EP0981546A1 - Proteines sm1 presentes a la surface des cellules hematopoietiques souches - Google Patents

Proteines sm1 presentes a la surface des cellules hematopoietiques souches

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Publication number
EP0981546A1
EP0981546A1 EP98920093A EP98920093A EP0981546A1 EP 0981546 A1 EP0981546 A1 EP 0981546A1 EP 98920093 A EP98920093 A EP 98920093A EP 98920093 A EP98920093 A EP 98920093A EP 0981546 A1 EP0981546 A1 EP 0981546A1
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EP
European Patent Office
Prior art keywords
cells
smi
antibody
protein
hematopoietic stem
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98920093A
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German (de)
English (en)
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EP0981546A4 (fr
Inventor
Peter M. C. Wong
Siu-Wah Chung
Xiaodong Han
Hong Chen
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Stemcell Therapeutics LLC
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Stemcell Therapeutics LLC
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Publication of EP0981546A1 publication Critical patent/EP0981546A1/fr
Publication of EP0981546A4 publication Critical patent/EP0981546A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • the present invention relates to a protein, referred to here as "SMI," in a form that is substantially purified from other proteins.
  • SMI protein, referred to here as "SMI,” in a form that is substantially purified from other proteins.
  • SMI is present on the surface of human and mouse hematopoietic stem cells and primitive progenitor cells, but is absent from those of other cells including FDC-Pl myeloid progenitor cells, EL4 T-cells, WEHI-3 myelomonocytic cells, and 70Z/3 pre-B lymphoid cells, or from differentiated hematopoietic cells of human cord blood or mouse bone marrow.
  • the present invention further relates to methods of using anti-SMI antibody to produce an enriched hematopoietic stem cell population.
  • All circulating blood cells develop from pluripotent stem cells through the process of hematopoiesis.
  • Hematopoietic stem cells are undifferentiated cells capable of self -renewal and differentiation into committed progenitor cells of the myeloid, erythroid, megakaryocytic and lymphoid blood cell lineages.
  • a thorough analysis of hematopoietic stem cells is fundamental to a comprehensive understanding of the developmental biology of the hematoly phoid system. Relatively little is known, however, about hematopoietic stem cells.
  • hematopoietic stem cells are capable of long-term reconstitution of the hematolymphoid system of lethally- irradiated recipients in vivo .
  • They also can differentiate into short-term hematopoietic stem cells, called day 12 spleen colony- forming units (CFU-S), which can be observed in in vivo assays for spleen foci formation.
  • CFU-S spleen colony- forming units
  • hematopoietic stem cells Efforts to characterize hematopoietic stem cells in more detail have been hampered primarily because of the proportionately minute amount (less than 0.01%) of hematopoietic stem ceils as compared with all cells, even in blood cell -forming organs such as bone marrow or the fetal liver. Li & Johnson, Blood 85:1472-1479 (1995). Accordingly, the elucidation of physical characteristics unique to hematopoietic stem cells is desirable as a means to produce enriched stem cell populations. For example, see Spangrude etai., Blood 78:1395-1402 (1991).
  • All known hematopoietic stem cell enrichment protocols involve cell -separation methods based mostly on the selection for cell surface markers or other physical means, such as density gradient centrifugation, counter flow centrifugal elutriation, and cell sorting based on light scattering properties.
  • Bertoncello et al . Expt. Hematol . 13:999-1006 (1985); Mulder & Visser, Expt. Hematol. 15:99-106 (1987); Ploemacher & Brons, Expt. Hematol . 17:263-271 (1989); Szilvassy et al . , PNAS 86:8798-8802 (1989) .
  • hematopoietic stem cells express cell surface differentiation antigen (Thy-l) and stem cell antigen- l (Sca-l). They do not, however, express the lineage markers (Lin) characteristic of B cells (B220) , granulocytes (Gr-1), myelomonocytic cells (Mac-1) and T cells (CD4, CD8)) . Spangrude et al . , supra .
  • the reportedly most widely used hematopoietic stem cell enrichment protocol involves the use of monoclonal antibodies against Thy-l and Sca-1. Orlic et al . , supra. Only a subset, however, of Thy-1 + , Sca-l "*" and Lin " cells are able to repopulate lethally- irradiated recipients long-term. Smith et al . , PNAS 88:2788-2792 (1991). Selection based on Thy-l and Sca-1 expression thus does not produce a pure hematopoietic stem cells population.
  • hematopoietic stem cell enrichment techniques such as those which involve the use of monoclonal antibodies against protein tyrosine kinases such as the W locus gene product, c - ki t, and fetal liver kinase-2 ( flk- 2 ) apparently are unable to distinguish between hematopoietic stem cells and progenitor cells. See, for example, Matthews et al . , Cell 65:1143-1152 (1991) .
  • Another example of a cell surface marker associated with hematopoietic stem cells is CD34.
  • CD34 A membrane phosphoglycoprotein, CD34 exists on hematopoietic stem cells, committed progenitor cells of all hematopoietic cell lineages, early multipotent hematopoietic progenitor cells, and endothelial cells.
  • CD34 + cells have been estimated to be about 2.5% of total bone marrow cells, Osawa et al . , Science 273:242 (1996) , and 1-4% in humans and baboons.
