EP0716570A1 - Cellules souches hematopoi tiques humaines modifiees genetiquement et leur descendance - Google Patents

Cellules souches hematopoi tiques humaines modifiees genetiquement et leur descendance

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Publication number
EP0716570A1
EP0716570A1 EP94928018A EP94928018A EP0716570A1 EP 0716570 A1 EP0716570 A1 EP 0716570A1 EP 94928018 A EP94928018 A EP 94928018A EP 94928018 A EP94928018 A EP 94928018A EP 0716570 A1 EP0716570 A1 EP 0716570A1
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European Patent Office
Prior art keywords
cells
cell
composition according
receptor
stem cells
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EP94928018A
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German (de)
English (en)
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EP0716570A4 (fr
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James Mule
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Systemix Inc
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Systemix Inc
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Publication of EP0716570A4 publication Critical patent/EP0716570A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the field of this invention is expansion of human hematopoietic stem cells and their genetic modification.
  • lymphoid lines comprising B cells and T cells, produces antibodies, regulates cellular immunity, and detects foreign agents such as disease-causing organisms in the blood.
  • the myeloid lineage which includes monocytes, granulocytes, and megakaryocytes, monitors the blood for foreign bodies, protects against neoplastic cells, scavenges foreign materials, and produces platelets.
  • the erythroid lineage includes red blood cells, which carry oxygen.
  • stem cells progenitor populations
  • stem cells progenitor populations
  • These rare primitive cells approximately 0.01% of bone marrow cells
  • Stem cells differentiate into multipotent progenitor cells and ultimately into each of the mature hematopoietic lineages.
  • stem cells are believed to be capable of generating long term hematopoiesis when transplanted into immunocompromised hosts.
  • the stem cell was originally defined by the capacity to self-renew and to give rise to progeny that are the committed precursors for all hematopoietic lineages.
  • progenitor cell compartment into stem cell and committed progenitor cells that these compartments constitute a hierarchy or continuum of cell types whose maturation is characterized by decreases in both pluripotentiality and the ability to repopulate the hematopoietic system of serially transplanted animals.
  • Stem cells constitute only a small percentage of the total number of hematopoietic cells. Hematopoietic cells are identifiable by the presence of a variety of cell surface "markers.” Such markers may be either specific to a particular lineage or progenitor cell or may be present on more than one cell type. Currently, it is not known how many of the markers associated with differentiated cells are also present on stem cells. One marker which was previously indicated as being present solely on stem cells, CD34, is also found on a significant number of lineage committed progenitors.
  • Table 1 summarizes probable phenotypes of stem cells in fetal, adult, and mobilized peripheral blood.
  • myelomonocytic stands for myelomonocytic associated markers
  • NK stands for natural killer cells
  • AMPB adult mobilized peripheral blood.
  • the negative sign or, uppercase negative sign, ( " ) means that the level of the specified marker is undetectable above Ig isotype controls by FACS analysis, and includes cells with very low expression of the specified marker.
  • hHSC human hematopoietic stem cell
  • TCR T cell antigen receptor
  • TCR-2 is a heterodimer of 2 disulfide-linked transmembrane polypeptides ( ⁇ and ⁇ )
  • TCR-1 is structurally similar but consists of y and ⁇ polypeptides.
  • the a and ⁇ or and ⁇ _. polypeptides form a heterodimer which contains an antigen recognition site.
  • These heterodimers recognize antigen in association with MHC molecules on the surface of antigen-presenting cells. All of these peptides contain a variable region which contributes to the antigen recognition site and a constant region which forms the bulk of the molecule and includes the transmembrane region and cytoplasmic tail.
  • Both receptors are associated with a complex of polypeptides making up the CD3 complex.
  • the CD3 complex is made up of the 7, f and e transmembrane polypeptides.
  • the CD3 complex mediates signal transduction when T cells are activated by antigen binding to the TCR.
  • a variety of these and related molecules have been cloned and expressed in various T cell lines. Kuwana et al. (1987) Biochem. Biophvs. Res. Comm. 149:960-968; Gross et al. (1989) Trans. Proc. 2 ⁇ :127-130; Becker et al. (1989) Cell 58:911-921; Gross et al. (1989) Proc. Natl. Acad. Sci.
  • TCR-2 bearing cells can be subdivided further into two distinct non-overlapping populations; the TH subset which is generally CD4 + and the Tc/s subset which is generally CD8 + .
  • CD4 + T cells recognize antigens in association with MHC class II molecules
  • CD8 + T cells recognize antigens in association with MHC class I molecules.
  • the CD4 + set can be further divided functionally into: 1) those cells which positively influence the immune response of T cells and B cells, i.e., the helper cell function; and 2) cells inducing suppressor/cytotoxic functions in CD8 + cells, i.e., the suppressor inducer function.
