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  • 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
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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  • 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
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
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  • C12N2501/39—Steroid hormones

Definitions

• This invention relates to regulation of hematopoiesis, and particularly to regulating production of hematopoietic stem cells.
• TGF-3 The transforming growth factor betas (TGF-3) are a family of cytokines for which a major function is inhibition of cell growth and differentiation. There is evidence that TGF-J inhibits cell proliferation by extending or arresting the Gl phase of the cell cycle (A.B. Roberts et al . (1985), Proc. Nat 'l . Acad. Sci . USA, Vol. 82, pp. 119 ff.). TGF-3 has been shown to be a negative regulator of hematopoiesis in long term bone marrow (LTBM) cultures.
• LTBM long term bone marrow
• TGF-j ⁇ has been shown to act directly on an enriched population of high proliferation multipotential hematopoietic progenitors from bone marrow (J.R. Keller et al . (1990), Blood, Vol. 75, pp. 596 ff.). In addition, there is also evidence that TGF- ⁇ also affects the mesenchymal stromal elements of these cultures (J.D. Cashman et al . (1990), Blood, Vol. 75, No. 1, pp. 96 ff.) .
• TGF-jS has a role in hematopoietic growth regulation, and that addition of an agent that neutralizes the activity of TGF- ⁇ types 1, 2, and 3 (that is, an agent that operates as a TGF- ⁇ antagonist) can stimulate production of hematopoietic stem cells in vitro and in vivo.
• long term survival of lethally-irradiated animals can be improved by administering to the animals bone marrow cells that have been cultured in the presence of an effective amount of an agent that neutralizes the biological activity of TGF-3 types 1, 2 and 3 (that is, an agent that operates as a TGF-/3 antagonist) to stimulate production of hematopoietic stem cells in the culture.
• an agent that neutralizes the biological activity of TGF-3 types 1, 2 and 3 that is, an agent that operates as a TGF-/3 antagonist
• 15 types 1, 2 and 3 that is, an agent that operates as a TGF- ⁇ antagonist.
• a chemotherapeutic drug e .g. , 5-fluorouracil
• the invention features a method for expanding growth of hematopoietic stem cells in culture, including a step of admixing a TGF- ⁇
• the term "expanding growth of hematopoietic stem cells” is used herein to mean an increase in the numbers of hematopoietic stem cells in the culture.
• the TGF-0 antagonist includes a
• the TGF- ⁇ antagonist includes an antibody or a combination of antibodies, more preferably a monoclonal antibody or a combination of monoclonal antibodies, that neutralizes TGF- ⁇ types 1, 2, and 3.
• a "TGF-J antagonist”, as that term is used herein, is a substance that inhibits, or a combination of substances that together inhibit, the biological activity of TGF-/3.
• a molecule, such as an anti-TGF-/3 antibody, that "neutralizes" TGF-/3, as that term is used herein, is a substance that inhibits the in vitro biological activity of TGF-/S.
• a most preferred TGF-3 antagonist is a monoclonal antibody that cross-reacts with and neutralizes TGF-jS types 1, 2, and 3.
• TGF-jS antagonist is a monoclonal antibody ("lDll.l ⁇ ") produced by a murine hybrido a cell line, 1D11.16, as described in copending U.S. patent application Serial No. 07/759,109, filed September 6, 1991, which is hereby incorporated herein by reference in its entirety.
• the invention features a method for treating an animal, by culturing bone marrow cells in vitro, admixing a TGF-J antagonist with the culture to stimulate production of hematopoietic stem cells, and administering cells from the culture to the animal.
• the bone marrow cells are cells that were derived from an animal of the same species as the animal to be treated, and more preferably are cells that were harvested from the animal to be treated.
• the invention features a method for treating an animal, by administering a TGF-jS antagonist to the animal.
• Dexter cultures have been treated with a monoclonal antibody, 1D11.16, which neutralizes the biological activity of TGF-jS types 1, 2 and 3, or with a control antibody.
• the composition and cellularity of the nonadherent cell populations in these cultures were assessed weekly.