  • Civin et al . J. Immunol . 133:157 (1984); Civin et al . , Exp . Hematol . 15:10 (1987); Berenson et al . , J. Clin . Invest . 81:951 (1988) .
  • Hematopoietic stem cells have been estimated to constitute less than 0.1% of total bone marrow cells. Thus, selection based on CD34 alone does not yield a pure population of true hematopoietic stem cells.
  • CD34 has been targeted in combination with other cell surface markers for stem cell purification. These markers include the so-called lineage- specific antigens, such as HLA-DR, Thy-l, CD33, MDR-1, c - ki t , CD45 and CD38.
  • lineage- specific antigens such as HLA-DR, Thy-l, CD33, MDR-1, c - ki t , CD45 and CD38.
  • CD34VCD38 cells were found to comprise less than 0.1% of total human bone marrow cells, Civin et al . , Blood 88:4102 (1996), and CD34 + Thy-1 " "Lin- cells to comprise 0.05% to 0.1% of human fetal bone marrow cells. Baum et al . , PNAS 89:2804 (1992).
  • CD34- enriched cells do not constitute a pure population of true hematopoietic stem cells.
  • Emerson, supra. The recent establishment of a ceil line from a lethally- irradiated recipient mouse reconstituted with fetal liver cells previously transduced with a rearranged retroviral genome has been reported. Wong et al . , supra .
  • BL3 cells exhibit all of the functional hematopoietic stem cell properties, i . e . , they can reconstitute lethally- irradiated recipients long-term, they give rise to pre-CFU-S and colony- forming cells and they develop "cobblestones" upon association with stromal cells.
  • BL3 cells also express a transcription factor, GATA-1, known to be expressed in hematopoietic stem cells. Sposi et al., PNAS 89:6353-6357 (1992).
  • BL3 cells are embryonic in origin, having derived from fetal liver cells of 12 -day old mouse embryos. BL3 cells thus may possess different cell surface markers than adult hematopoietic stem cells. Jordan et al . , supra; Spangrude et al . , supra .
  • the present inventors have provided SMI protein substantially purified from other proteins, where SMI has a molecular weight of about 230 kDa, as measured by immunoprecipitation and SDS-PAGE, is present on the surface of human and mouse hematopoietic stem cells and primitive progenitor cells, but is absent from the surface of other cells, such as FDC-Pl myeloid progenitor cells, EL4 T-cells, WEHI-3 myelomonocytic cells, and 70Z/3 pre-B lymphoid cells, or from differentiated hematopoietic cells of human cord blood or mouse bone marrow.
  • the objectives also are achieved by an antibody against SMI and the use of the antibody to enrich for hematopoietic stem cells.
  • anti-SMI antibody is used to prepare a composition enriched for hematopoietic stem cells according to the invention.
  • the inventive methodology comprises the steps of (a) providing antibody that binds SMI, (b) immobilizing the antibody on a support platform such that the antibody retains its SMI-binding capability, then (c) bringing a mixed population of cells containing putative hematopoietic stem cells into contact with the antibody such that the stem cells adhere to the support platform, and 'd) removing nonadherenr cells, whereby a population enriched for hematopoietic stem cells remains adhered to the support platform.
  • kits for preparing a composition enriched for hematopoietic stem cells comprising (i) an antibody that binds SMI and
  • bone marrow can be obtained from a HLA- identical or nearly identical donor.
  • Bone marrow cells can then be contacted with the antibodies of the kit.
  • Cells isolated in this manner may be subjected to growth factors and cytokines to achieve a sufficiently pure population of hematopoietic stem cells suitable for transplantation into human patients.
  • a methodology for detecting in a sample a hematopoietic factor that binds SMI comprises (a) contacting a sample suspected of containing said growth factor with labeled-SMI, and (b) detecting the binding of the hematopoietic factor with labeled-SMI.
  • kits for the detection of a hematopoietic factor that binds SMI comprising labeled-SMI, and further comprising written instructions for the use of the kit.
  • One other embodiment of the invention includes methods of amplifying or expanding in vi tro human SMI cells.
  • SMI cells may be suspended in liquid media with additional growth factors and cytokines.
  • SMI cells may be grown on or within an adherent layer of mixed stroma ceil preparations, with or without the addition of growth factors and cytokines.
  • Yet another embodiment of the invention provides an isolated DNA molecule encoding SMI.
  • a particular embodiment of the invention provides an isolated DNA molecule that includes the following nucleotide sequence
  • TTGCCAGCAA CAACACTAAA TGAATCTACA GATCCTGGCA GTTCCATCCC CTGTTTTTCA CAGCAAACTG TTGATTCTTC TGAGGCAAAG CAAGAACTAA AAACTGTATG TATACGAGAT TCACAGTCAA TTCTTGTTAG GACTCCAGGT GGGAACACTG GAGTTGTAAA AGTACAAACT AATCCGGAAC AAAATTCACC CAACAGTTTA TCTTCAAGTT CTGTTTTCAC CTTTACACCT CAATTTCAGG
  • Yet another particular embodiment of the invention provides an isolated DNA molecule that includes the following nucleotide sequence (SEQ. ID. No. 2): GAATTCTTTT TATTGCAAAC AAACGTCTAA ATTAATTTCT CCACCCACTT
  • FIG. 1 DNA sequence of mouse SMI gene.