  • CD4 + cells expressing natural killer (NK) cell markers produce the lymphokine interleukin-2 (IL-2) , and do not proliferate in response to antigens and mitogens.
  • NK natural killer
  • IL-2 lymphokine interleukin-2
  • recent in vitro studies on CD4 + clones in mouse and man have defined two separate populations (THI and TH2) based on the production of different lymphokines.
  • CD8 + T cells can also be subdivided by a number of criteria and a variety of monoclonal antibodies into specific functional subsets. For example, cells which recognize antigen in association with MHC molecules and produce IL-2 (CD28 + ) and cells which do not recognize antigen in association with MHC molecules or produce IL-2 (CDllb + ) .
  • CD3 + /TCR-1 + cells represent a minority of circulating T cells which are also CD4", CD8". These cells home in to surface epithelia such as the epidermis and mucosal epithelia and are termed intra-epithelial lymphocytes (IEL) . In interstitial mucosal epithelium TCR-1 + cells also express CD8.
  • CD2 is also found on about 50% of CD3"NK cells.
  • CD5 molecules are expressed on all T cells, and also on a subpopulation of B cells which are involved in autoantibody production.
  • B lymphocytes represent about 5-15% of the circulating lymphoid pool and are classically defined by the presence of endogenously produced immunoglobulins (antibody) . These molecules are inserted into the surface membrane where they act as specific antigen receptors. They are detected on the surface of mature cells by staining cells suspensions with fluorochrome- labelled specific antibodies to the appropriate immunoglobulin of the species under investigation.
  • the majority of human peripheral blood B lymphocytes express both surface IgM and IgD molecules, which share the same specificity on the same cell. Very few cells express surface IgG, IgA or IgE in the circulation although these are present in larger numbers in specific locations in the body, for example, IgA-bearing cells in the intestinal mucosa.
  • the present invention provides genetically modified stem cells and lymphoid progenitor cells that express recombinant DNA encoding an antigenic specificity region and a signal transducing region. These recombinant DNAs include recombinant TCRs and chimeric TCRs.
  • the recombinant TCRs are full length ⁇ and ⁇ chains of known antigen binding specificity.
  • the chimeric TCRs are genetically engineered such that they contain a functional signal transducing region and a functional antibody-derived antigen binding site.
  • T cell specific expression of the recombinant DNA enables the T cell progeny of the transduced stem cells to bind to antigen and exert T cell functions including, but not limited to, cytolysis and B cell helper activity.
  • the transduction of stem cells introduces genes encoding the recombinant DNA.
  • T cells with the recombinant DNA has the potential to broaden the immunotherapy of viral infections, including, but not limited to, human immunodeficiency virus (HIV) and hepatitis B, and to extend and specifically target cancer treatment to multiple cancer histologies.
  • HIV human immunodeficiency virus
  • hepatitis B hepatitis B
  • stem cells The unigue characteristics of stem cells distinguish them from other cells which have been successfully genetically engineered. Efforts to genetically engineer the progeny of stem cells frequently encounter lack of transmission of functional expression of the introduced construct in cells, intermittent expression, and the like. Therefore, even where one has demonstrated the successful integration of the construct in such cells, subsequent growth of the progeny cells and their differentiation may result in the failure of the construct to function.
  • inducible promoters expression of various protein products can be achieved at selected levels of differentiation or in selected cell lineages, or even in response to particular chemicals, such as chemoattractants, particular ligands, and the like. Also, as there is better understanding of the manner in which stem cells differentiate to specific lineages, particular lineages, such as megakaryocytes, subsets of T cells, monocytes, and the like can be produced in culture.
  • T cell gene therapy requires ex vivo expansion of T cells with cytokines.
  • the modified T cells Upon re-infusion, the modified T cells often do not home properly to their target organs and may become trapped in (and cleared by) the lungs, liver or spleen. This improper homing may be due to alteration of the membranes during the ex vivo processing, downregulation of homing receptors, or the like.
  • Ex vivo expansion of T cells is costly, cumbersome and time consuming and thus less than ideal for treatment.
  • T cells have a finite life span, the process of transduction of mature T cells and infusion must be repeated, whereas stem cells self-renew and, therefore, transduced HSCs are able to differentiate to replenish lost T cells.
  • the recombinant TCR can be put under the control of a T cell specific promoter so that it is only expressed in T cells.
  • the promoter could be Granzyme A, which would cause the recombinant TCR to be expressed predominantly in NK cells and cytotoxic T lymphocytes (CTLs) .
  • CTLs cytotoxic T lymphocytes
  • modified T cells recognizing the target antigen should be relatively amplified. Also, it may be possible to get a stronger response from the T cells derived from transduced stem cells. If more mature T cells are transduced with the recombinant TCR, they may have a dampened response if they are "memory" cells (i.e. previously exposed to antigens) and, therefore, "biased.” Another advantage to genetically modified HSC over mature T cells would be the ability to express the recombinant TCR in more than one hematopoietic lineage. For example, since macrophages are known to have the ability to engulf tumor cells, it may be useful to express the recombinant TCR in macrophages.