• Treatment with anti-TGF-3 Ab resulted in a 5-9 fold increase in nonadherent cells in the cultures as compared to either the control or untreated cultures by week four.
• the majority of these cells were granulocyte/ macrophage lineage cells as assessed by histologic and flow cytometric analysis. There was also a significant increase in megakaryocytes in cultures treated with anti-TGF-/S Ab.
• TGF- ⁇ l , TGF-32 and TGF-33 or with a control antibody We have examined the resulting cell populations histologically and by flow cytometric analysis. By disrupting the negative growth control exerted by endogenous TGF-jS on hematopoietic progenitors a dramatic nonadherent cell outgrowth is seen. Phenotypically, most of the nonadherent cells that exhibit increased growth in neutralizing antibody treated cultures are those of the monocyte and granulocyte lineages and megakaryocytes. Analysis of both adherent and nonadherent cells indicates a similar increase in CFU-S stem cells.
• Fig. 1 is a histogram showing results of treatment of cells in Dexter long term bone marrow culture with an anti-TGF-3 antibody.
• Nonadherent cells from triplicate cultures were mixed and counted at weekly intervals, beginning one week after initiation of the culture (solid bars) and continuing for 5 weeks (week 2, dark crosshatched bars; week 3, shaded bars; week 4, light crosshatched bars; week 5, open bars) .
• Cell counts in cultures treated with anti-TGF-S monoclonal antibody were at a maximum 8 times as great at 4 weeks as in cultures receiving a control antibody, or in cultures receiving no treatment. Data in the Fig. represent 5 separate experiments.
• Fig. 2 is a histogram showing results of treatment of cells in Dexter long term bone marrow culture with various quantities of anti-TGF-S antibody.
• Nonadherent cells from triplicate cultures were mixed and counted at weekly intervals, beginning one week after initiation of the culture.
• Cell counts increased in cultures treated with anti-TGF-3 antibody (5 ⁇ g/ml, light shaded bars; 10 ⁇ g/ml, light crosshatched bars; 25 ⁇ g/ml, clear bars; 50 ⁇ g/ml, dark shaded bars) in dose dependent fashion.
• Cell numbers in untreated cultures (solid bars) or cultures treated with 50 ⁇ g/ml control antibody (dark crosshatched bars) increased little during this culture period.
• Data in Fig. 2 represent 3 separate experiments.
• Fig. 3 is a histogram showing results of initiating treatment of cells in Dexter long term bone marrow cultures with anti-TGF-/3 antibody at the time of initiation of the cultures or at various times (1, 2, or 3 weeks) following initiation of the cultures.
• Nonadherent cells from triplicate cultures were mixed and counted. Cultures were set up untreated or with 25 ⁇ g/ml control antibody (crosshatched bars) or anti-TGF-3 antibody (solid bars) .
• Nonadherent cell growth increased only in cultures to which anti-TGF- ⁇ antibody was added at initiation.
• Figs. 3 is a histogram showing results of initiating treatment of cells in Dexter long term bone marrow cultures with anti-TGF-/3 antibody at the time of initiation of the cultures or at various times (1, 2, or 3 weeks) following initiation of the cultures.
• Nonadherent cells from triplicate cultures were mixed and counted. Cultures were set up untreated or with 25 ⁇ g/ml control antibody (cross
• 4.1 - 4.3 are prints of photomicrographs (10X magnification, Giemsa stain) showing samples of nonadherent cell populations cytospun onto glass slides from 5 week cultures.
• a modest yield of maturing granulocyte/macrophage lineage cells and occasional erythroid elements appear untreated cultures (Fig. 4.1) and in cultures treated with a control antibody (Fig. 4.2).
• a markedly higher yield of granulocyte/macrophage lineage, and numerous megakaryocytes (*) appear in cultures treated with anti-TGF-3 antibodies (Fig. 4.3).
• Figs. 5.1 - 5.3 are prints of photomicrographs showing stromal layers of 3 week old Dexter cultures.