  • FIG. 1 Immunoprecipitation of SMI surface protein specifically on BL3 cells. Immunoprecipitation of 35 S-methionine labeled cells with SMI antibody indicates that of the tested samples, only BL3 cells expressed SMI protein on the cell surface.
  • FIG. 3 Southern blot analysis on CFU-S DNA from recipients of 100 and 1,000 SM1 + cells. SM1 + cells in the mouse bone marrow was estimated initially to be about 10%. To investigate whether hematopoietic stem cells reside in a subset of SMl + cells, cells were depleted that were positive for lineage specific markers, i . e . ,
  • CD4 T helper cells
  • CD8 T killer cells
  • Gr-1 Gr-1
  • FIG. 4 Two color- fluorescence activated cell sorter (FACS) analysis of mouse bone marrow cells.
  • SM1 + cells from mouse bone marrow was estimated initially to be about 10%.
  • SMI lineage specific markers
  • CD4 T helper cells
  • CD8 T killer cells
  • Gr-1 granulocytes
  • TER119 erythroid cells
  • Mac-1 Mac-1
  • SMI " cells. FACS analysis was performed on mouse bone marrow cells by using PE (polyerythrin) conjugated antibodies directed against all the lineage specific markers and FITC-conjugated SMI antibody.
  • PE polyerythrin
  • Figure 5 Nucleotide sequence of a human SMI gene.
  • Figure 6 FACS analysis of human cord blood cells, double stained with PE-conjugated lineage specific antibodies and FITC-conjugated anti-SMI antibodies.
  • SMI protein
  • the present invention relates to antibodies against SMI.
  • antibodies against SMI In addition to their use for the enrichment for hematopoietic stem cells, such antibodies could represent research and diagnostic tools in the study of hematopoietic factors and the development of antibody conjugated therapeutic agents for the treatment of diseases.
  • pharmaceutical compositions comprising antibodies against SMI may represent effective therapeutics.
  • Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, and fragments of polyclonal and monoclonal antibodies. The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al.
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas , selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e . g. , Coligan et al . , sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al . , Purification of
  • Multiplication in vi tro may be carried out in suitable culture media such as Dulbecco ' s Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth- sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • suitable culture media such as Dulbecco ' s Modified Eagle Medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth- sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • Production in vi tro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies.
  • Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture.
  • Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e . g. , syngeneic mice, to cause growth of antibody-producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
  • antibodies of the present invention may also be derived from subhuman primate antibody.
  • General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al . , International Patent Publication WO 91/11465 (1991) , and Losman et al . , Int . J. Cancer 46:310 (1990), the respective contents of which are hereby incorporated by reference.
  • a therapeutically useful anti-SMI antibody may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts .
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
  • General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al . , PNAS 86:3833 (1989) , which is hereby incorporated in its entirety by reference.
  • Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al . , METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al . , Arm . Rev. Immunol . 12: 433 (1994) , which are hereby incorporated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA) .
  • antibodies of the present invention may be derived from a human monoclonal antibody.
  • Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody- secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described by Green et al . , Nature Genet . 7:13 (1994); Lonberg et al . , Nature 368:356 (1994); and Taylor et al .
  • Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab 1 monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of V H and V L chains. This association may be noncovalent, as described in Inbar et al . , PNAS 69:2659 (1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross -linked by chemicals such as glutaraldehyde . See, e . g. , Sandhu, supra .
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • These single- chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide .
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli .
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains .
  • Methods for producing sFvs are described, for example, by Whitlow et al . , METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird et al . , Science 242:423-426
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991) .
  • SMI protein isolation and characterization of SMI protein was achieved through the establishment of a monoclonal antibody against SMI.
  • To prepare specific monoclonal antibodies a general procedure as described in Harlow & Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratories (1988) ) , which is incorporated herein by reference.
  • Three male Lew/hsd rats (Animal Center of Fox Chase Cancer Institute, Philadelphia, PA) were each immunized subcutaneously by injecting 5xl0 7 BL3 cells suspended in 0.5 ml PBS mixed with complete Freund's adjuvant. Pre-immune sera were collected prior to the injection.
  • Three weeks later, the rats were boosted subcutaneously with a dose of lxlO 8 BL3 cells.
  • Immune antisera were collected after the second and third boosting injections and were tested by live- cell enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) . All sera were tested positive on BL3 cells and negative on EL4 cells (a T cell line) .
  • ELISA live- cell enzyme-linked immunosorbent assay
  • IP immunoprecipitation
  • the titer of the antisera ranged from 1:1,000 to
  • BL3 cells Three days before fusion, another 100 million BL3 cells, with no adjuvant, were injected intravenously into one positive rat. On the third day, the rat was sacrificed by carbon dioxide asphyxiation, its spleen was removed, and a single cell suspension was prepared in Dulbecco's Modified Eagle Medium (DMEM) + 2% fetal calf serum (FCS) . Splenic cells and YB2/0 myeloma cells were mixed at a ratio of 10 to 1 and fused in the presence of 50% polyethylene glycol (PEG) . Hybridoma cell clones were selected by culturing the cell mixture in HAT selection medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS 2% fetal calf serum
  • the hybridomas were screened for the production of antibody specific for BL3 cells.