  • Substantially homogeneous hHSCs can be maintained in long-term cultures and expanded in number in appropriate media, optionally in conjunction with hematopoietic factors such as LIF, stem cell factor, IL3, IL6, IL7, IL11, GCSF, GMC, EPO, MlP-l ⁇ and IFN7, under otherwise conventional conditions.
  • the hHSCs may be maintained in culture for long periods of time, as demonstrated by their capacity to continually differentiate into multilineage progeny.
  • hHSC genetic modification of the hHSC can be accomplished at any point during their maintenance by transducing a substantially homogeneous stem cell composition with a recombinant DNA construct described herein.
  • a retroviral vector is employed for the introduction of the DNA construct into the hHSC host.
  • the resulting cells may then be grown under conditions similar to those for unmodified hHSC, whereby the modified hHSC may be expanded and used for a variety of purposes.
  • the hHSC which are employed may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonatal, or adult; and obtained from liver, bone marrow, blood or any other conventional source.
  • the manner in which the stem cells are separated from other cells, whether of the hematopoietic or of other lineage is not critical to this invention. Conveniently, the cells may be separated as described in U.S. Patent No. 5,061,620.
  • the substantially homogeneous composition of hHSC may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with stem cells. At such time as a specific marker is identified for hHSC, binding of an antibody to such marker may provide the desired composition.
  • a large proportion of the differentiated cells may be removed by initially using a relatively crude separation, where major cell population lineages of the hematopoietic system, such as lymphocytic and myelomonocytic, are removed, as well as minor populations, such as egakaryocytic, mast cells, eosinophils and basophils. Usually, at least about 70 to 90 percent of the hematopoietic cells will be removed.
  • a prior separation may be employed to remove erythrocytes, by employing Ficoll-Hypaque separation. The gross separation may be achieved using methods known in the art including but not limited to magnetic beads, cytotoxic agents, affinity chromatography or panning.
  • Antibodies which find use include antibodies to lineage specific markers which allow for removal of most, if not all, mature cells, while being absent on hHSC.
  • a negative selection may be carried out, where antibodies to specific markers present on dedicated cells are employed.
  • these markers include, but are not limited to, CD3 ' , CD7 “ , CD8 ' , CD10-, CD14 ' , CD15-, CD19 “ , CD20 “ , CD33 “ and glycophorin A; preferably including, but not limited to, at least CD3 " , CD8 “ , CD10", CD19 “ , CD20 “ and CD33 " ; and normally including at least CD10", CD19 “ and CD33 " . See Table l.
  • the hematopoietic cell composition substantially depleted of dedicated cells may then be further separated using a marker for Thy-1, whereby a substantially homogeneous stem cell population is achieved.
  • a substantially homogeneous stem cell population is achieved.
  • exemplary of this stem cell population is a population which is CD34 + Thy-1 + , which provides an enriched stem cell composition.
  • An hHSC composition is characterized by being able to be maintained in culture for extended periods of time, being capable of selection and transfer to secondary and higher order cultures, and being capable of differentiating into the various lymphocytic and myelomonocytic lineages, particularly B and T lymphocytes, monocytes, macrophages, neutrophils, erythrocytes and the like.
  • the hHSC may be grown in culture in an appropriate nutrient medium, including, but not limited to, conditioned medium, a co-culture with an appropriate stromal cell line, adhesion molecules, or a medium comprising a synthetic combination of growth factors which are sufficient to maintain the growth of hematopoietic cells.
  • an appropriate nutrient medium including, but not limited to, conditioned medium, a co-culture with an appropriate stromal cell line, adhesion molecules, or a medium comprising a synthetic combination of growth factors which are sufficient to maintain the growth of hematopoietic cells.
  • stromal cell lines For conditioned media or co-cultures, various stromal cell lines may be used. Since human stromal cell lines are not required, other stromal cell lines may be employed, including but not limited to rodentiae, particularly murine. Suitable murine stromal cell lines include AC3 and AC6, which are described in hitlock et al. (1987) Cell 4jJ:1009-1021. Other stromal cell lines may be developed, if desired. Preferably, the stromal cell line used is a passage of AC6, AC6.21 (otherwise referred to as SySl) .
  • tissue culture plates or flasks may be employed where confluent stromal cell layers may be maintained for extended periods of time without passage, but with changing of the tissue culture medium about every five to seven days.
  • the hHSC may be grown in co-culture by placing the hHSC onto the stromal cell lines, either directly or separated by a porous membrane. For example, about 3 x 10 4 to 3 x 10 5 cells/ml are placed on a confluent stromal cell layer.
  • the media employed in the co-culture may be any convenient growth medium, including but not limited to RPMI-1640 and IMDM either individually or in combination, where appropriate antibiotics to prevent bacterial growth, e.g.
