WO1991018972A1 - Culture de cellules de moelle osseuse utilisees en immunotherapie adoptive - Google Patents

Culture de cellules de moelle osseuse utilisees en immunotherapie adoptive Download PDF

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WO1991018972A1
WO1991018972A1 PCT/US1991/003555 US9103555W WO9118972A1 WO 1991018972 A1 WO1991018972 A1 WO 1991018972A1 US 9103555 W US9103555 W US 9103555W WO 9118972 A1 WO9118972 A1 WO 9118972A1
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cells
bone marrow
bioreactor
interleukin
growth promoting
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PCT/US1991/003555
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Richard Allan Knazek
William Robert Kidwell
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Cellco, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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

Definitions

  • This invention is directed to methods for culturing bone marrow cells jLn vitro in a hollow fiber bioreactor, to the cultured bone marrow cells and to methods of adoptive immunotherapy using the cultured bone marrow cells.
  • the bone marrow cells that are cultured in the bioreactor are obtained from normal individuals and from individuals suffering from various diseases including leukemias, metastatic cancers, and genetic disorders.
  • Adoptive immunotherapy is the passive transfer to an individual, who is suffering from an acquired or inherited disease, of immunologically active cells, which have been removed from the individual or from a donor. Often the immunologically active cells are manipulated and/or modified in vitro prior to transfer to the recipient.
  • the transferred cells are used therapeutically to treat the disease via destruction of the affected cells of the recipient by virtue of specific interaction with the affected cells of the recipient, by replacement or supplementation of the affected cells or by furnishing therapeutically effective substances or immunologically active cells to the recipient.
  • lymphoid cells are being explored as a means for introducing genetically engineered DNA into an individual for genetic therapy or to deliver therapeutic agents (see, e.g.. Genetic Eng. News. Vol. 9, No. 3, March 1989 and p. 133 in Business Week/ May 1, 1989) .
  • lymphocytes will be removed from the patient and DNA that encodes a wild-type protein for which the individual is deficient or that encodes a therapeutically effective anticancer or antiviral agent, such as interferon or tumor necrosis factor (TNF) , will be introduced into lymphocytes in a manner such that the encoded protein will be expressed.
  • a therapeutically effective anticancer or antiviral agent such as interferon or tumor necrosis factor (TNF)
  • Lymphocytes are not ideal candidates for genetic therapy because they are differentiated end-
  • An undifferentiated stem cell which is self-renewing, such as a pluripotent stem cell from the bone marrow, is a more suitable candidate for genetic therapy. Because of difficulties in culturing such cells, it has not, however, as yet been possible to introduce heterologous DNA into such cells. Bone marrow, hematopoiesis and lymphopoiesis.
  • Bone marrow of adult animals which is found within all of the hollow bones of the body, serves as source of transplantable pluripotent stem cells.
  • Pluripotent stem cells (often abbreviated CFU-S) , which also exist in the liver and spleen of adult mammals, have the ability to both proliferate and to differentiate into multipotent cells (see, e.g.. Schrader et al. (1978) J. Exp. Med. 148; 823) .
  • Multipotent cells potentially, which can develop into fewer lineages than the pluripotentent stem cells and, are, thus of restricted potency, have the ability to both proliferate and differentiate into cells of more restricted potency (see, e.g. Fig. 1, Dexter (1987) British Med.
  • erythroid, myeloid, and lymphoid cells are derived from stem cells that give rise to erythrocytes, granulocytes/macrophages, and lymphocytes, respectively. These latter cells represent the end-stage of differentiation and appear to lack self-renewal potential.
  • the process by which the undifferentiated •pluripotent stem cells of the bone marrow develop into the various component cell types of the blood is called hematopoiesis.
  • the pluripotent stem ceils proliferate and differentiate on a framework, called the stromal network, that contains fat cells, fibroblasts, macrophages, blood vessels and sinusoids that coalesce into the marrow venous drainage system.
  • a single pluripotent stem cell can give rise to cells of any lineage.
  • the more differentiated progenitor cells present in bone marrow can be identified by their respective physical properties, such as distinctive colony phenotype, sedimentation velocity, and density.
  • progenitor cell which gives rise to erythroid cells is called a "burst forming unit" (BFU-E) because of the distinctive multicentric colonies of erythroid cells that form when BFU-Es are cultured on agar.
  • BFU-E burst forming unit
  • the cells can also be identified by the distinctive array of cell surface antigens carried by each cell type so that monoclonal antibodies that specifically recognize a particular cell surface antigen can be used to identify a particular cell type or a particular differentiated state.
  • the presence and numbers of pluripotent stem cells in a bone marrow sample can be assessed by colony formation assays.
  • the length of time it takes for colonies to form and the type of cell formed are indicative of both the presence and rela ⁇ tive numbers of pluripotent cells present in a sample.
  • Lymphopoiesis refers to the process by which CFU-S differentiate into lymphocytes. Lymphocytes first appear in the yolk sac and liver of a developing embryo.
  • T and B cells are the principal classes of lymphocytes and the thymus and bone marrow are the primary lymphatic organs. The spleen and lymph nodes are the secondary lymphatic organs. Both T and B lymphocytes, which are ubiquitous in the blood, lymph and connective tissues, are regenerated in the bone marrow from pluripotent stem cells. Prothymocytes are produced in the bone marrow and migrate to the thymus where, under the influence of thymus-produced hormones and growth factors, they proliferate and differentiate into T-cell subpopulations.
  • helper T cells T h
  • suppressor T cells T s
  • CTLs cytotoxic killer cells
  • T h mouse T helper cells
  • T suppressor T s T suppressor cells
  • CTL cytotoxic lymphocytes
  • T-cell precursors After trafficking through the thymus, T-cell precursors develop into immunologically active effector and regulatory T-cells. Mature T-cells release factors that regulate growth and differentiation of both B and T cells in the bone marrow.
  • B and T lymphocytes which differ in some surface antigens, appear morphologically similar. They are small, motile, non-phagocytic cells. Antigenic stimulation induces the secretion of lymphokines and leads to changes in the morphology of specific lymphocyte subpopulations. For example, specific binding of antigen to the cell surface stimulates the transformation of small lymphocytes into large ones. Some of these large lymphocytes, of B lineage, differentiate into mature plasma cells, which are active in the synthesis and secretion of immunoglobin (Ig) .
  • Ig immunoglobin
  • B cells mature in the bone marrow.
  • the earliest identifiable stage of B cell differentiation (in mice) is the pre-B cell, which has immunoglobin in the cytoplasm but little, if any, on the cell surface.
  • DNA rearrangement is a necessary step for transcription of immunoglobin genes and, thus, Ig.
  • the presence of cytoplasmic Ig indicates that the DNA that encodes the Ig has already undergone rearrangement by this early stage of differentiation.
  • B lymphocyte developmental pathway Cellular intermediates in the B lymphocyte developmental pathway are distinguished by differences in the organization of the genes that encode the single heavy and light Ig chains, the type of Ig chain (isotype) that is expressed, the amount of Ig that is expressed, and the cell surface antigens that are expressed.
  • B cell maturation is antigen-independent until the mature B cell stage, after which stage, development requires interaction with appropriate mitogens, antigens, T-cell- produced factors, and macrophages.
  • Antigen-stimulated mature B cells develop into either activated, memory or plasma cells.
  • B and T cells are necessary. Restoration of immunologic function in animals, whose immune systems have been destroyed by ra-diation or other treatments, can be achieved by the injection of either bone marrow or thymus cells, however, antibody response is greatest if both cell types can be regenerated.
  • the phenotypes of B, as well as T cells are determined by the characteristic enzymes or cell surface antigens. B and T cells differ in surface molecules, called differentiation antigens, many of which are "alloantigens", which are encoded by allelic genes and, thus, differ among individuals in the same species. For example, alloantigens can be elicited by injecting mice of a strain that lacks a particular alloantigen with cells from a strain that possess it.