  • BL3 cells or other control cells such as EL4, FDC-Pl and
  • WEHI-3 cells were incubated with 80 l of hybridoma supernatant at 4°C for 30 minutes, and after washing twice were further labeled with FITC-conjugated goat anti-rat IgG+M secondary antibody under the same conditions. After washing, the cells of each clone were then screened by light microscopic examination. The antiserum was used as a positive control and pre- immune serum or some hybridoma supernatants were used as negative controls. Cells from three out of 170 hybridomas were shown to be specific for BL3 cells. Of these three, one recognized a molecule designated SMI. By a standard immunodiffusion assay, the SMI monoclonal antibody has been shown to be an immunoglobulin IgM allotype.
  • the isolation of SMI cDNA was performed by first constructing a Lambda gtll cDNA phage expression library. The construction of the cDNA library was done as follows. To isolate poly (A) RNA, total RNA was extracted using phenol/chloroform/Guanidine thiocyanate method. Sambrook et al . , MOLECULAR CLONING 2nd ed. (Cold Spring Harbor Laboratory Press 1989) . Cells (5xl0 8 to 10x10 s ) were lysed in 10 ml of 4M GTC solution (25 mM sodium citrate, 85 mM sodium lauryl sarcosine, 4M Guanidine thiocyanate and 0.1 M 2 -mercaptoethanol) .
  • 4M GTC solution 25 mM sodium citrate, 85 mM sodium lauryl sarcosine, 4M Guanidine thiocyanate and 0.1 M 2 -mercaptoethanol
  • DNA was sheared by passing through an 20 Gauge needle. The volume was increased to 20 ml by adding 10 ml of 4M GTC solution. 2 ml of 4M NaAc (pH 4.0) was added and mixed well before equal volume of DEPC-H 2 0 saturated-phenol was added. After the mixture was mixed thoroughly, 10% of final volume of chloroform was added and mixed vigorously again. The mixture was allowed to sit on ice for 15 minutes, and then centrifuged for 20 minutes at 2500 g (5000 rpm in Sorvall RC-5B centrifuge with Sorvall SA600 rotor) . The top aqueous phase containing RNA was transferred to a new tube.
  • RNA pellet was obtained after centrifugation at 2500 g for 20 minutes and dissolved in 0.4 ml of 4M GTC solution. The RNA was precipitated again with 10 ' ⁇ l of 1M HAc and 300 ⁇ l of ethanol . The final RNA pellet was dissolved in 0.5 ml of lmM EDTA/0.05% SDS and stored at -70°C.
  • Poly (A) RNA was selected by passing through two rounds over an oligo dT- cellulose column from Collaborative Research.
  • the IX binding buffer consists of 20 mM sodium phosphate and 0.5 M NaCl .
  • the amount of poly (A) RNA selected was about 5% of total RNA applied with a ratio of 0.D . 260 /O .D . 2g0 of 2.0.
  • the poly (A) RNA was aliquoted, mixed with one tenth volume of 3M NaAc and three times volume of ethanol , and stored at -70°C.
  • RNA was reverse transcribed into cDNA by superscript II reverse transcriptase (GibcoBRL) with oligo dT and random hexamer as primer following BRL's instructions. About 30% of poly (A) RNA was converted into cDNA.
  • the synthesized cDNA:RNA hybrid was size- fractionated through Sepharose CL-4B column (Pharmacia) to remove small cDNA.
  • Three ⁇ g of first strand cDNA:RNA hybrid was used for second strand cDNA synthesis. RNA strand was replaced with DNA strand by using RNAse H, DNA polymerase I, E.
  • EcoRI recognition sites in dsDNA were methylated by EcoRI methylase (Promega) to prevent digestion by EcoRI to be carried out in a later step.
  • Three different EcoRI linkers (8mer, lOmer, and 12mer) were used for ligation with ds cDNA in 100:1 molar ratio of linker : cDNA to create three different reading frames for translation of any cDNA in the library. After ligation, EcoRI digestion was performed to generate EcoRI cohesive ends in each cDNA molecule. Excess EcoRI linkers were removed by size-fractionation through a Sepharose CL-4B column.
  • the (doubled- stranded) ds cDNA with EcoRI sites were ligated with ⁇ gtll/EcoRI vector (Stratagene) and packaged into phage particles using phage package extracts (Stratagene) following the vendor's instructions.
  • the size of cDNA library was determined by titering the packaging mixture, i.e., infection of bacteria Y1088 with diluted packaging mixture. A total of 2 ⁇ g of ⁇ gtll/EcoRI vector and 0.45 ⁇ g of ds cDNA were used for ligation. For the HL60 sample, five packaging extracts were used.
  • the size of the ⁇ gtll-HL60 cDNA library is 1.35xl0 6 pfu.
  • the size of the ⁇ gtll-BL3 cDNA library is 1.5xl0 7 pfu.