  • penicillin peneptomycin
  • pen/strep penicillin, streptomycin (pen/strep) and other additives, such as pyruvate (0.1-5 mM) , glutamine (0.5-5 mM) , 2-mercaptoethanol (1-10 x 10 "5 M, 2-ME) and from about 5-15%, preferably about 10% of serum, e . g. fetal calf serum (FCS) .
  • FCS fetal calf serum
  • LIF may be added in from about 1 ng/ml to 100 ng/ml, more usually 5 ng/ml to 30 ng/ml if the cells are to be expanded prior to transduction.
  • Other factors may also be added, including but not limited to, interleukins, colony stimulating factors, steel factor. Of particular interest are LIF, IL-3, IL-6, GM-CSF and MlP-l ⁇ .
  • the factors which are employed may be naturally occurring or synthetic, e . g. prepared recombinantly, and may be human or of other species, e .g. murine, preferably human. The amount of the other factors will generally be in the range of about 1 ng/ml to 100 ng/ml.
  • the concentration will be in the range of about 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 100 ng/ml; for IL-6, the concentration will be in the range of about 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml, and for GM-CSF, the concentration will generally be 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml.
  • the stem cells are optionally expanded prior to or after transduction. During expansion, the growth factors may be'present only during the initial course of the stem cell growth and expansion, usually at least 24 hours, more usually at least about 48 hours to 4 days or may be maintained during the course of the expansion.
  • the stem cells are cultured with or without LIF or other factors in an appropriate medium, transduced with the appropriate vector, cultured for approximately 72 hours and reintroduced into the host.
  • a retroviral vector will be employed, however any other suitable vector may be used. These include, but are not limited to, adenovirus, adenoassociated virus and artificial chromosomes derived from yeast. Combinations of retroviruses and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells.
  • amphotropic virus-producing cell lines including, but not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437): PA317 (Miller et al. (1986) Mol. Cell. Biol. 1:2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci USA 85:6460-6464).
  • Possible methods of transduction include, but are not limited to, direct co-culture of hHSC with producer cells e.g. by the method of Bregni et al. (1992) Blood JP_:1418-1422 or culturing with viral supernatant alone with or without appropriate growth factors and polycations e.g. by the method of Xu et al. (1994) Exp. Hemat. 11:223-230; and Hughes et al. (1992) J. Clin. Invest. 89:1817.
  • the constructs employed will normally include a marker gene, which allows for selection of cells into which the DNA has been integrated, as against cells which have not integrated the DNA construct.
  • Various marker genes include, but are not limited to, antibiotic resistance markers, such as resistance to G418 or hygromycin. Less conveniently, negative selection may be used, including, but not limited to, where the marker is the HSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir.
  • selections could be accomplished by employment of a stable cell surface marker to select for transgene expressing hHSC by FACS sorting.
  • the constructs can be prepared in a variety of conventional ways. Numerous vectors are now available which provide the desired features, such as long terminal repeats, marker genes, and restriction sites, which may be further modified by techniques known in the art.
  • the constructs will encode a signal peptide sequence in addition to the antigenic specificity region and cytoplasmic signalling sequence, to ensure that the recombinant TCR is properly processed post- translationally and expressed on the cell surface.
  • the construct is under the control of a T cell specific promoter. Suitable T cell specific promoters include, but are not limited to, GranzymeA and CD8.
  • the signal transducing region and antigenic specificity region are both obtained from TCRs ("classic TCR”) .
  • constructs encode chimeric polypeptides comprising the signal transducing region obtained from a T cell specific receptor or the FC receptor and an antigen binding portion of an immunoglobulin (“chimeric TCR”) .
  • the recombinant classic TCRs are functional, preferably full length, TCR a and ⁇ or 7 and ⁇ polypeptides which have been derived from a T cell with known antigenic specificity.
  • Suitable sources of antigen-specific TCRs include, but are not limited to, cytotoxic T lymphocytes, T helper cells and natural killer cells.
  • the polypeptides may be recombined so as to form a single functional polypeptide with the V ⁇ and V3 regions forming the antigen binding site.
  • the V ⁇ and V/3 regions from different TCRs may be recombined to endow the TCR with a different specificity.
  • the chimeric molecule contains an antigen binding sequence from an antibody, a transmembrane sequence and a sequence that can transduce a T cell specific function.
  • Suitable signal transducing regions can be obtained from T cells including, but not limited to, the 7 chain of the F c receptor, the TCR f chain, and the IL-2 receptor ⁇ , ⁇ or 7 chains.
  • Suitable antigen binding domains may be derived from antibodies that specifically bind the target antigen. These include, but are not limited to, antibodies that recognize viruses, bacteria and cancer cells.
  • the functional portion of the chimeric molecule is the constant region of a FC7 or f polypeptide and the antigen binding domain is a variable region of an antibody.