  • T-cells when stimulated by an antigen, produces cell surface molecules that are encoded by a gene locus called the major histocompatibility complex (MHC) in mice.
  • MHC major histocompatibility complex
  • Subsets of T h cells have surface antigens that are encoded by different portions of the MHC locus.
  • B cells also exhibit cell surface receptors that are encoded by the MHC-locus.
  • MHC-encoded receptors restrict which antigens elicit an immune response by controlling the interaction of T cells with B cells. The specificity of the restriction is determined by the thymic microenvironment (the haplotype of the MHC locus) in which the T cells mature, not by the haplotype of the T cell's MHC locus.
  • the immune response of most lymphocytes depends upon the specific binding of antigen and the interaction with regulatory lymphocytes and acrophages.
  • the specificity of the T cell response to anti-gen depends, not on recognition of cell-surface antigen alone, but on recognition of the antigen plus products of the MHC locus on the same cell surface. This specificity is called MHC restriction.
  • lymphokines which are soluble mediating factors, play a role in communication among the cell populations.
  • T h cells produce a variety of lymphokines in response to antigenic challenge.
  • the production of lymphokines by these cells is dependent upon the antigen but is independent of T-cell proliferation.
  • T-cell proliferation in response to antigenic challenge represents the proliferation of only a specific subpopulation of T-cells in response to a T h cell-produced lymphokine.
  • the lymphokine interleukin-1 facilitates or induces the production of other lymphokines, such as interleukin-2 (IL-2) , by T h cells.
  • IL-2 in turn promotes T-cell proliferation and promotes the differentiation and amplification of certain T-cell subpopulations, including cytotoxic killer cells and tumor infiltrating lymphocytes (TILs) .
  • Another such growth substance, erythropoietin which is induced in vivo by anemia or hypoxia, is required in vitro for the differentiation of erythroid precursor cells (BFU-E) into non-nucleated hemoglobin- producing cells (red blood cells) .
  • Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates the growth of granulocyte/macrophage colonies, which in turn produce other regulatory proteins.
  • cytokines are factors, such as lymphokines or monokines, that are produced by cells that affect other cells
  • lymphokines are substances that are produced and secreted by activated T lymphocytes and that affect other cell types.
  • interleukin-3 which stimulates proliferation and development of multipotent stem cells and colony forming cells (CFCs or CFUs) of more restricted potency
  • CFCs or CFUs colony forming cells
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • GM- CSF granulocyte- acrophage colony stimulating factor
  • interleukin-6 which acts on B-cells
  • interleukins and colony stimulating factors have also been produced using recombinant DNA technology.
  • Clinical trials are presently underway to assess the effectiveness of these factors in promoting hematopoiesis in vivo following bone transplantation.
  • recombinant human GM-CSF which was administered to patients beginning three hours after bone marrow infusion, accelerated myeloid recovery compared to untreated controls (see, Brandt et al. (1988) New Engl.
  • GM-CSF has also been shown to stimulate hematopoiesis and induce a fivefold to tenfold increase in circulating blood leukocytes; G-CSF induces an increase in the numbers of stem cells, committed myeloid progenitors, and circulating blood neutrophils;
  • IL-3 stimulates stem cell proliferation in vivo: and IL-
  • lymphokines such as interleukin-2 (IL-2)
  • IL-2 interleukin-2
  • IL-2 mediate specific expansion of subpopulations of lymphoid cells that bear specific phenotypic surface markers and that specifically recognize certain antigens on the surfaces of affected cells
  • Incubation of resting lymphocytes, which are obtained from tumor bearing hosts, including human and murine hosts, in the presence of IL-2 for three to four days results in the expansion of subpopulations of lymphocytes that are capable of lysing natural killer cell (hereinafter NK)- resistant tumor cells, but not normal cells (see, e.g..
  • NK natural killer cell
  • LAK lymphokine activated killing
  • TIL lymphokine activated killing
  • TIL cells are lymphocytes that infiltrate into tumors, against which a host's immune system is mounting an immunological response, and can be isolated therefrom (see, e.g.. Yron et al.. supra. _ . TIL cells are found to have greater specificity than LAK cells for autologous cells and greater efficacy than LAK cells in adoptive immunotherapy of cancer (see, e.g.. Yron et al.. supra.) . TIL cells have been obtained from resected human tumors, including cancers of the kidney, colon, and breast, melanomas, and sarcomas.
  • TIL cells also show promise for use in methods of genetic therapy (see, e.g. Culliton (1989) , "News and Comment” in Science 244: 1430-1433 and Knazek et al.. supra... They provide a source of autologous cells that can be modified by the insertions of DNA encoding a desired protein, cultured, and reintroduced into the patient.
  • the desired protein may be a therapeutically effective protein, such as tumor necrosis factor, which is used in cancer therapy, CD4 receptor to which HIV binds, an enzyme, for which the treated host is deficient, or a it may be a marker protein, whereby the fate of the TIL cells in the treated host may be studied.
  • lymphokines have primarily been used to generate such subpopulations of lymphoid cells
  • lymphokines such as IL-4, IL-6 and other interferons, and TNF have also been shown to be to be useful in the production of in vitro expanded lymphoid cells and may also prove to be useful in expanding specific subpopulations of lymphoid cells.
  • IL-4 (also called BSF-1) is a glycoprotein that is derived from T cells and has been shown to induce LAK activity if the lymphoid cells are first stimulated with IL-2, but is inhibitory if the cells are not pre- stimulated (Kawakami et al. (1989) J. of Immunol. 142: 3452-3461) IL-4 also has been shown to be capable of stimulating the growth of TIL cells both alone and in conjunction with IL-2. IL-4 appears to enhance the growth of TIL cells and concomitantly inhibit the growth of NKHI + cells, which are responsible for non-specific killer activity (Lotze fe al. (1989) at pp. 167-179 in Human Tumor Antigens and Specific Tumor Therapy. Alan R. Liss, Inc., see, also, Kawakami et al.. (1988) J. of Exp. Med. 168: 2183-2191.).
  • disorders of bone marrow cells or bone marrow function and development is implicated in the pathology of numerous diseases, including: leuke ias, metastatic cancers, AIDS and other immunodeficiencies, allergies, inherited diseases and others.
  • diseases such as leukemia, often result from the depletion, surplus, or absence of certain subpopulations of bone marrow cells or from abnormal cells that develop in certain subpopulations (see, e.g. TABLE I, infra. ; see, also, Scientific American Medicine, Rubenstein and Federman, eds. (April, 1990) Section, 5, Chapter VIII, p. 11).
  • Immune system cell imbalances and defects can arise from defects in the regulation of growth and differentiation of cells in the bone marrow.
  • Leukemia involves the proliferation of a clone of abnormal hematopoietic cells.
  • leukemic cells exhibit poor responsiveness to normal regulatory mechanisms, a diminished capacity for normal cell differentiation, the ability to expand at the expense of normal myeloid or lymphoid lines, and the ability to suppress or impair normal myeloid or lymphoid cell growth.
  • Leukemic cells are identified by the particular type of hematopoietic cell that is involved.
  • myeloid leukemias involve cells derived from myeloid stem cells and lymphoid leukemias involve abnormalities in the cells derived from lymphoid stem cells.
  • CML chronic myelogenous leukemia
  • CML chronic myelocytic leukemia
  • chronic granulocyti ⁇ leukemia which is considered a prototypical stem cell disease
  • Philadelphia chromosome a chromosomal abnormality that is specific for CML, is found in erythroid, granulocytic and megakaryocytic cells lines.
  • An abnormal leukemic stem cell arises and gives rise to abnormal red cells, neutrophils, eosinophils, basophils, monocyte- acrophages, platelets, T cells and B cells.
  • the Philadelphia chromosome is microscopically visible.
  • the Philadelphia chromosome results from a reciprocal translocation between chromosomes 9 and 22.
  • Chromosome 22, which is shortened and usually readily identifiable, is the Philadelphia chromosome.
  • the c-abl oncogene from chromosome 9 is adjacent to the breakpoint cluster region, bcr, gene of chromosome 22.