  • the libraries were amplified once by infection of bacteria Y1088. To determine the average size of cDNAs in the library, 18 phage clones were randomly picked up for analysis. Phage DNA was extracted and digested with EcoRI to release the cDNA inserts. The average size of cDNAs was obtained by dividing the total size of EcoRI fragments from all 18 phage DNA samples with 18, giving a value of 1.4kb.
  • SMI monoclonal antibody (MAb) first antibody to be used for gene screening were predetermined by incubating serially diluted antibody supernatant, as well as supernatant of YB2/0 myeloma line (negative control) , with lysates of BL3 cells in parallel with that of E. coli as a control.
  • SMI monoclonal antibody has been shown to be the IgM form.
  • optimal amount of alkaline phosphatase conjugated second antibody was also predetermined.
  • Alkaline phosphatase conjugated anti- rat light chains ( K and ⁇ ) monoclonal antibody from Sigma and alkaline phosphatase conjugated anti-rat IgM ( ⁇ -chain specific) antibody from Rockland were tested for specific interaction with SMI MAb.
  • Rat IgM (from Rockland) and E. coli phage lysate (from Stratagene) were used as negative controls.
  • Five fold serial dilutions of each protein were made in blocking solution from 10 ⁇ g/ml of starting concentration to 2 ⁇ g/ml, 0.4 ⁇ g/ml and 0.08 ⁇ g/ml. One ⁇ l of each solution was spotted onto a nylon membrane.
  • the cDNA library then was screened with anti-SMI antibody under optimized conditions according to manufacturer's instruction (Stratagene, La Jolla, CA) .
  • a loop of Y1090R bacteria grown in LB plate with 50 ⁇ g/ml of ampicillin was inoculated into 15 ml of LB supplemented with 0.2% maltose and 10 mM MgS0 4 .
  • the culture was incubated at 37°C with shaking until the O.D. 600 reaches 0.5-1.0.
  • the bacteria were pelleted and resuspended in lOmM MgS0 4 to 0.5 O-D. ⁇ /ml.
  • a 0.6 ml aliquot of bacteria was mixed with ⁇ gtll-BL3 library phage stock containing 50,000 pfu and incubated at 37°C for 15 minutes.
  • Eight ml of top agar (0.7% agarose in NZCYM) was added to the mixture and plated onto a 150 mm NZCYM plate. Twenty such plates were prepared and were incubated at 42 °C for 3.5 to 4 hours until clear plaques grew up.
  • Dry nylon membranes pretreated with 10 mM IPTG were applied onto the plates and the plates were incubated at 37°C for 3.5 hours to transfer the plaques onto the membranes .
  • the membranes were removed from the plates and washed in TBST (20 mM Tris.Cl pH 7.5/150 mM NaCl/0.05% Tween 20) 4 times for 15 minutes per wash. They were further blocked in blocking solution (1% BSA in TBS (20 mM Tris.Cl pH 7.5/150 mM NaCl) ) for at least 1 hour to prevent nonspecific signals. After that, 10 -fold diluted SMI monoclonal antibody culture supernatant was added into blocking solution at 8 ml/membrane and incubated with agitation at room temperature for 3 hours.
  • the membranes were washed 5 times in TBST for 5 minutes per wash and incubated in fresh blocking solution containing secondary antibody conjugated with alkaline phosphatase (Rockland, anti-Rat IgM( ⁇ ) -AP, 1:10,000 dilution) at room temperature for 3 hours with gentle shaking. Finally, the membranes were washed in TBST and incubated in color development solution (1:50 dilution of NBT/BCIP stock solution from BMB with 0.1M Tris.Cl pH 9.5/50 mM MgCl 2 /0.1M NaCl) for 5-10 minutes in the dark, and the results were recorded. From about 10 million plaques screened, ten strongly positive clones were identified.
  • alkaline phosphatase Rockland, anti-Rat IgM( ⁇ ) -AP, 1:10,000 dilution
  • yeast glucoamylase precursor accesion #P08640
  • glycoprotein X precursor Ace #P28968
  • yeast alpha-agglutinin attachment subunit precursor Ace #P32323
  • spore coat protein sp96 Ace #1103869
  • Bovine herpesvirus gp80 Ace #z84818, e3004708
  • integumentary mucin c.l Q05049
  • microfilarial sheath protein Ace # 1163086, U43510
  • SMI protein was characterized by immunoprecipitation according to the following procedure. Twenty million BL3 cells were harvested and washed twice with P2 buffer (PBS plus 2% FCS) . The cell pellet was resuspended with 0.5 ml P2 buffer and incubated with 10 ⁇ g IgG for two hours at 4°C. The cells were washed twice with P2 and lysed with the same lysis buffer as described for western blot. The cell lysates were placed on ice for 30 minutes, spun and the supernatants transferred into the tubes containing 40 ⁇ l Protein A-agarose suspension (50% volume swollen agarose, BMB) . They were incubated for a further two hours at 4°C.
  • P2 buffer PBS plus 2% FCS
  • the present invention in one aspect relates to SMI protein, substantially purified from other proteins that has a molecular weight of about
  • the invention also includes peptide fragments of SMI. Such peptide fragments could represent research and diagnostic tools in the study of hematopoietic stem cell development.