  • the variable region may be either the V H or V L regions or a single chain recombinant thereof.
  • the T cell progeny of the stem cells containing the recombinant classic TCR polypeptides are "MHC restricted", that is, they will only recognize antigen in the presence of MHC. Thus, when using these cells to treat a patient, the TCRs must be of the same haplotype of the host. It is well within the skill of one in the art to match the haplotype of the host and the TCR.
  • the T cell progeny of the stem cells containing the chimeric TCR molecules will recognize antigen in the absence of MHC and thus will not be MHC restricted. These molecules are suitable for use in all hosts regardless of haplotype.
  • the hHSC may be genetically modified so that upon reintroduction into the host and subsequent differentiation, T cells are produced that are specifically directed against any antigen.
  • suitable antigens include, but are not limited to, those found on virally infected cells, and specific cancer cells. More specifically, suitable antigens include, but are not limited to, viral coat proteins and specific surface proteins of cancer cells.
  • modified T cells can be used to treat any disease or cancer to which there is available a specific antibody.
  • Monoclonal antibodies directed against many different tumor antigens are available and can be used to generate tumor-specific chimeric receptors.
  • the T cells will home to the cancer and effect T cell activity including but not limited to cytolysis and/or B cell helper activity. These activities will be effective in eradicating cancer cells.
  • the specificity is for viral proteins
  • the T cells will home to virally infected cells and effect cell lysis to prevent further replication of the virus.
  • the effect may be potentiated by the systemic co-administration of recombinant cytokines (e.g. IL-2 or IL-3 by either boosting reactivity, function or causing expansion of the relevant cells in situ.
  • recombinant cytokines e.g. IL-2 or IL-3 by either boosting reactivity, function or causing expansion of the relevant cells in situ.
  • a T cell line can be transduced with the constructs and assayed for activity resulting specifically from the genes expressed by the construct. For instance, for testing for activity of a construct encoding a cancer cell specific recombinant polypeptide, the transduced T cell line would be assayed for its ability to lyse cancer cells expressing the protein recognized by the recombinant polypeptide but not cells that do not express this polypeptide. Once activity of the recombinant polypeptide is confirmed, the DNA construct can be used to transduce stem cells. Also included are the stem cells containing the DNA constructs described herein.
  • the vectors containing the polynucleotides of interest can be introduced into the stem cells by any of a number of appropriate means, including, but not limited to, electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; infection or transduction (where the vector is an infectious agent, such as a retroviral genome) .
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances
  • microprojectile bombardment e.g., a retroviral genome
  • cell immunotherapy involves removal of bone marrow or other source of stem cells from a human host, isolating the stem cells from the source and expanding the stem cells. Meanwhile, the host may be treated to partially, substantially or completely ablate native hematopoietic capability. The isolated stem cells may be modified during this period of time, so as to provide for stem cells having the desired genetic modification. After completion of the treatment of the host, the modified stem cells may then be restored to the host to provide for the new capability. The methods of stem cell removal, host ablation and stem cell repopulation are known in the art. If necessary, the process may be repeated to ensure the substantial repopulation of the modified stem cells. Immunotherapies using modified stem cell populations to produce T cells with new specificities circumvent the cumbersome ex vivo process of isolating, expanding and transducing mature T cells.
  • a vector-specific probe may be used to verify presence of the vector in the transduced stem cells or their progeny. Proper expression of the construct on the cell surface may be detected by, for example, an anti- idiotypic antibody or by assays for T cell activation which will depend on the recombinant TCR binding to the target antigen (e.g., cytokine release).
  • the cells may be grown under various conditions to ensure that they are capable of maturation to all of the hematopoietic lineages while maintaining the capability, as appropriate, of the introduced DNA.
  • Various tests in vitro and in vivo may be employed to ensure that the pluripotent capability of the stem cells has been maintained.
  • fetal thymus is isolated and cultured for 4-7 days at about 25°C, so as to deplete substantially the lymphoid population.
  • the cells to be tested for T cell activity are then microinjected into the thymus tissue, where the HLA of the population which is injected is mismatched with the HLA of the thymus cells.
  • the thymus tissue may then be transplanted into a scid/scid mouse as described in U.S. Patent No. 5,147,784, particularly transplanting under the kidney capsule.
  • assays of the thymus fragments injected with the cells can be performed and assessed for donor derived T cells.
  • the modified stem cells may be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least 1 x 10 5 cells will be administered, preferably 1 x 10 6 or more. The cells may be introduced by injection, catheter, or the like. If desired, factors may also be included, including, but not limited to, interleukins, e.g. IL-2, IL-3, IL-6, and IL-11, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM- CSF, interferons, e.g. 7-interferon, erythropoietin. The following examples are provided to illustrate but not limit the invention.
  • the vector used was Sp6 ⁇ 7, described in Eshhar et al. (1993) Proc. Natl. Acad. Sci. USA 90:720-724, which has the variable regions of the anti-TNP antibody Sp6 and the 7 chain of the Fc receptor.