  • the oncogene and bcr gene encode a chimeric bcr/c-able mRNA that encodes a tyrosine kinase activity, which is produced in CML patients who do not appear to have a Philadelphia chromosome.
  • the translocation must, however, be present and should be detectable by hybridization of chromosome 22 DNA with probes that span the breakpoint.
  • aplastic anemia cyclic neutropenia
  • Blackfan-Diamond syndrome pure red-cell aplasia some neutropenias and certain immune deficiency disorders.
  • Bone marrow cells are destroyed by gamma or X-ray irradiation. If an animal is irradiated such that only the bone marrow cells are destroyed, the pluripotent stem cells (CFU-S) , which occur in the spleen and liver, can repopulate the bone marrow and immune system and the animal does not necessarily die. If the entire body of the animal is irradiated, death is inevitable.
  • CFU-S pluripotent stem cells
  • Bone marrow transplantation involves removal of a small amount of bone marrow from the pelvic bone and long bones of the donor and the intravenous introduction of donated marrow into a recipient, who has first been treated with radiation and toxic chemicals to destroy his or her bone marrow and immune cells. Bone marrow from a matched donor is then injected, and, the pluripotent stem cells within the donor marrow can reconstitute the immune system.
  • bone marrow transplants There are several general types of bone marrow transplants: autologous transplants in which the patient's own marrow is removed, treated, and returned to the individual and allogenic transplants involving the transplantation of matched marrow from an identical twin, a sibling or an unrelated, but matched, donor. Autologous bone marrow transplants are performed to reconstitute the marrow of patients who have become severely immunologically deficient secondary to high dose chemotherapy and/or radiation therapy used in treating certain types of cancer, which include some types of lymphomas and testicular and ovarian carcinomas and which, generally do not metastasize to the bone marrow early in the course of the disease.
  • an aliquot of bone marrow can be removed and saved, prior to treatment of the patient, for subsequent infusion.
  • 400 to 800 ml. of marrow is aspirated and frozen until therapy has been completed.
  • the stored marrow is reinfused into the patient, who may, however, remain immuno-suppressed for several months until his or her immune system becomes reconstituted.
  • Allogenic transplants which pose substantially greater risk to the patient, are used when an autologous transplant cannot be used or is unavailable, such as in cases of patients who have genetic defects or metastatic spread of a malignancy to the bone marrow.
  • the donor is matched closely to the human leukocyte antigen (HLA) phenotype.
  • Human leukocyte antigens are encoded by the major histocompatibility complex genes, which are analogous to the MHC locus in mice, and are present on the cell surfaces. Matching the antigens of the donor with those of the recipient lessens the likelihood of host and/or graft rejection. The closer the HLA match the greater the likelihood of engraftment.
  • the likelihood of engraftment of slightly mismatched transplants can be increased by incubating the donor marrow in the presence of anti-T cell antibody, which destroys these mediators of rejection.
  • the allogenic recipient is commonly treated with agents, such as cyclosporin, to suppress the rejection.
  • treatments in which autologous transplants can be used are the most promising. Such treatments may involve removal of some bone marrow prior to irradiation and then treatment of that bone marrow aliquot with agents that preferentially destroy the diseased cells. After irradiation of the afflicted individual, the treated bone marrow is reinfused to repopulate the hematopoietic and immune systems with healthy cells. These treatments can be used, for example, for treating cancer and potentially lethally irradiated victims of nuclear accidents. This method has been used to treat individuals suffering from metastatic breast cancer, which typically metastasizes to the bone marrow.
  • the donor marrow After transplantation, the donor marrow must reconstitute the immune system of the recipient.
  • the process of marrow engraftment and hematopoietic reconstitution takes nearly three weeks and complete regeneration of the immune system can take many months.
  • the recipient Prior to hematopoietic reconstitution, the recipient is at risk of contracting infections, other diseases, and, except for autologous or allogenic transplants from an identical twin, rejecting the donated marrow.
  • the marrow For successful transplantation, not only must the marrow be matched to the recipient, it must contain a sufficient number of pluripotent stem cells to reconstitute the immune system of the recipient sufficiently fast before the recipient succumbs to infection.
  • pluripotent stem cells proliferate.
  • any manipulation causes the pluripotent stem cells to irreversibly commit to a particular lineage. In vitro bone marrow cultures.
  • hematopoietic progenitors In the agar culture system, the number of colonies that develop from a given concentration of hematopoietic cells depends upon the concentration of CSF (high concentrations inhibit colony formation and low concentrations are insufficient) and upon the quality (lot) of horse serum used. In this system hematopoietic cells proliferate for only a week to ten days and differentiation to committed hematopoietic progenitors (GM-CFUs) continues for 2 to 3 weeks (see, e.g.. Dexter et ______ (1976) Methods in Cell Biol. 14.: 387).
  • CSF high concentrations inhibit colony formation and low concentrations are insufficient
  • Colony formation in agar or other semi-solid culture medium has been used to assay for the concentrations of various committed progenitor cells in an aliquot of bone marrow cells, since colony formation is proportional to the number of committed cells.
  • the type of colony formed is a function of the type of CSF activity that is added to the agar.
  • thymus is known to influence hematopoiesis of bone marrow cells. It has been shown that incubation of bone marrow cells with either thymocytes (see. Miller (1973) J. Immunol. Ill: 1005) or thymic factors (see. Miller (174) J. Immunol. 113: 110) in 20% fetal calf serum (FCS) , minimal essential medium (MEM) , vitamins, non-essential amino acids, and antibiotics produces functional T cells that are able to "help" B cells in exhibiting an anti-sheep red blood cell response in vitro. This method for maintaining stem cell proliferation and hematopoiesis in culture was the first somewhat successful method.
  • Suspensions of thymus cells are incubated in Dexter medium (supra.) in glass culture bottles or flasks. After several days the cultures consists of a population of cells that adhere to the glass surfaces and a population of cells in the overlying medium, which is decanted.
  • the adherent cells are a mixture of cell types, including fibroblastic, epithelioid and phagocytic cells.
  • bone marrow cells are then added to the cultures. Initially the overlying bone marrow cells are primarily granulocytes in various stages of maturation. After two weeks, however, the culture becomes either one that produces primarily granulocytes (G-type) or one that produces primarily macrophages (M-type) . Commitment to either type occurs during the first week in culture. In the M-type cultures the number of CFU-S in the overlying medium decline and are gone by week five, GM-CFU decline in number and are gone by week 7 and granulocytes disappear. In the G-type cultures CFU-S persist for over 12 weeks, the percentage of mononuclear phagocytic cells decreases over time, and GM-CFU persist for at least 10 weeks.
  • G-type granulocytes
  • M-type macrophages
  • the bone marrow plus bone marrow liquid culture method which uses multiple inoculations of bone marrow into a liquid culture, was first described by Dexter and Lajtha in 1974 (see, Br. J. Haematol. 2 _ 525). Pooled murine femoral bone marrow cells are inoculated at a concentration of 10 6 nucleated cells per milliliter into non-siliconized tissue culture bottles that contain Fischer's medium and 20% horse serum. At first the success of the culture was dependent upon the lot of horse serum used, but later (see, e.g.. Greenberger et al. (1979) J. Exp. Haematol. Z: Supp. 5: 135) it was discovered by adding of hydrocortisone (final concentration about 10 "7 M) to the medium the dependence upon the particular lot of horse serum was eliminated and any lot of horse or fetal calf serum could be used to maintain the cultures.
  • hydrocortisone final concentration about 10 "7 M
  • the adherent layer is composed of several phenotypically distinguishable cell types, including endothelial-appearing cells, adipocytes, and reticular cells (see, e.g. Hines (1983) Blood ⁇ '. 397) . It forms during the first three weeks of culture, before the second inoculation. Within two to three weeks after inoculation, the adherent layer appears to consist of a multi-layer pavement-like structure of endothelial cells, large branching dendritic cells, foci of lipid-filled adipocytes and some macrophages. CFU-S, present in the second inoculum, migrate to this layer and form membrane- functional complexes with cells in the adherent layer. Committed progenitors and mature hematopoietic cells are continuously released into the overlying medium (see, Dexter (1982) J. Cell. Phys. Supp. 1: 87).