  • compositions comprising isolated and purified peptide fragments of SMI may represent effective therapeutics against various diseases such as acquired immunodeficiency syndrome (AIDS) .
  • AIDS acquired immunodeficiency syndrome
  • a search in GeneBank using SMI DNA sequence indicates that it has weak sequence homology to one encoding a receptor molecule.
  • chemokine/cytokine receptor molecules have been implicated in the process of human immunodeficiency virus (HIV) infection, and HIV viral entry is thought to require more than one receptor molecule.
  • HIV human immunodeficiency virus
  • SMI cytokines or chemokines binding to their corresponding receptors.
  • SMI likewise may be a novel receptor, such that binding by its ligand would block HIV viral entry and, hence, render target cells resistant to HIV infection.
  • the invention relates not only to fragments of naturally- occurring SMI but also to SMI mutants and chemically synthesized derivatives of SMI.
  • changes in the amino acid sequence of SMI are contemplated in the present invention.
  • SMI can be altered by changing the D ⁇ A encoding the protein. Preferably, only conservative amino acid alterations are undertaken, using amino acids that have the same or similar properties.
  • Illustrative amino acid substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.
  • variants and fragments of SMI can be used in the present invention.
  • Variants include analogs, homologs, derivatives, muteins and mimetics of SMI.
  • Fragments of the SMI refer to portions of the amino acid sequence of SMI.
  • the variants and fragments can be generated directly from SMI itself by chemical modification, by proteolytic enzyme digestion, or by combinations thereof. Additionally, genetic engineering techniques, as well as methods of synthesizing polypeptides directly from amino acid residues, can be employed.
  • Non-peptide compounds that mimic the binding and function of SMI can be produced by the approach outlined in Saragovi et al . , Science 253: 792-95
  • Mimetics are molecules which mimic elements of protein secondary structure. See, for example, Johnson et al . , “Peptide Turn Mimetics,” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al . , Eds. (Chapman and Hall, New
  • peptide mimetics can be considered to be the equivalent of SMI itself.
  • Variants and fragments also can be created by recombinant techniques employing genomic or cDNA cloning methods. Site- specific and region-directed mutagenesis techniques can be employed. See CURRENT PROTOCOLS IN MOLECULAR BIOLOGY vol. 1, ch. 8 (Ausubel et al . eds . , J. Wiley & Sons 1989 & Supp.
  • BIOLOGY vols. 1 & 2 , supra. Protein sequencing, structure and modeling approaches for use with any of the above techniques are disclosed in PROTEIN ENGINEERING, loc . ci t . , and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 & 2 , supra .
  • CFU-S- forming cells are multipotent hematopoietic progenitors capable of reconstituting lethally- irradiated recipient mice short-term.
  • CFU-S spleen- focus assays were performed as described in Wong et al . (1994) , supra, using donor adult bone marrow cells.
  • Donor mice were inbred male C57BL/6J (Jackson Laboratory, Bar Harbor, ME) , and recipient mice were female of the same strain.
  • To prepare labeled bone marrow cells male mice were sacrificed by cervical dislocation, and bone marrow cells were as we described previously (Wong et al . (1994), supra) .
  • MNC Mononuclear cells
  • BM bone marrow
  • LSM lymphocyte- separation medium
  • FITC-conjugated antibody Abs
  • mice For recipient female mice, each of them was irradiated with a dose of 9.5 Gy prior to engraftment with SMI sorted cells. Irradiation was done by using a cesium source Mark 1 (model 30-1) irradiator (JL Shepherd
  • each dissected CFU-S focus was placed into an Eppendorf tube containing 0.5ml of PBS, and single cell suspension was prepared by repeated pipetting. The cells were washed once with PBS and lysed in DNA extraction buffer. This was followed by treatment with 100 ⁇ g/ml RNAse at 37°C for one hour, and 100 ⁇ g/ml proteinase K at 56°C for 3 hours. DNA then was extracted twice with phenol/chloroform and precipitated with 2M ammonium acetate and 2X volume of absolute ethanol .
  • DNA was dissolved thereafter in 0.4 ml of TE buffer and the concentration of DNA was determined.
  • the pY2 probe was used. This probe has been shown to be relatively specific for the Y chromosome in male cells.
  • RNA was then neutralized with 0.1 volume of 2M ammonium acetate pH 7.0, and blotted onto NYTRAN nylon filter. Four- fifth of a sample was used for hybridization with pY2 probe and one fifth with a GAPDH probe. Positive samples would then be used for southern blot analysis to confirm the presence of Y- specific band using the pY2 DNA fragment as probe.
  • For southern blot analysis typically 10 ⁇ g of DNA was digested with BamHl restriction enzyme, and the digested DNA was processed, transferred to nylon filter and hybridized with a random primer- labeled pY2 probe.
  • SM1 ⁇ cells in the mouse bone marrow was estimated to be about 1-5%.
  • lineage specific markers i.e., CD4 (T helper cells) , CD8 (T killer cells) , Gr-1 (granulocytes) , TER119 erythroid cells), Mac-1 (macrophages) and B220 (pre B cells) .
  • Lin “ cells (for lineage negative) were further divided into SM1 + and SMI " cells.