  • the vector was transfected into the packaging cell line PA317. Approximately 200,000 cells of the CD34 hi Lin ⁇ phenotype were seeded onto irradiated Sp6 ⁇ 7 producer cells (3K rads) .
  • CD34 hi Lin " stem cells were isolated from fetal bone marrow by the method described by DiGiusto et al. (1994) Blood 84:421-432. "Lin-" refers to cells lacking lineage-specific markers; in this case, Lin- refers to cells lacking CD2, 14, 15, 16, 19 and glycophorin A.
  • the fetal bone marrow stem cell source was determined to GAPA + .
  • the CD34 hi Lin " cells were seeded into 2 wells of a 12- well dish containing Sp6 ⁇ 7 producer cells in 2 ml of hitlock-Witte media containing LIF (50 ng/ml) and IL-6 (10 ng/ml) .
  • TNP-PE phycoerythrin
  • CD4-FITC CD4-FITC
  • CD8-TriColor CD8-TriColor
  • TNP-PE propidiu iodide was -22-
  • the other half of the cells were stained with: (1) CD4-PE; (2) CD8-Tricolor; (3) GAPA-FITC; propidium iodide was included in the final suspension for live/dead discrimination.
  • the labelled antibodies were obtained from Becton Dickinson. The results obtained are presented in Table 2 where NI stands for not injected and ND stands for not determined due to insufficient ceils.
  • GAPA-FITC recognized the donor cells.
  • An autologous hHSC population is genetically modified such that differentiated T cells derived from the population express TCRs that enable the cell to recognize and destroy HIV infected cells.
  • a segment of DNA is engineered to encode a chimeric TCR. Eshhar et al. (1993) Proc. Natl. Acad. Sci. USA 90:720-724.
  • the variable portion of the receptor consists of the variable region (Fv) of an i munoglobulin that recognizes peptides of the viral envelope of HIV (e.g. gpl60) .
  • the signal transduction region consists of either the f chain of the TCR/CD3 complex or the 7 chain of the Fc receptor (FcR) ; the TCR a or ⁇ chains could also be used as the signal transducers.
  • the Fv portion is expressed as a single chain (scFV) , with the V L domain bridged to the V H domain of the antibody via a flexible linker.
  • the recombinant TCR is fused to the SC kappa leader peptide to ensure translocation to the cell membrane according to the method described by Eshhar et al.
  • the resulting recombinant protein has the following structure (amino- to carboxy-terminus) : SC kappa leader peptide-V L -V H - chain of the TCR, or y chain of the FcR.
  • TCR ⁇ or ⁇ chains may also be used as the signaling molecules.
  • the DNA encoding the receptor is inserted into the LXSN bicistronic retroviral vector according to the method described by Miller and Rosman (1989)
  • BioTechniques 2 ⁇ 980 under the transcriptional control of the LTR from the Moloney Murine Leukemia Virus, along with the neomycin phosphotransferase gene under the transcriptional control of the SV-40 early region promoter. Transcription of the chimeric receptor is under the control of an endogenous T cell promoter. Using this system, is possible to package scFv-f" and scFv-7 transcripts into retroviral particles and transduce hHSCs, selecting for G418 resistance.
  • the host is treated to partially, substantially or completely ablate native hematopoietic capability.
  • the modified stem cells are then restored to the host to provide for the recognition and destruction of HIV infected cells. If necessary, the process is repeated to ensure the substantial absence of the original host cells and the substantial population of the modified stem cells.
  • T cells derived from gene- modified hHSC that mature in a patient after the myeloablative regimen are endowed with specificity to HIV-1.
  • hHSC is co-transduced with vectors encoding TAR decoys or rev mutants.
  • Gene-modified hHSC is admixed with unmodified hHSC to endow the patient with a broad T cell repertoire. Patients may then be given recombinant interleukin-2 systemically to expand the HIV-1 reactive T cell population.
  • An autologous hHSC population is genetically modified such that differentiated T cells derived from the population express chimeric TCRs that enable the cell to recognize and destroy specific types of cancer cells.
  • Construction of Vector A segment of DNA is engineered to encode a chimeric TCR as described above.
  • the variable portion of the receptor consists of the variable region (Fv) of an immunoglobulin that recognizes peptides found on the surface of a particular cancer cell type.
  • An example is the monoclonal antibody MOvl ⁇ which is specific for human ovarian carcinoma. Hwu et al. (1993) J. Exp. Med. 178:361-366.
  • the signal transduction region consists of either the f chain of the TCR or the y chain of the FcR.
  • the Fv portion is expressed as a single chain (scFV) , with the V L domain bridged to the V H domain of the antibody via a flexible linker as described by Eshhar et al.
  • the recombinant TCR is fused to the SC K leader peptide to ensure translocation to the outer cell membrane.