  • Adherent layers from other tissues do not develop adipocytes, which may, therefore, be responsible or contribute to the sustenance of hematopoiesis. It is the addition of hydrocortisone the medium that induces differentiation of pre-adipocytes into adipocytes. As discussed above, hydrocortisone or an appropriate lot of horse serum is needed to achieve sustained hematopoiesis. It has also been found (see, Greenberger et al. (1979) , supra.. that infection of Dexter cultures with murine sarcoma virus, which infects pre-adipocytes, blocks hematopoiesis. Thus, it appears that the adipocytes play an important role in sustaining hematopoiesis.
  • the adherent layer appears to produce at least some factors that participate in sustaining hematopoiesis.
  • the rate of proliferation of CFU-S cycles over time. This cycling appears to be associated with the production of molecules in the adherent layer that either stimulates or inhibit DNA synthesis in CFU-S.
  • the concentration of stimulatory material increases relative to the concentration of the inhibitory material shortly after the cells are fed. Several days later the relative concentrations are reversed. The cycling can be altered by adding either factor to the medium.
  • long term Dexter cultures reconstitute the immune system (see, Schrader et al. (1978) , supra.) .
  • the B cell progenitors and Thyl " cytotoxic cells are specifically expanded if an aliquot of the culture is transferred to lymphocyte-conditioned medium (see, Dorshkine et al. (1982) J. Immunol. 129: 2444) .
  • the Dexter culture system microenvironment does not, however, produce functional mature T-lymphoid cells.
  • the Dexter method for long term culture of murine bonemarrow cells fails to achieve sustained hematopoiesis of human bone marrow specimens. It is found that cultures of human bone marrow cells steadily decline in viability (see, e.g.. Greenberger et al. (1979) Exp. Haematol. 7_ (Supp. 5) : 135) . In human bone marrow cultures the adherent layer does not develop properly. It develops very slowly, few foci of adipocytes are observed, it becomes overgrown with fibroblasts and activated macrophages, and the cells tend to pile up rather than spread out, leading to necrosis and detachment of the cells (see, e.g...
  • lymphocytes unlike in murine bone marrow cultures, in human bone marrow cultures lymphocytes, particularly T cells, persist. Modification of the Dexter method, has, however, produced human bone marrow cultures that have been maintained for as long as 20 weeks (see, Greenberger et al. (1981) Blood 58- 724, Moore ___ ⁇ * al. (1980) Blood 55.: .682) . These modifications include: growth at 37° C, rather than 33° C, which is optimal for murine cultures, and elimination of the second inoculum of bone marrow cells.
  • This invention significantly improves the procedure for culturing bone marrow cells in vitro by providing an improved method for culturing said cells that can be adapted to methods in which bone marrow cells may be cleared of diseased cells or modified by introduction of heterologous DNA.
  • FIGURES presents a scheme of the pathways by which stem cells differentiate and develop into the various lineage restricted cell types, which are of limited potency, from which the end state differentiated cells, which lack or are of severely restricted potency, develop (see. Dexter (1987) supra.) .
  • Figure 2 is a scanning electron micrograph of normal bone marrow cells growing in between the hollow fibers of the CELLMAXTM 100 bioreactor.
  • adoptive immunotherapy is a therapeutic method, whereby cells of the immune system are removed from an individual, cultured and/or manipulated in vitro, and introduced into the same or a different individual as part of a therapeutic treatment for an acquired or inherited disease.
  • bone marrow includes any cells are that are derived from or are part of the bone marrow and also includes other substances derived from or components of the bone marrow.
  • hematopoietic and lymphoid cell progenitors including pluripotent stem cells, stromal cells, which include adipocytes, fibroblasts and endothelial cells, and the bone marrow extracellular matrix, which includes laminin, collagen, and glycosaminoglycans to which some growth factors that are produced by stromal cells and hematopoietic cells bind.
  • stromal cells which include adipocytes, fibroblasts and endothelial cells
  • the bone marrow extracellular matrix which includes laminin, collagen, and glycosaminoglycans to which some growth factors that are produced by stromal cells and hematopoietic cells bind.
  • bone marrow cells are derived from an individual suffering from leukemia or other cancer, bone marrow cells include any leukemic or other cancerous cell present in the bone marrow.
  • culturing of bone marrow cells refers to the introduction of bone marrow cells into a suitable medium at an appropriate temperature, generally about 32-37° C, in suitable tissue culture medium and the maintenance or increase in the relative proportion of pluripotent stem cells under these conditions in vitro for periods of time of days up to months.
  • bone marrow cells are cultured i vitro in order to provide a source of healthy pluripotent stem cells that are used in methods of treatment that require bone marrow transplantation.
  • the bone marrow cells that are produced in accordance with the methods disclosed of this invention are herein - referred to as in vitro cultured bone marrow cells.
  • the bone marrow cells may be cultured in the presence of chemical agents, other cells, such as TIL cells, or in the presence of growth promoting substances that expand particular subpopulations of the bone marrow cells, including TIL cells.
  • TIL cells may be cultured in the presence of a ⁇ yt ⁇ kine, such as IL-2
  • the in vitro expanded subpopulations of cells that are produced include activated lymphoid cells and, depending upon the source thereof and the cytokine used, may include LAK cells and TIL cells.
  • growth promoting substances include, but are not limited to cytokines, such as IL-2, IL-1, IL-6 and IL-4 or mixtures thereof.
  • the in vitro expanded subpopulation of cells that is produced may include CTL, LAK, and/or TIL cells.
  • Bone marrow cells may also be genetically engineered to express heterologous gene products by culturing such cells in the presence of an effective concentration of a recombinant vector or recombinant viral vector, whereby heterologous DNA included in the vector becomes stably incorporated into the bone marrow cells and the products expressed by the bone marrow cells, particularly pluripotent or multipotent stem cells.
  • neoplastic cells include any type of transformed or altered cell that exhibits characteristics typical of transformed cells, such as a lack of contact inhibition and the acquisition of tumor- specific antigens. Such cells include, but are not limited to leukemic cells and cells derived from a tumor.
  • neoplastic disease is any disease in which neoplastic cells are present in the individual afflicted with the disease. Such diseases include, any disease characterized as cancer.
  • heterologous DNA is DNA that encodes proteins that are not normally produced in vivo by the cells. Examples of such proteins include traceable foreign marker proteins, such as a protein that confers neomycin resistance, and therapeutically effective substances, such as anti-cancer agents.
  • Cells may be genetically engineered to contain and to express DNA encoding drug resistance or drug sensitivity, such as methotrexate resistance, so that, when such DNA is expressed, such cells may be selectively expanded or destroyed in vivo.
  • genetic therapy may be used to correct genetic disorders.
  • the cells of an individual who suffers from an inherited or acquired genetic defect, such as 3-thalassemia, may be genetically engineered to correct the defect by incorporation of DNA that encodes a normal version of the defective gene.
  • lymphoid cells include lymphocytes, macrophages, and monocytes that are derived from any tissue in which such cells are present. In general lymphoid cells are removed from an individual who is to be treated.
  • the lymphoid cells may be derived from a tumor, peripheral blood, or other tissues, such as the lymph nodes and spleen that contain or produce lymphoid .cells.
  • therapeutically useful subpopulations of in vitro expanded bone marrow or - lymphoid cells are cells that are expanded upon exposure of bone marrow or lymphoid cells to a growth promoting substances, such as lymphokines, when bone marrow or lymphoid cells are cultured in vitro. For example, culturing bone marrow cells in the presence of IL-2, preferentially expands lymphocyte subpopulations present in then inoculum.