  • FACS analysis was performed on mouse bone marrow cells by using PE (polyerythrin) conjugated antibodies directed against all the lineage specific markers and FITC-conjugated SMI antibody.
  • Figure 4 indicates the result of such a two-color analysis.
  • SMI * /Lin ' - sorted cells were plated into semi-solid methylcellulose clonogenic culture familiar to one skilled in the art. The details of the assay are described by Han et al . , PNAS, 92:11014 (1995), which is incorporated herein by reference.
  • About 1,000 cells were plated into each dish under the conditions in which either pokeweed-mitogen stimulated spleen cell conditioned medium (SCM) or BL3 conditioned medium (BLCM) was present. Seven and twelve days later, the numbers and types of hematopoietic colonies were recorded.
  • Compact colonies are those tight -appearing aggregates of undifferentiated cells with an estimated average size of 50-200 cells. Characterization of human SMI DNA
  • a ⁇ gtll cDNA library was constructed using mRNA of HL60 cell line, which is a human myelomonocytic leukemic cell line and which expresses three mRNA specific to the mouse SMI DNA. Construction of HL60 cDNA library is similar to what was done on the construction of BL3 ⁇ gtll cDNA library, as already described. A 1.5kb EcoRI mouse SMI fragment was used to screen the HL60 cDNA library. Several positive clones were obtained. DNA of two clones were sequenced and one region with the sequence as shown in Figure 5 was found to be common to both DNA samples. A search in the EST library of GenBank indicates that this sequence is homologous to a homo sapiens cDNA (for example, accession number H98251) ; no known function for this cDNA has ever been reported. Expression of human SMI gene
  • mRNA in human cells hybridized positively with the mouse SMI probe. Among these species, only one may be responsible for cell surface expression of the human SMI protein.
  • the three species of mRNA may be related by way of differential splicing, accounting for the fact that common RNA sequence is shared among these species of mRNA.
  • these species of mRNA may represent the product of three distinct genes that are members of a single gene family.
  • HL60 cells expressing all three species of SMI mRNA also express the SMI on their cell surface; whereas K562 cells expressing only the 4kb species did not express the SMI protein on their surface. SMI protein also is detected weakly in another cell line J45.
  • Lysates of various cell lines were immunoprecipitated with SMI antibody and the immunoprecipitates were resolved on SDS-PAGE.
  • BL3 cell lysate was used as a positive control.
  • Five million cells per sample were labeled with 35 S -methionine (0.25 mCi) for 1 hour and then immunoprecipitated for 2 hours at 4°C with 20 ⁇ g SMI antibody.
  • the cells were then washed with PBS and lysed in 0.5 ml of IP buffer (130mM NaCl, lOmM Tris.Cl pH 7.5, 5mM EDTA, 1% Triton X-100 and protease inhibitors) .
  • IP buffer 130mM NaCl, lOmM Tris.Cl pH 7.5, 5mM EDTA, 1% Triton X-100 and protease inhibitors
  • SMI monoclonal antibody also can recognize human hematopoietic cells ( Figure 6) .
  • FACS analysis was therefore carried out to examine the proportion of cells that express SMI molecule on their cell surface. To do that 1 million mononuclear cells from human cord blood were first stained with a mixture of antibodies, which contain rat-anti-CD38 , rat-anti-glycophorin A and/or anti-CD33 and anti-HLA-DR, together with PE conjugated anti -rat antibodies. These antibodies detect lineage specific antigens and the cells bearing these antigens are called Lin + cells.
  • Reanalyzing and re-plotting area A shows that SM1 + Lin ' cells constitute about 0.3% of the whole cord blood mononuclear blood sample (shown as 0.4% in compartment 4).
  • SMI antibody alone 1% of human cord blood mononuclear cells are found to carry the SMI antigen.
  • Hematopoietic stem cells have been found to be present in human cord blood at a very high frequency.
  • CD34 antigen which has also been used for hematopoietic stem cell enrichment, Hayloek et al . , Blood 80:1405 (1992) , has been shown to occur on about 2% of cord blood cells, Broxmeyer et al . , PNAS 86:3828 (1989), 2% of bone marrow cells and 0.2% of peripheral blood cells. Bender et al . , Blood 77 :2591-2596 (1991) .
  • SM1 + /Lin " - enriched cord blood cells constituting 0.3% of the total cord blood mononuclear cell population, were examined by the clonogenic assay.
  • One thousand sorted cells were plated in methylcellulose culture in the presence or absence of conditioned medium from 5367 cells derived from a patient with a bladder carcinoma; the conditioned medium (CM) is known to contain various hematopoietic growth factors capable of stimulating primitive hematopoietic stem/progenitor cell growth. Broxmeyer et al . , supra . After 10 days of incubation in the presence 10% 5367CM, blast colonies containing cells dispersed diffusely could be observed (Table 2) . In the absence of 5367CM, no colonies were observed. These data indicate that SMI + /Lin ' -enriched cell population contains primitive hematopoietic stem/progenitor cells.
  • anti -SMI antibody is used to prepare a composition enriched for hematopoietic stem cells .