• Fig. 5.1 Few hematopoietic progenitor cells are attached to the adherent layer in untreated cultures (Fig. 5.1). A slightly greater number of hematopoietic progenitor cells are attached to the adherent layer in culture treated with a control antibody (MOPC 21C) (Fig. 5.2). A markedly greater number of progenitor cells are attached in hematopoietic islands in the adherent layer in cultures treated with anti-TGF-S antibody (Fig. 5.3).
• Fig. 6 is a histogram showing the number of Day 14 CFU-S colonies induced by 2 X 10 5 cells is the same for all Dexter culture conditions tested (untreated; treated with control antibody; treated with anti-TGF-jS antibody) . Data shown represent three experiments in which similar results were seen. v. Best Mode of Carrying out the Invention
• mice Animals Balb/c mice (Jackson Laboratories) , 6-8 weeks old, were used in all the examples.
• Monoclonal antibodies 1D11.16 and MOPC 21C and 1410 KG7 were purified over a protein A column (J.W. Goding et al . (1976), Jour. Immunol . Methods , Vol. 42, pp. 17 ff.) from ascites fluid. SDS PAGE gels were run to assess purity (U.K. Laemmli (1970), Nature , Vol. 227, pp. 680 ff.) .
• 1D11.16 is an IgGl antibody which neutralizes the biological activity of TGF-jSl, TGF-j82 and TGF-S3, was produced in Balb/c mice immunized with TGF-/32 generally as described in J.R. Dasch et al . (1989), Jour. Immunol . , Vol. 142, No. 5, pp. 1536 ff. MOPC 21C, a murine IgGl plasmacytoma of unknown specificity, was obtained from ATCC for use as a control antibody (P.M. Knopf et al .
• Antibodies were added at the start of culture unless otherwise noted. Cultures were refed weekly by removal of 4 ml of medium, followed by addition of 4 ml of medium supplemented with antibody at the original concentration. In a kinetics experiment antibody was added to cultures starting at culture initiation and at 1, 2, and 3 weeks after initiation.
• Nonadherent cells were adhered to glass slides using a cytospin, and stained on the slides with a Wright-Giemsa stain. Cell populations that were adherent in culture were stained with a Wright-Giemsa stain directly in the tissue culture flask.
• Flow cytometric analysis Populations of nonadherent cells were phenotypically analyzed using a Facstar fluorescence activated cell sorter (Becton Dickinson) . The cells were stained at a concentration of 1 ⁇ g antibody per 1 x 10 6 cells, on ice, for 30 minutes. The cells were washed and 10,000 cells were analyzed for each sample. Anti-TGF- ⁇ treatment stimulates growth of nonadherent cells in Dexter cultures
• 25 ⁇ g/ml neutralizing antibody wee administered to the cultures at the time of initiation of the culture, or at 1, 2, or 3 weeks after initiation.
• Fig. 3 shows, antibody must be present at the start of culture to induce an increase in the nonadherent cell populations.
• Nonadherent cell counts in 4 week old cultures treated with anti-TGF-/3 showed a six-fold increase in cell number when antibody was introduced at culture initiation and a three fold increase when antibody was introduced after 1 week of incubation. No effect was seen if antibody was added later than 1 week after culture initiation.
• Cytospins of nonadherent cell populations from these cultures show an increase in cells of the granulocyte/macrophage lineage, and an appearance of numerous megakaryocytes in anti-TGF-3 treated cultures over time (Figs. 4.1 - 4.3).
• Differential counts of cytospins from 5 week cultures revealed a shift in the anti-TGF-jS treated cultures to a more granulocytic population (80 %) , and fewer macrophages (15 %) .
• the method of the invention can be used for greatly expanding the numbers of a variety of types of hematopoietic stem cells in culture, without substantially altering the cell types in the culture.
• the invention thus provides means for obtaining hematopoietic stem cells in high number in culture.
• the invention provides for means for improving or restoring hematopoietic function in an animal.
• an animal such as a human patient may be subject to a form of medical treatment, such as radiation treatment or chemotherapy, that effectively destroys hamatopoietic function.