  • a target antigen is an antigen that is present on the surface of a cancerous cell that is specifically recognized by a subpopulation of in vitro expanded lymphoid cells. Such cancerous cells may be found in the bone marrow of patients suffering from metastatic tumors.
  • tumor-specific in vitro expanded lymphoid cells are cells that specifically recognize target antigens that are present on or in tumor cells.
  • TIL cells are tumor specific lymphoid cells.
  • a tumor-specific antigen is an antigen that is disposed on the surface or inside of a tumor cell.
  • Tumor specific antigens may be used in purified form, on irradiated tumor cells, or they may be obtained by purifying them from tumor cells or by synthesizing them in vitro by methods, such as genetic engineering.
  • a growth promoting substance is a substance, that may be soluble or insoluble, that in some manner participates in or induces cells to differentiate, activate, grow and/or divide. Growth promoting substances include mitogens and cytokines. Examples of growth promoting substances include the fibroblast growth factors, osteogenin, which has been purified from demineralized bone (see, Luyten, F. P. et al. (1989) J. Biol. Chem.
  • epidermal growth factor the products of oncogenes, the interleukins, colony stimulating factors, and any other of such factors that are known to those of skill in the art.
  • Recombinantly-produced growth promoting substances such as recombinantly-produced interleukins, are suitable for use in this invention.
  • Means to clone DNA encoding such proteins and the means to produce biologically active proteins from such cloned DNA are within the skill in the art. For example, interleukins 1 through 6 have been cloned.
  • Various growth promoting substances and combinations thereof may be used to expand desired subpopulations of lymphoid cells.
  • a mitogen is a substance that induces cells to divide and in particular, as used herein, are substances that stimulate a lymphocyte population in an antigen-independent manner to proliferate and differentiate into effector cells. Examples of such substances include lectins and 1ipopolysaccharides.
  • a cytokine is a factor, such as lymphokine or monokine, that is produced by cells that affect the same or other cells.
  • a lymphokine is a substance that is produced and secreted by activated T lymphocytes and that affects the same or other cell types. Tumor necrosis factor, the interleukins and the interferons are examples of lymphokines.
  • a monokine is a substance that is secreted by monocytes or macrophages that affects the same or other cells.
  • an effective number of in vitro expanded lymphoid cells is the number of such cells that is at least sufficient to achieve a desired therapeutic -effect, when such cells are used in a particular method of adoptive immunotherapy.
  • an effective number of TILs may be added to a bone marrow culture in a bioreactor or mixed with the bone marrow cells prior to or upon inoculation into a bioreactor in order to clear the bone marrow of all cancerous cells.
  • an effective amount of growth promoting substance is an amount that is effective in inducing a particular subpopulation of bone marrow cells.
  • an effective amount of IL-2 may be an amount that is effective in inducing activation and/or proliferation of TIL cells in the bone marrow inoculum', whereby all cancerous cells in the bone marrow are inactivated.
  • a hollow cell fiber culture system consists of a hollow fiber bioreactor as well as pumping means for perfusing medium through said system, reservoir means for providing and collecting medium, and other components, including electronic controlling. recording or sensing devices.
  • a hollow fiber bioreactor is a cartridge that consists of a multitude of semi- permeable tube-shaped fibers encased in a hollow shell. The terms hollow fiber reactor and hollow fiber bioreactor are used interchangeably.
  • a "B3" bioreactor cartridge is one that contains a plurality of fibers consist of semi-permeable DEAE-cellulose fibers that have a nominal molecular weight cut-off of about 3,000 Daltons.
  • a type "A” cartridge includes fibers that are constructed of polyolefin and whose fiber walls have pores of about 0.5 microns diameter.
  • the extra fiber space (EFS) is the space in which the cells grow within the shell of the hollow fiber bioreactor that is external to the semi- permeable fibers.
  • the EFS is alternatively referred to as the extra capillary space (ECS) .
  • the EFS cell supernatant is the medium in which the cells in the EFS are growing. It contains secreted cellular products, diffusible nutrients and any growth promoting or suppressing substances, such as lymphokines and cytokines, produced by the cultured bone marrow cells or added to the EFS or tissue culture medium.
  • the particular components included in the EFS is a function not only of what is inoculated therein, but also of the characteristics of the selected hollow fiber.
  • a hollow fiber bioreactor or hollow fiber bioreactor cartridge consists of an outer shell casing that is suitable for the growth of mammalian cells, a plurality of semi-permeable hollow fibers encased within the shell that are suitable for the growth of mammalian cells on or near them, and the EFS, which contains the cells and the EFS cell supernatant.
  • Tissue culture medium perfuses through the fiber lumens and is also included within the shell surrounding said fibers.
  • the tissue culture medium which may differ in these two compartments, contains diffusible components that are capable of sustaining and permitting proliferation of any CFU-S in the bone marrow cells.
  • the medium is provided in a reservoir from which it is pumped through the fibers.
  • the flow rate can be controlled varied by the varying the applied pressure.
  • the EFS or perfusing medium may additionally contain an effective amount of at least one growth promoting or suppressing substance that specifically promotes the expansion or suppression of at least one subpopulation of the bone marrow cells, such as TIL cells, in which the effective amount is an amount sufficient to effect said specific expansion.
  • EFS conditioned medium is the EFS cell supernatant after it has been centrifuged to remove any cells and particulate matter and dialyzed against tissue culture medium.
  • tissue culture medium includes any culture medium that is suitable for the growth of mammalian cells and in which bone marrow cells remain viable in vitro.
  • AIM-V and Iscove's medium
  • the medium may be supplemented with additional ingredients including serum, serum proteins, growth suppressing, and growth promoting substances, such as cytokines, and selective agents for selecting genetically engineered or modified cells.
  • complete AIM-V is a tissue culture medium that consists of the proprietary formula AIM-V (GIBCO, Grand Island, N.Y.) and also contains 10 ⁇ g. genta icin/ml. (GIBCO), 50 ⁇ g. streptomycin/ml. (GIBCO), 50 ⁇ g penicillin/ml. (GIBCO), 1.25 ⁇ g. fungizone/ml.
  • AIM-V supernatant is prepared as described in Muul et al. (1986) J. Immunol. Methods 88: 265) . Briefly, LAK AIM-V supernatant is prepared by growing peripheral blood lymphocytes in AIM-V or other suitable tissue culture medium in the presence of IL-2 for 2 to 3 days and removing the cells by centrifugation o obtain the supernatant.
  • suitable tissue culture media are well- known and readily available to those of skill in the art and may be readily substituted for AIM-V.
  • a medium that consists of a 50-50 mixture of complete AIM-V and RPMI having 10% heat-inactivated human serum, and further supplemented with LAK supernatant may be used.
  • Hollow fiber bioreactors (abbreviated herein as HF) are known to those of skill in the art (see, e.g.. Knazek et al.. U.S. Patent Nos. 4,220,725, 4,206,015, 4,200,689, 3,883,393, and 3,821,087, which disclosures are herein incorporated by reference thereto) .
  • Hollow fiber bioreactors have been used for the growth of mammalian cells and for the production of biologically active products that are secreted thereby (see, e.g...
  • Hollow fiber bioreactors have, not however, heretofore been used for the selective growth of biologically active cells, such as the in vitro expanded lymphoid cells of this invention, which cells are used in vivo in methods of adoptive immunotherapy.
  • the hollow fiber bioreactor that is contemplated for use in the practicing this invention contains a multitude of tube shaped semi-permeable membranes (hereinafter called fibers) that are encased in a hollow shell. Cultured cells grow and fill the spaces between the fibers and are fed by passage of nutrients through the fiber walls from medium that is perfuses through the lumina of said membranes.
  • An example of a hollow fiber - bioreactor that may be used in practicing this invention is the hollow fiber bioreactor, B3, Cellco Advanced Bioreactors, Inc., Kensington, MD, or the hollow fiber bioreactor, B4,Cellco Advanced Bioreactors, Inc., Kensington, MD, (see U.S. Application Serial(see U.S. Application Serial No. 07/238,445, supra.