  • the resulting recombinant protein has the following structure (amino- to carboxy-terminus) : SC K leader peptide-V L -V H -f chain of the TCR, or y chain of the FcR.
  • the DNA encoding the receptor is inserted into the LXSN bicistronic retroviral vector as described by Miller and Rosman, under the transcriptional control of the LTR from the Moloney Murine Leukemia Virus, along with the neomycin phosphotransferase gene under the transcriptional control of the SV-40 early region promoter. Transcription of the chimeric receptor is under the control of an endogenous T cell promoter. Using this system, is possible to package scFv-f and scFv-7 transcripts into retroviral particles and transduce hHSCs, selecting for G418 resistance.
  • CD34 + Thv-1 + Population Bone marrow or mobilized peripheral blood is removed from the human host using standard techniques.
  • CD34 + Thy-1 + cell populations are isolated and expanded using the techniques outlined above.
  • the infection is performed by introducing approximately 10 4 CD34 + Thy-1 + cells and 1 x 10 6 CFU of the virus in 1 ml of long-term culture medium ("LTCM") comprising 10 ng/ml of LIF, IL-3, IL-6, and GM- CSF.
  • LTCM long-term culture medium
  • the cell mixture is maintained for at least 24 hours, and the medium diluted to 10 ml with LTCM and 100 ⁇ l of the medium introduced into wells in which confluent layers of the AC6.21 stromal line is present.
  • the cells from 5-15 wells, each of the cells with and without virus, are then introduced into a methylcellulose culture and maintained for 2 weeks. At the end of this time, all cells are collected from each methylcellulose culture and analyzed using Southern and Northern analysis to confirm successful gene insertion and transcriptional expression. Cell Therapy.
  • the host is treated to substantially or completely ablate native hematopoietic capability. After completion of the treatment of the host, the modified stem cells are then restored to the host to provide for the recognition and destruction of specific types of cancer cells. If necessary, the process is repeated to ensure the substantial absence of the original host cells and the substantial population of the modified stem cells.
  • T cells derived from gene-modified hHSC that mature in a patient after myeloablative regimen are endowed with specificity to the carcinoma to which the binding region of the antibody has specificity.
  • the T cells In the case of antibody MOV18, the T cells have specificity for ovarian carcinoma. Further expansion (or activation of) the relevant T cell population could be achieved by the systemic administration to the patient of appropriate recombinant cytokines (e.g. IL-2, IL-7) .
  • An autologous hHSC population is genetically modified such that differentiated T cells derived from the population express recombinant TCRs that enable the cell to recognize and destroy specific types of cancer cells.
  • the segments of DNA which encode the TCR and ⁇ chains are obtained from a T cell clone expressing a TCR of the desired specificity.
  • the genes are then cloned as described below.
  • the genes may be cloned into the same vector, if so only one transduction event need occur; or into separate vectors with the concomitant need for separate transduction events to take place.
  • the DNA encoding the receptor is inserted into the LXSN bicistronic retroviral vector as described by Miller and Rosman, under the transcriptional control of the LTR from the Moloney Murine Leukemia Virus, along with the neomycin phosphotransferase gene under the transcriptional control of the SV-40 early region promoter. Transcription of the genes is under the control of an endogenous T cell promoter. Using this system, is possible to package TCR ⁇ and ⁇ transcripts into retroviral particles and transduce hHSCs, selecting for G418 resistance.
  • CD34 + Thv-1 + Population Bone marrow or mobilized peripheral blood is removed from the human host using standard techniques.
  • CD34 + Thy- 1 + cell populations are isolated and expanded using the techniques outlined above.
  • the infection is performed by introducing approximately 10 4 CD34 + Thy-1 + cells and 1 x 10° CFU of the virus in 1 ml of long-term culture medium ("LTCM") comprising 10 ng/ml of LIF, IL-3, IL-6, and GM- CSF (or additional cytokines) .
  • LTCM long-term culture medium
  • the cell mixture is maintained for at least 24 hours, and the medium diluted to 10 ml with LTCM and 100 ⁇ l of the medium introduced into wells in which confluent layers of the AC6.21 stromal line is present.
  • each of the cells with and without virus are then introduced into a methylcellulose culture and maintained for 2 weeks. At the end of this time, all cells are collected from each methylcellulose culture and analyzed using Southern and Northern analysis to confirm successful gene insertion and transcriptional expression.
  • the host is treated with high-dose chemotherapy which may substantially or completely ablate native hematopoietic capability.
  • the modified stem cells are then restored to the host to provide for the recognition and destruction of specific types of cancer cells.
  • unmodified HSC may be administered simultaneously with the modified HSC to reconstitute multilineage hematopoietic function. If necessary, the process is repeated to ensure the substantial absence of the tumor cells and the substantial population of the modified stem cells.