• a form of medical treatment such as radiation treatment or chemotherapy
• bone marrow cells are removed prior to the medical treatment and brought into culture; the TGF-/3 antagonist is then admixed to the culture to expand the numbers of the hematopoietic stem cells, according to the method of the invention.
• the hematopoietic stem cells from the culture are returned to the patient.
• a TGF- ⁇ antagonist can according to the invention be administered directly to an animal that is in need of improvement or restoration of hematopoiesis, in an amount effective to stimulate expansion of hematopoietic stem cells in vivo.
• TGF- ⁇ is a potent modulator of hematopoietic stem cell growth and differentiation.
• CFU-GEMM multipotential progenitor cells
• HPP-CFC high proliferative potential colony forming cells
• More mature hematopoietic stem cells such as those stimulated to form colonies in response to G-CSF, M-CSF and GM-CSF, have been reported to be either stimulated or unaffected by treatment with TGF-/3 (see, e .g. , S.E.W. Jacobsen et al . (1991), supra ; J.R. Keller et al. (1989), supra ; J.R. Keller et al. (1992), supra ; W. Piacibello et al . (1991), supra ; K. Fan et al . (1992), Blood, Vol. 79, No. 7, pp. 1679 ff.; W.C. Hooper et al .
• the Dexter-type long term bone marrow culture system provides a model which has both bone marrow stroma and hematopoietic stem cells in various stages of differentiation. After the formation of an adequate stromal layer, Dexter cultures support the growth of hematopoietic stem cells of the granulocyte, monocyte/macrophage, and megakaryocyte lineages and of HPP-CFC. In addition, cells in these cultures are capable of producing colonies in the spleens of irradiated animals (CFU-S) (see, e .g. , P.J. Quesenberry et al . (1989), Ann . N. Y. Acad . Sci . , Vol. 554, pp.
• CFU-S irradiated animals
• TGF-S has been shown to have a profound effect on the ability of these cultures to maintain growth of any of these hematopoietic lineages. This effect is mediated through direct interaction with both the stromal microenvironment and the hematopoietic stem cells (see, e .g. , S.I. Hiyashi et al . (1989), supra ; Z.C. Han et al. (1991), supra ; P.W. Kincade (1990), Adv. Cancer Res . , Vol. 54, pp. 235 ff.; D.J. Kuter et al . (1992), Blood, Vol. 79, No. 3, pp. 619 ff.; F.W.
• This effect may be mediated through the stromal layer, as kinetic studies revealed that neutralization of endogenous TGF-jS must occur during the first week of culture when the stromal layer is forming. Later addition of anti-TGF-/3 did not release the progenitor cells from normal growth controls and no increase in cell numbers was seen. Treatment with anti-TGF-S did not phenotypically alter nonadherent cell types supported by the Dexter cultures. Granulocyte and monocyte/macrophage lineage cells were identified, both histologically and by flow cytometric analysis, to be the primary cell types present with or without anti-TGF-jS treatment. The major observed effect of abrogating TGF-jS in these cultures is extensive proliferation.
• TGF- ⁇ has been shown to be able to inhibit cell cycling of CFU-S and CFU-S colony formation (see, e . g. , A. Migdalska et al . (1991), Growth Factors, Vol. 4, pp. 239 ff.; G.J. Graham et al . (1990), Prog. Growth Factor Res . , Vol. 2, pp. 181 ff.; J. Hampson et al . (1991), Exp. Hematol . , Vol. 19, pp. 245 ff.).
• the animal model used in our examples provides an understanding of TGF-/3 suppression in a well defined bone marrow culture system. Neutralization of TGF-3 under
• TGF- ⁇ antagonists stimulates production of hematopoietic stem cells in vitro and in vivo
• Treatment with either anti-TGF-jS mAb or soluble TGF-jS receptor resulted in a dose-dependent increase in survival over animals that received 5- flourouracil alone.
• examination of bone marrow cells in mice at 2 weeks following 5-fluorouracil treatment showed a dose-dependent increase in cell number after anti-TGF-jS mAb or soluble TGF- ⁇ receptor treatment.

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