  • the bioreactor, B3 contains about 6000 tube-shaped, semi-permeable membranes, which provide a 1.1 m 2 surface area.
  • the fibers which are each approximately 250 ⁇ m in diameter, are pulled through a polycarbonate tube that is about 12 inches in length, and sealed at each end in such a manner that liquid only flows through the lumina of the fibers to exit at the opposite end of the shell.
  • the fiber walls nominally restrict passage to substances having molecular weights less than a desired cut-off range.
  • the fibers divide the cartridge into the extra-fiber space (EFS) , typically about 50 ml. in volume, and the volume within the fiber lumina.
  • EFS extra-fiber space
  • Minimal bulk flow of liquid occurs within the extra-fiber space, which is also referred to as the extra-capillary or shell-side space.
  • growth promoting substances or vectors may be bound to the fibers, introduced into the EFS, or included in the perfusing medium.
  • the fibers are selected as a function of the components of the perfusing medium to which they must be permeable and as a function of the components of the EFS.
  • the fibers may be selected so that exogenous growth promoting substances can bind thereto.
  • Binding may be irreversible and may be accomplished by the use of cross-linking agents, such as glycosaminoglycans, or other methods known to those of skill in the art or binding may be reversible, such as by absorption of the antigen or substance to the fiber.
  • cross-linking agents such as glycosaminoglycans, or other methods known to those of skill in the art
  • binding may be reversible, such as by absorption of the antigen or substance to the fiber.
  • Glycosaminoglycans to which colony stimulating factors and other growth factors bind in vivo (see, e.g., Gordon et al. (1987) Nature 326: 403-405), may be bound to the fibers or to stromal cells in the bioreactor.
  • Both GM- CSF and IL-3 specifically bind to the glycosaminoglycan, heparin sulfate, which is a part of the bone marrow stromal extracellular matrix.
  • the growth factor which is then added to the bioreactor, binds to the glycosaminoglycans, assumes an active conformation by virtue of this binding, and thereby mimics its in vivo activity.
  • glycosaminoglycans can be added to the bioreactor with stromal cells. Growth promoting substances, including colony stimulating factors, osteogenin or other growth factors, are bound to the hollow fibers and to the fibers via the glycosaminoglycan or other cross-linking agent.
  • the hollow fiber bioreactor is a component of a hollow fiber cell culture system.
  • a typical hollow fiber cell culture system such as the CELLMAXTM 100 hollow fiber cell culture system (Cellco Advanced Bioreactors, Inc., Kensington, MD.), which is described in Knazek et al.. U.S. Patent Application No. 07/238,445, supra..
  • Tissue culture medium which may, for example, include growth promoting substances, such as IL-2, and/or recombinant vectors, is drawn from the reservoir, pumped through the lumina of the hollow fibers, and then passed through the gas exchange tubing in which it is re- oxygenated and its pH readjusted prior to returning to the reservoir for subsequent recirculation.
  • THe order of sequences may be altered without substantially changing teh functionality.
  • the flow rate can be increased as the number of cells increases with time. Typically the initial flow rate of the medium is adjusted to about 40 ml./min.
  • the direction of perfusion of the medium through the hollow fiber lumina may be periodically and automatically reversed, typically every ten minutes, in order to provide a more uniform distribution of nutrient supply, waste dilution, and cells within the space surrounding the hollow fibers.
  • the entire system is sterilized prior to cell inoculation and is designed for operation in a standard air-C0 2 tissue culture incubator.
  • the cells settle onto the surface of the hollow fibers, through which nutrients pass to feed the cells and through which metabolic waste products pass and are diluted into the large volume of the recirculating perfusate.
  • the selected fiber should be semi-permeable to permit the passage of nutrients into the EFS and should be of a material on which or in the vicinity of which the cells are able to grow.
  • the fibers are made of material, such as DEAE-cellulose or polypropylene, that is semi-permeable or porous and suitable for the growth of mammalian cells.
  • material such as DEAE-cellulose or polypropylene
  • cellulosic hollow fibers 12 inches in length, whose walls nominally restrict diffusion to substances having a molecular weight less than 3000 Daltons are suitable for use in practicing this invention.
  • components of bone marrow or bone marrow cultures such as stromal cells or glycosaminoglycans, including heparin, to which growth factors adsorb, may be bound to the fibers, either reversibly or irreversibly. Endogenously produced growth factors will then bind to the fibers.
  • exogenous growth factors such as the interleukins, colony stimulating factors, and osteogenin
  • the bone marrow cells are then introduced into the EFS and are cultured in an environment that mimics the in vivo environment. Binding may be reversible, such as by adsorption, or irreversible if a cross-linking agent is used to permanently affix the component or the growth promoting substance to the fiber.
  • the growth promoting substance may also be included in the perfusate and/or in the EFS.
  • a suspension of cells is inoculated into the extra-fiber space (EFS) of a hollow fiber bioreactor typically through one of two side ports.
  • EFS extra-fiber space
  • the lumina are perfused with cell culture medium and the cells are maintained in vitro for the desired period of time.
  • EFS Upon inoculation into the EFS it is important that an adequate supply of oxygen is provided to the cells in order to prevent hypoxia, which predisposes stem cells to commit irreversibly to the erythropoietic pathway.
  • Relatively low flow rates are used in order to prevent the bone marrow cells and/or the adherent stromal cells, which are loosely adherent, from washing off the fibers as a result of EFS bulk flow and to prevent displacement of diffusible or poorly diffusible paracrine secreted Products from the microenvironment of the target cells, thereby preventing them from being maintained, suppressed or stimulated.
  • a small circulating volume of medium should be used initially so that the diffusible nutrient paracrine factors are not diluted to too great an extent.
  • Relatively low incubator temperatures may be needed (32° C to 33° C) for maintenance of the culture.
  • the EFS should not be disturbed. Removing the marrow from the EFS to examine the cells disturbs the microenvironment and may trigger the commitment process.
  • the methods of this invention may be used for culturing bone marrow obtained from healthy donors or from patients suffering from disorders, such as cancers that have not metastasized to the bone marrow, that do not involve the bone marrow.
  • Such bone marrow herein referred to as - normal marrow, may be used for autologous or allogenic transplants.
  • a sufficient amount of bone marrow which is usually washed and separated from erythrocytes using standard clinical methods, is inoculated into a hollow fiber bioreactor, such as the CELLMAX TH bioreactor, and cultured until it is needed for transplantation, such time is generally at least two to four weeks, but may be substantially longer.
  • the proportion of pluripotent stem cells remains substantially constant or increases compared the proportion of such cells in the inoculum.
  • the proportion of stem cells initially present in the bone marrow usually does not substantially decrease.
  • the methods of this invention may be used for clearing leukemic and/or other cancerous cells from the marrow in vitro.
  • malignant cells are present in the marrow it is may be advantageous to destroy such cells by chemotherapy and/or radiation therapy.
  • a sample of marrow Prior to treatment, a sample of marrow, of about, although not limited to, 400 to 800 ml., is aspirated from the patient. If desired, erythrocytes can be removed using standard well-known methods. After aspiration, the marrow can be treated with agents that destroy diseased cells, after which treatment the remaining cells are inoculated into a hollow fiber bioreactor.
  • the marrow can be introduced into a hollow fiber culture bioreactor without treatment.
  • marrow is obtained from a patient suffering from a leukemia, such as CML, which exhibits detectable phenotypic or genotypic markers, such as chromosomal translocations that may be visible upon microscopic inspection or detected by methods such as hybridization with probes that span the breakpoint (see, e.g.. U.S. Patent No. 4,701,409 to Croce et al... After aspiration the marrow is inoculated into a hollow fiber bioreactor, such as the CELLMAXTM 100 bioreactor, and is cultured.
  • the leukemic cells are cleared from the culture.
  • the cultured cells are then harvested from the bioreactor and reinfused into the patient.
  • the bone marrow cells may be co-cultured with or pretreated with TIL and/or LAK cells, which are prepared from the same patient or which are known to specifically react with the leukemic cells of the patient.
