in the epidermis and mucous membranes. Interstitial dendritic cells populate most organs such as the heart, lungs, liver, kidney, and gastrointestinal tract.
  • Interdigiting dendritic cells are present in T-cell areas of the secondary lymphoid tissue and the thymic medulla. Circulating dendritic cells include "veiled cells" which constitute about 0.1% of the blood leukocytes. In general, dendritic cells are covered with a maze of long membrane processes resembling dendrites of nerve cells. Due to their long dendritic processes, dendritic cells have been challenging to study using conventional procedures for isolating lymphocytes and accessory immune-system cells. Dendritic cells tend to express high levels of both class II MHC molecules and the co-stimulatory B7 molecule.
  • Follicular dendritic cells are also included. Follicular dendritic cells do not express class II MHC molecules and therefore do not function as antigen presenting cells for T H -cell activation. They are located exclusively in the follicles of the lymph nodes and express high levels of membrane receptors for antibody and complement. Binding of circulating antibody-antigen complexes by these receptors is thought to facilitate B-cell activation in lymph nodes.
  • the invention also contemplates a preparation containing hematopoietic progenitor cells, wherein the hematopoietic progenitor cells have been previously treated with a shiga-like toxin and, advantageously, purged substantially of shiga-like toxin sensitive cells.
  • the hematopoietic progenitor cells are from the blood or bone marrow of a human.
  • the preparation is suitable for infusion into a mammal.
  • the mammal is suffering from a hematological disorder.
  • Hematopoietic progenitor cells (HPC) and colony forming cells may be enriched after treatment with a shiga-like toxin (SLT).
  • Mobilized mononuclear cells (MNC) were treated with 5 ⁇ g/ml SLT for 60 minutes, washed and cultured.
  • treated or untreated cells were cultured in growth media with appropriate growth factors for 7 days, followed by harvest of cultured cells and staining with CD34-PE and CD45-QR.
  • Mobilized MNC were enriched HPC by negative selection after antibody coating and magnetic bead depletion.
  • Table 1 shows that pretreatment with a Shiga-like toxin increased the percent of CD34+45 10 cells in HPC enriched cultures.
  • the number of CD34+45 10 cells in the Shiga-like toxin pretreated culture was more than doubled.
  • SLT treatment resulted in a quintupling of the CD34+45 10 cells in HPC enriched cultures.
  • an enriched HPC fraction seventeen percent of the cells display the CD34+CD45 10 phenotype of HPC cells.
  • an enriched HPC lymphoma cell culture pretreated with SLT thirty nine percent of the cells display the CD34+CD45 10 phenotype.
  • a control myeloma cell culture from an enriched HPC fraction only five percent of the cells display the CD34+CD45 10 phenotype of HPC cells.
  • twenty-eight percent of the cells display the CD34+CD45 10 phenotype.
  • HPC may be exhibit increased proliferation after treatment with SLT through a direct stimulatory effect on the HPC themselves.
  • SLT treatment may deplete an inhibitor of HPC proliferation, thereby allowing HPC proliferation as an indirect effect of SLT.
  • An indirect effect may also occur if SLT alters the balance between negative and positive regulators of HPC proliferation, thereby unbalancing the normal equilibrium in favor of increased numbers of HPC.
  • CFU colony forming units
  • the model postulating an inhibitor might also predict that the inhibitor is relatively infrequent and is less easily detected in cultures receiving only 100-200 cells for generating CFU (microtiter wells) but more easily detected in cultures receiving 1000 cells (such as the dishes). It is also possible that the small wells can only support so many colonies, preventing the detection of enriched numbers that might otherwise result in this experimental system during the postulated rapid phase in colony formation after the supposed inhibitor dies. If there were, for example, sufficient space and factors in the wells for cultures to continue generating colonies for a longer period of time, it is postulated that the results would be comparable to that in the dishes.
  • SLT pretreatment may alter regulation of HPC and their clonal expansion as colonies in ways that are beneficial to the growth and differentiation of CFU, with a consequent enrichment in hematopoiesis.
  • SLT pre-treatment provides a one step method for purging malignant cells which at the same time enriches HPC, with the added benefit that hematopoiesis may proceed even more efficiently in vivo.
  • SLT treatment may function as a hematopoietic progenitor cell enrichment/selection procedure as well as a purging method.
  • SLT- IB binding was detected using SLT-1B-FITC or SLT-IB-Alexa.
  • CD77 was detected by indirect immunofluorescence using a FITC or a PE conjugated second stage antibody.
  • B cells and plasma cells from freshly prepared blood and bone marrow samples from myeloma patients were tested for surface CD77 by using anti-CD77 antibodies. The samples showed that the cells lacked CD77 expression. However, it has been shown that myeloma B and plasma cells are killed by the SLT toxin despite this apparent lack of CD77 and an inability to bind the B subunit.
  • Plasma cells (CD38-hi or BB4+) from the bone marrow of patients with multiple myeloma were found to lack detectable binding of the SLT- IB subunit. These results were consistent for each of the ten samples tested. B cells from the peripheral blood of myeloma patients were also tested for SLT- IB binding. It was found that circulating B cells (CD 19+) generally lacked detectable binding of the SLT- IB subunit. However, when the CD 19+ cells were treated with SLT, it was found that the malignant cells were killed, despite the apparent lack of CD77 and detectable SLT- IB binding.
  • the pattern of SLT-mediated killing of freshly isolated lymphoma, B-CLL and myeloma cells is very different from that reported for Burkitt's cell lines or breast cancer cell lines (see, e.g., Lacasse et al. Blood 88(5) 1561-7 (1996); Lacasse et al. Blood 94(8) 2901-10 (1999)).
  • Burkitt's cell lines cell killing was detectable 2 to 3 days post toxin exposure.
  • significant killing was only detectable 6 to 10 days after toxin exposure.
  • the toxin exposure period was limited to only one hour at the initiation of culture, which was followed by removal of all unbound toxin before the culture period.
  • the 6-10 day period for cell killing is not limited to freshly isolated or prepared CD77 negative cells.
  • CD77+ follicular lymphoma cells were exposed to toxin, the cells were killed 6-10 days after toxin exposure rather than the 2-3 day window predicted from the data with Burkitt's lymphoma cell lines.