  • This is achieved by providing antibody that binds SMI, immobilizing anti -SMI antibody on a support platform such that the antibody retains its SMI-binding capability, then bringing a mixed population of cells into contact with the antibody, where the mixed population contains hematopoietic stem cells, such that the stem cells adhere to the support platform, and removing nonadherent cells, so that a population enriched for hematopoietic stem cells remains adhered to the support platform.
  • support platform is meant any solid support such as beads, hollow fiber membranes, resins, plastic petri dishes, or an antibody against the anti-SMI antibody.
  • the antibodies may be conjugated with markers such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the remaining cells.
  • the invention relates to a kit for detecting a hematopoietic factor that binds to SMI.
  • hematopoietic factor is meant any protein associated with hematopoiesis.
  • This kit comprises the antibody of the present invention, and also can comprise a detectable label and a set of written instructions for using such a kit.
  • a kit may comprise a receptacle being compartmentalized to receive one or more containers such as vials, tubes and the like, such containers holding separate elements of the invention.
  • SMI is used in a method of detecting in a sample a hematopoietic factor that binds SMI . Such methods may be used to detect and evaluate factors associated with the regeneration, differentiation, and maturation of hematopoietic cells.
  • 3M1, and SM1 + cells may be used in assays to determine the activity of media, such as conditioned media, and to evaluate fluids for cell growth activity, involvement with dedication of particular lineages, or the like.
  • This in vi tro assay involves contacting a sample suspected of containing a hematopoietic factor that binds SMI with detectably labeled-SMI. The hematopoietic factor is then detected.
  • sample is meant any cell culture medium or any body fluid or tissue, including blood, urine, saliva, spinal fluid, semen, peritoneal fluid, and tissue from any part of the body.
  • Such assays may involve binding SMI to a solid surface. Many methods for immobilizing biomolecules on solid surfaces are known in the art.
  • the solid surface may be a membrane ( e . g. , nitrocellulose), a microtiter dish or a bead.
  • the bound molecule may be covalently or noncovalently attached through unspecific bonding.
  • the manner of linking a wide variety of compounds to various surfaces is well-known and well-documented in the literature. See, for example, Chibata, Immunological Enzymes, Halsted Press (1978), and Cuatvecasos, J " . Biol . Chem. 245:3059 (1970), the respective contents of which are incorporated herein by reference.
  • SMI is labeled by methods well-known in the art.
  • a common method involves the use of radioisotopes such as 3 H, 125 I, 35 S, 14 C or 32 P. Detection is accomplished by autoradiography.
  • Non- radioactive labels include the covalent binding of biotin to the compound of the present invention. Biotin is then bound to an anti-ligand such as streptavidin, which is either inherently labeled or bound to a signal system, such as a detectable enzyme, a fluorescent or chemiluminescent compound.
  • SMI SMI cells
  • SMI SMI cells
  • Such mechanisms may, for example, involve any molecule or factor, hematopoietic or not, that is associated with or interferes in SMI mediated signal- ransduction.
  • Hematopoietic cells purified according to the present invention can also be used in a method of gene therapy.
  • Such methods may comprise gene constructs, which include those mediated by viruses (e.g., retrovirus, adenovirus , adeno-associated virus, Epstein-Barr virus, hepatitis virus, lentivirus) , and non-virally mediated methods such as gene transfer into the purified cells.
  • viruses e.g., retrovirus, adenovirus , adeno-associated virus, Epstein-Barr virus, hepatitis virus, lentivirus
  • non-virally mediated methods such as gene transfer into the purified cells.
  • retrovirally-mediated gene transfer are known in art Bodine et al . , PNAS, 86:8897-8901 (1989), but heretofore it has not been possible to use such homogenous population of cells having SMI as the cells transfected. Such transfected cells can then be used for therapeutic applications.
  • diseases such as B- thalassemia, sickle cell anemia, adenosine deaminase deficiency, etc.
  • diseases such as B- thalassemia, sickle cell anemia, adenosine deaminase deficiency, etc.
  • Other indications of gene therapy include introduction of viral or bacterial resistance genes, antisense sequence or ribozyme to prevent the proliferation of the pathogen in the SMI hematopoietic cells.
  • diseases associated with an overproduction of a particular secreted product such as hormone, enzyme, or the like, the SMI hematopoietic cells may also be inserted with a ribozyme, antisense, or other inhibiting factor to inhibit the particular disease.

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Abstract

L'invention porte sur une protéine dite 'SM1' d'un poids moléculaire de 230 kDa mesuré par immunoprécipitation et SDS-PAGE. Les protéines SM1 sont présentes à la surface des cellules hématopoïétiques souches de l'homme et de la souris, et sur les cellules ascendantes primitives, mais absentes de la surface des autres cellules telles que les cellules ascendantes FDC-P1 du myloïde, les cellules T EL4, les cellules myélomonocytiques WEHI-3 et des cellules lymphoïdes pré-B, ou l es cellules hématopoïétiques prédifférenciées du sang de la moelle épinière humaine ou de la moelle de souris. Les anticorps anti SM1 peuvent servir à élaborer une préparation enrichie en cellules hématopoïétiques souches.
EP98920093A 1997-05-06 1998-05-01 Proteines sm1 presentes a la surface des cellules hematopoietiques souches Withdrawn EP0981546A4 (fr)

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