  • the TIL and or LAK cells may be induced upon inoculation of the bone marrow into the bioreactor by the addition of an effective concentration of a growth promoting substance, such as IL-2, to the EFS and/or perfusate.
  • the bone marrow cells may be treated with or cultured in the presence of chemotherapeutic agents that destroy the cancerous cells or that enhance the ability of immune cell in the marrow to destroy cancer- cells.
  • the cells or agents may - be introduced into the perfusing medium or into the EFS. If introduced into the EFS, the cells or agents may be bound, reversibly or irreversibly, to the surfaces of the fibers.
  • the methods of this invention are used to clear solid tumor cells from bone marrow.
  • the marrow from a patient suffering from metastatic cancer is introduced into a bioreactor and is cultured in the presence of a mitogen, such as an interleukin, that specifically expands tumor- specific T-lymphocytes (see, co-pending U.S. Patent Application No. 07/407,456 to Knazek et al., supra...
  • the tumor-free bone marrow cells are harvested and reinfused into the patient, who has been treated with chemotherapy and/or radiation to destroy tumor cells.
  • marrow containing metastatic tumor cells is withdrawn from the patient and inoculated into the EFS of a CELLMAXTM bioreactor.
  • IL-2 is also inoculated into the EFS and/or is included in the perfusing medium.
  • - TIL and/or LAK cells are inoculated into the EFS.
  • bone marrow cells are harvested from the bioreactor and reinfused into the patient.
  • bone marrow cells are transfected with a recombinant vector, which includes DNA encoding a gene product and which is capable of expressing this DNA in mammalian cells, to produce recombinant bone marrow stem cells for gene therapy.
  • a gene product is permanently provided to the recipient.
  • the preparation and selection of the vector and DNA encoding at least one gene product is within the level of skill in the art.
  • the vector will be a viral vector that can be replicated and packaged by selected target cells but not by bone marrow cells.
  • the gene product may be a therapeutic product, such as an anti-cancer or anti-viral agent; it may be a product, such as adenosine deaminase or immunoglobulin, that the recipient fails to produce or produces in a mutated defective form because of a genetic defect; it may be a marker, such as DNA that encodes neomycin or methotrexate resistance, whereby the reinfused bone marrow cells may be selected or detected, or any other gene product.
  • the selected recombinant vector is introduced by transfection or any other method known to those of skill in the art into a convenient target host cell, which is capable of replicating and packaging the vector at high titer.
  • the target host cell is then cultured in a bioreactor, such as the CELLMAXTM bioreactor for a time and under conditions whereby the vector is released into the EFS, which then contains high titers of the recombinant vector.
  • the EFS is then harvested batchwise, periodically, or continuously by connecting it to the EFS of a second bioreactor.
  • Bone marrow cells are removed from a donor, who is preferably the intended recipient of the cultured modified bone marrow cells, and introduced into the second bioreactor, such as the CELLMAXTM bioreactor.
  • the harvested EFS that contains the recombinant viral vector is introduced into the EFS of the bioreactor that contains the bone marrow cells or is mixed with the bone marrow cells prior to introduction into the bioreactor. This step can be repeated a plurality of times in order to insure that a high percentage of the pluripotent stem cells take up or are transfected with the recombinant DNA.
  • the EFS that contains the vector may be included in the perfusing medium, if hollow fibers having a sufficiently large pore size to permit diffusion of the vector into the EFS.
  • the bone marrow cells are cultured for at least about two to four weeks, whereby stem cells that contain the recombinant vector in a stable manner are produced and/or maintained.
  • the cultured bone marrow cells are then harvested and infused into a recipient.
  • the recipient is treated with chemotherapy and/or radiation to destroy his or her bone marrow cells, prior to infusion of the recombinant stem cells.
  • the recipient may be treated with the drug prior to infusion and/or after infusion of the selective-marker-modified bone marrow cells.
  • bone marrow cells must be removed from an individual.
  • Such individual is generally the patient who is to be treated using an adoptive immunotherapeutic method or a matched donor.
  • the cells obtained from the patient or donor are suspended in any cell culture medium that is suitable for sustaining the growth of such mammalian cells. Such media are readily available and the choice of an appropriate medium is well within the level of skill in the art.
  • the cells may be treated to remove the erythrocytes and are then suspended in the tissue culture medium at a suitable concentration, which is about, but is not limited to, 10 s to 10 7 cells per ml.
  • a suitable concentration which is about, but is not limited to, 10 s to 10 7 cells per ml.
  • a sufficient volume of cells to fill the EFS of a bioreactor cartridge is inoculated into the pre- sterilized cartridge and placed in an incubator at an appropriate temperature, generally about 32° C to about 37° C and maintained under these conditions for up to several months.
  • the conditions, including temperature and media are selected whereby the relative proportion of pluripotent stem cells remains constant or increases. After about 3 to 7 days in the EFS erythroid colonies appear, which is evidenced by the appearance of reddening of the previously white streaks of cells.
  • the culture medium is continuously perfused through the hollow fiber bioreactor by means of externally applied pressure, such as a pump.
  • a glass reservoir, the hollow fiber bioreactor, and pumping means are connected by tubing, typically silicone rubber, aa hollow fiber oxygenator or other means of. oxygenating media known to those of skill in the art, which simultaneously serves as a membrane gas exchanger to replenish oxygen and, if the medium is buffered with bicarbonate, to maintain the pH via C0 2 transport into the perfusion medium.
  • Medium that is buffered with systems other than bicarbonate do not necessarily require C0 2 in the incubator.
  • the perfusate can be replaced. Typically, it is replaced about once a week. Care must be taken not to disturb the cells in the EFS. Disturbances to these cell may cause them to become committed multipotent and end-stage cells.
  • the perfusing medium can be replenished by replacing the reservoir bottle with one containing fresh medium. After growth of the cells has been established, the cells can be harvested by gently shaking the bioreactor and pouring the suspended cells into a side port bottle.
  • the EFS cell supernatant which is rich non-or poorly-diffusible cellular products, including useful biologically active agents, cytokines and lymphokines produced by the cultured cells, can be .recovered for further processing in order to purify or partially purify said biologically active agents.
  • the cells can be spun down using a centrifuge or by any other - means known to those of skill in the art to yield a cell pellet and the EFS cell supernatant, which is enriched in biologically active molecules, such as growth-promoting substances.
  • the harvested cells can be assayed for the presence of normal pluripotent stem cells using standard semi-solid colony assays.
  • the harvested cells can also be transplanted into a recipient.
  • the EFS cell supernatant may be dialyzed against fresh tissue culture medium in order to produce EFS conditioned medium.
  • the conditioning factors may also be isolated or partially purified using standard well-known protein purification methods.
  • bone marrow cells are harvested from a patient suffering from CML or from a healthy bone marrow donor. All operations in which the cells are manipulated are performed using sterile techniques in a laminar flow hood. The erythrocytes are removed using standard clinical methods and the remaining cells and suspended in suitable tissue culture medium, such as AIM-V, at a density of about 10 7 and 10 8 cells per ml. About 50 ml.
  • the suspension is inoculated into a single bioreactor cartridge.
  • the hollow fiber culture system Prior to use the hollow fiber culture system is steam autoclaved, continuously perfused with 1.3 liters of recirculating deionized water, drained, flushed, and perfused with the selected tissue culture medium in both the EFS and perfusate pathways.
  • the inoculated bioreactor is transferred to a * standard incubator where it is perfused with medium. If the cells are to be modified for use in genetic therapy, a high concentration of the selected recombinant vectors, containing the heterologous DNA, is added to the EFS.
  • the vector may be added to the EFS continuously or a plurality of times during the incubation period or it may be added to the EFS via the perfusing medium if hollow fibers that have a sufficiently high pore size to permit diffusion of the vector are selected.