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Abstract

L'invention concerne des procédés permettant d'enrichir les cellules souches hématopoïétiques dans une culture avec une toxine de type Shiga (Shiga-like toxin), et de purger cette culture de manière à séparer les cellules normales ou malignes sensibles à cette toxine de type shiga, qui sont dépourvues de récepteurs CD77 ou de récepteurs de la sous-unité SLTB.
PCT/US2000/011927 1999-05-03 2000-05-02 Toxine de type shiga utilisee en tant qu'agent d'enrichissement et de purge WO2000066714A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995022349A1 (fr) * 1994-02-22 1995-08-24 Geva, Ruth Compositions pharmaceutiques de verotoxines et traitements medicaux utilisant ces compositions
US5801145A (en) * 1996-02-09 1998-09-01 Ontario Cancer Institute Method for selectively purging CD77+ cells from bone marrow
US5866115A (en) * 1994-04-14 1999-02-02 Klinikum Der Albert-Ludwigs-Universitat Freiburg Process for preparing dendritic cells, cells thus produced and containers for carrying out this process

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995022349A1 (fr) * 1994-02-22 1995-08-24 Geva, Ruth Compositions pharmaceutiques de verotoxines et traitements medicaux utilisant ces compositions
US5866115A (en) * 1994-04-14 1999-02-02 Klinikum Der Albert-Ludwigs-Universitat Freiburg Process for preparing dendritic cells, cells thus produced and containers for carrying out this process
US5801145A (en) * 1996-02-09 1998-09-01 Ontario Cancer Institute Method for selectively purging CD77+ cells from bone marrow

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRAY M R ET AL: "Expression of the Shiga-like toxin I receptor CD77 on human breast carcinomas, follicular lymphomas and multiple myelomas and absence of expression on CD34+ human hematopoietic cells: Implications for tumor cell purging." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL, vol. 39, March 1998 (1998-03), page 63 XP002153267 89th Annual Meeting of the American Association for Cancer Research; New Orleans, 28 March-1 April 1998 *
LACASSE E C ET AL: "Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence from CD34+ hematopoietic stem cells: Implications for ex vivo tumor purging and autologous stem cell transplantation." BLOOD, vol. 94, no. 8, 15 October 1999 (1999-10-15), pages 2901-2910, XP002153268 *

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