  • T cells derived from gene-modified hHSC that mature in a patient after myelosuppressive or myeloablative regimen are endowed with the specificity to which the binding region of the recombinant TCR has specificity. Further expansion (or activation of) the relevant T cell population could be achieved by the systemic administration to the patient of appropriate recombinant cytokines (e.g. IL-2, IL-7) .
  • appropriate recombinant cytokines e.g. IL-2, IL-7 .

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Abstract

Des cellules souches hématopoiëtiques humaines expriment un ADN, qui est un produit d'assemblage obtenu par recombinaison et qui est capable de coder une molécule de recombinaison contenant une région de transduction de signaux et une région de spécificité antigénique, et permettent de produire des lymphocytes T avec des spécificités modifiées.
EP94928018A 1993-09-03 1994-09-06 Cellules souches hematopoi tiques humaines modifiees genetiquement et leur descendance Withdrawn EP0716570A4 (fr)

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US5635386A (en) 1989-06-15 1997-06-03 The Regents Of The University Of Michigan Methods for regulating the specific lineages of cells produced in a human hematopoietic cell culture
US5763266A (en) * 1989-06-15 1998-06-09 The Regents Of The University Of Michigan Methods, compositions and devices for maintaining and growing human stem and/or hematopoietics cells
US5437994A (en) * 1989-06-15 1995-08-01 Regents Of The University Of Michigan Method for the ex vivo replication of stem cells, for the optimization of hematopoietic progenitor cell cultures, and for increasing the metabolism, GM-CSF secretion and/or IL-6 secretion of human stromal cells
US5605822A (en) * 1989-06-15 1997-02-25 The Regents Of The University Of Michigan Methods, compositions and devices for growing human hematopoietic cells
US5399493A (en) * 1989-06-15 1995-03-21 The Regents Of The University Of Michigan Methods and compositions for the optimization of human hematopoietic progenitor cell cultures
US6319494B1 (en) 1990-12-14 2001-11-20 Cell Genesys, Inc. Chimeric chains for receptor-associated signal transduction pathways
US6407221B1 (en) 1990-12-14 2002-06-18 Cell Genesys, Inc. Chimeric chains for receptor-associated signal transduction pathways
US5972627A (en) * 1994-06-15 1999-10-26 Systemix, Inc. Method of purifying a population of cells enriched for dendritic and/or lymphoid progenitors and populations of cells obtained thereby
US5985660A (en) * 1994-06-15 1999-11-16 Systemix, Inc. Method of identifying biological response modifiers involved in dendritic and/or lymphoid progenitor cell proliferation and/or differentiation
US5830755A (en) * 1995-03-27 1998-11-03 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services T-cell receptors and their use in therapeutic and diagnostic methods
US6015554A (en) * 1995-06-07 2000-01-18 Systemix, Inc. Method of reconstituting human lymphoid and dendritic cells
US20020034819A1 (en) 1998-02-23 2002-03-21 Alan K. Smith Human lineage committed cell composition with enhanced proliferative potential, biological effector function, or both; methods for obtaining same; and their uses
US7723111B2 (en) 2001-03-09 2010-05-25 The United States Of America As Represented By The Department Of Health And Human Services Activated dual specificity lymphocytes and their methods of use
EP1627563A1 (fr) 2004-08-10 2006-02-22 Academisch Medisch Centrum bij de Universiteit van Amsterdam Moyens et méthodes de préparation de cellules stabilisées
EP1891209A1 (fr) * 2005-06-06 2008-02-27 Academisch Medisch Centrum bij de Universiteit van Amsterdam Moyens et procedes de generation d'un lymphocyte t dirige contre un antigene d'interet
USRE49583E1 (en) 2005-11-17 2023-07-18 Tet Systems Gmbh & Co. Kg Inducible expression systems
ATE512228T1 (de) 2005-11-17 2011-06-15 Tet Systems Gmbh & Co Kg Induzierbare expressionssysteme
JP5753339B2 (ja) 2005-12-09 2015-07-22 アカデミッシュ メディッシュ セントラム ビーアイジェイ ド ユニバーシテイト バン アムステルダム 抗体産生細胞の安定性に影響を与える手段および方法
WO2007067032A1 (fr) 2005-12-09 2007-06-14 Academisch Medisch Cemtrum Bij De Universiteit Van Amsterdam Moyen et methodes permettant d'influencer la stabilite de cellules
CA2768207C (fr) 2009-07-15 2019-12-03 Aimm Therapeutics B.V. Moyens et procedes de production d'anticorps a haute affinite
US9206247B2 (en) 2010-12-02 2015-12-08 Aimm Therapeutics B.V. Means and methods for producing high affinity antibodies
US10316289B2 (en) 2012-09-06 2019-06-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methods of producing T memory stem cell populations
CA2938193C (fr) 2014-01-31 2023-05-02 Aimm Therapeutics B.V. Moyens et procedes de production d'anticorps stables
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