  • TIL cells are desired, such as in embodiments in which the bone marrow is obtained from a patient suffering from metastatic cancer, they are added to the EFS or induced to proliferated or are activated by the addition of an appropriate growth-promoting substance, such as IL-2 or anti-CD3 monoclonal antibody, to the EFS and/or to the perfusing medium. Additionally, any desired chemotherapeutic agents that destroy malignant cells may be added to the EFS and/or perfusing medium.
  • Incubation continues for at least about one to thirty days. During the incubation period the reservoir containing the perfusing medium is replaced in order to maintain a sufficiently high concentration of glucose and other diffusible nutrients in the EFS and for waste removal.
  • the cells are harvested by shaking the hollow fiber bioreactor and draining the EFS. The cells are pelleted and the EFS cell supernatant collected for further processing. If the bone marrow cells were obtained from a healthy donor or from a patient suffering from a leukemia, the harvested cells will most likely be non- leukemic and should contain a greater proportion of pluripotent stem cells than did the inoculum.
  • the following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
  • EXAMPLE 1 The CELLMAX 100 bioreactor system was used for the bone marrow cultures with either a B3 or B4 hollow fiber bioreactor. Prior to use the silicone rubber tubing flow path was connected to the pump and reservoir and steam autoclaved with side port tubing and bottles at 121° C for 20 minutes. Each hollow fiber bioreactor cartridge was sterilely removed from its package and sterilely inserted into the sterilized silicone rubber tubing pathway. The side port bottles were attached to the side ports. Each bioreactor was also, on occasion, steam autoclaved simultaneously with the perfusion path after having been inserted into the perfusion flow path. During this procedure the cart-ridge was kept full of distilled water, because allowing the fiber alters their ability to support cell growth.
  • the distilled water in the EFS of the bioreactor was drained into sideport bottles, discarded, and replaced with complete culture medium.
  • the reservoir was also filled with complete medium and the entire system was perfused overnight in a humidified 5% C0 2 incubator at 37° C.
  • Media that were used include: AIM-V, Iscove's.
  • Bone marrow cells which were obtained from patients or paid volunteers and were cleared of red cells using standard clinical methods. The cells were inoculated into the EFS via the side port bottles to fill the EFS. The entire CELLMAXTM bioreactor unit was then put into the incubator, but not perfused for 15 hours in order to facilitate attachment of cells to the fibers.
  • Bone marrow cells were obtained from a patient having from CML. As determined by standard cytological techniques, virtually 100% of the cells exhibited the characteristic Philadelphia chromosome. The cells were inoculated into the bioreactor and cultured in AIM-V medium as described in Example 1 except that four days after inoculation, the incubator temperature was decreased from 37° C to 33° C.
  • EXAMPLE 3 Approximately 5 x 10 7 cells from bone marrow that had been harvested from a normal volunteer were inoculated into a B3 cartridge through which with Iscove's medium was perfusing. A colony-forming assay was also performed on an aliquot of bone marrow cells at the time of harvest from the patient. This revealed the following after 14 days of culture in agar: CFU-GM: 0.2 per 10 5 cells plated BFU-F: 0.1 per 10 s cells plated.

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Abstract

Procédés de culture in vitro de quantités thérapeutiques de cellules de moelle osseuse utilisés dans le domaine de la transplantation de moelle oseuse et en immunothérapie adoptive. On met en culture des cellules de moelle osseuse pouvant être autologues ou prélevées sur un donneur, dans un système de culture comprenant un bioréacteur à fibres creuses. On peut prétraiter les cellules de moelle osseuse à l'aide d'un agent chimiothérapeutique ou autre, ou on peut les mettre en culture en présence d'au moins un agent chimiothérapeutique ou autre, ou d'une substance stimulant la croissance. On peut introduire de l'ADN hétérologue dans les cellules de moelle osseuse pendant la culture. On peut utiliser les cellules de moelle osseuse cultivées selon les procédés de l'invention dans des techniques d'immunothérapie adoptive afin de traiter le cancer, des dérèglements génétiques ainsi que le SIDA.
PCT/US1991/003555 1990-05-30 1991-05-24 Culture de cellules de moelle osseuse utilisees en immunotherapie adoptive WO1991018972A1 (fr)

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US5512480A (en) * 1994-03-11 1996-04-30 Baxter International Inc. Flow-through bioreactor with grooves for cell retention
US5599705A (en) * 1993-11-16 1997-02-04 Cameron; Robert B. In vitro method for producing differentiated universally compatible mature human blood cells
US5728581A (en) * 1995-06-07 1998-03-17 Systemix, Inc. Method of expanding hematopoietic stem cells, reagents and bioreactors for use therein
WO2000050048A2 (fr) * 1999-02-26 2000-08-31 University Of Pittsburgh Of The Commonwealth System Of Higher Education Transplantation de moelle osseuse pour la regeneration et la reparation hepatique
US7326571B2 (en) 2003-07-17 2008-02-05 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
WO2012168295A1 (fr) 2011-06-06 2012-12-13 ReGenesys BVBA Multiplication de cellules souches dans des bioréacteurs à fibres creuses
AU2007265147B2 (en) * 2006-06-26 2013-11-07 Terumo Bct, Inc. Method of culturing mesenchymal stem cells
US10633625B2 (en) 2013-11-16 2020-04-28 Terumo Bct, Inc. Expanding cells in a bioreactor
US10669519B2 (en) 2010-10-08 2020-06-02 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
CN114891718A (zh) * 2022-06-08 2022-08-12 天康制药(苏州)有限公司 用于悬浮培养骨髓细胞的培养基、制备方法、应用和诱导骨髓源细胞分化为巨噬细胞的方法

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Cited By (20)

* Cited by examiner, † Cited by third party
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US5599705A (en) * 1993-11-16 1997-02-04 Cameron; Robert B. In vitro method for producing differentiated universally compatible mature human blood cells
US5512480A (en) * 1994-03-11 1996-04-30 Baxter International Inc. Flow-through bioreactor with grooves for cell retention
US5728581A (en) * 1995-06-07 1998-03-17 Systemix, Inc. Method of expanding hematopoietic stem cells, reagents and bioreactors for use therein
WO2000050048A2 (fr) * 1999-02-26 2000-08-31 University Of Pittsburgh Of The Commonwealth System Of Higher Education Transplantation de moelle osseuse pour la regeneration et la reparation hepatique
WO2000050048A3 (fr) * 1999-02-26 2001-02-01 Univ Pittsburgh Transplantation de moelle osseuse pour la regeneration et la reparation hepatique
US8790920B2 (en) 2003-07-17 2014-07-29 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
US7326571B2 (en) 2003-07-17 2008-02-05 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
AU2007265147B2 (en) * 2006-06-26 2013-11-07 Terumo Bct, Inc. Method of culturing mesenchymal stem cells
US11746319B2 (en) 2010-10-08 2023-09-05 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11773363B2 (en) 2010-10-08 2023-10-03 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US10669519B2 (en) 2010-10-08 2020-06-02 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US10870827B2 (en) 2010-10-08 2020-12-22 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
WO2012168295A1 (fr) 2011-06-06 2012-12-13 ReGenesys BVBA Multiplication de cellules souches dans des bioréacteurs à fibres creuses
EP3572497A1 (fr) 2011-06-06 2019-11-27 Regenesys bvba Expansion de cellules souches dans des bioréacteurs à fibres creuses
US10633625B2 (en) 2013-11-16 2020-04-28 Terumo Bct, Inc. Expanding cells in a bioreactor
US11708554B2 (en) 2013-11-16 2023-07-25 Terumo Bct, Inc. Expanding cells in a bioreactor
US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
CN114891718A (zh) * 2022-06-08 2022-08-12 天康制药(苏州)有限公司 用于悬浮培养骨髓细胞的培养基、制备方法、应用和诱导骨髓源细胞分化为巨噬细胞的方法
CN114891718B (zh) * 2022-06-08 2024-01-30 天康制药股份有限公司 用于悬浮培养骨髓细胞的培养基、制备方法、应用和诱导骨髓源细胞分化为巨噬细胞的方法

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