EP1017790A1 - Procedes et compositions utilises pour lier des cellules souches hematopoietiques - Google Patents

Procedes et compositions utilises pour lier des cellules souches hematopoietiques

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Publication number
EP1017790A1
EP1017790A1 EP98948540A EP98948540A EP1017790A1 EP 1017790 A1 EP1017790 A1 EP 1017790A1 EP 98948540 A EP98948540 A EP 98948540A EP 98948540 A EP98948540 A EP 98948540A EP 1017790 A1 EP1017790 A1 EP 1017790A1
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EP
European Patent Office
Prior art keywords
binding partner
stem cells
lectin
cells
binding
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EP98948540A
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German (de)
English (en)
Inventor
John L. Magnani
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GlycoTech Corp
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GlycoTech Corp
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Publication of EP1017790A1 publication Critical patent/EP1017790A1/fr
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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

Definitions

  • the present invention relates generally to methods and compositions for binding hematopoietic stem cells.
  • the invention is more particularly directed to methods for immobilizing, punfymg or labeling stem cells.
  • the invention is also directed to methods for targeting agents, such as therapeutic agents, to stem cells.
  • Gene therapy represents a promising approach to the prevention and treatment of a va ⁇ ety of diseases.
  • the application of genetic therapies has been limited by practical problems, such as the inability to target an agent specifically to a desired cell type or the limited hfespan of a genetically altered cell.
  • multipotent hematopoietic progenitor cells as vehicles for gene therapy could potentially overcome such difficulties.
  • stem cells which are characte ⁇ zed by a cell surface marker known as CD34, could be genetically modified in vitro, and then transplanted into a patient using routine techniques. Following transplantation, the transgenic cells can home directly to bone narrow and, if required, fully repopulate and eventually reconstitute all of the blood cells of the host. In this fashion, the desired gene product may be produced in a constitutively regenerating population of cells in vivo.
  • stem cells have been co-cultured with microvascular endothehal feeder cells (p ⁇ mary cells from the brain of non-human sources) and certain cytokines Under such conditions, an expanded population of stem cells has been shown to remain CD34 + and CD38 " while interacting with feeder cells (see Davis et al., Blood 55:1751- 61, 1995). Adhesion to these endothehal cells appears to be a c ⁇ tical requirement for maintaining the plu ⁇ potent CD34 phenotype.
  • this invention provides methods and compositions for binding hematopoietic stem cells
  • the present invention provides methods for immobilizing hematopoietic stem cells, comp ⁇ sing contacting a biological sample containing hematopoietic stem cells with a binding partner, wherein the binding partner binds to a sialylated carbohydrate chain comp ⁇ sing the structure NeuAc 2- 3Gal ⁇ l-4 Withm specific embodiments, the binding partner may be attached to a solid support before or after complex formation. Suitable binding partners include antibodies and antigen-binding fragments thereof and lectins.
  • the sialylated carbohydrate chain comp ⁇ ses the structure NeuAc ⁇ 2- 3(Gal ⁇ l-4GlcNAc ⁇ l-3) n , wherein n is an integer ranging from 1 to 100, and more preferably from 2 to 50.
  • methods for pu ⁇ fying hematopoietic stem cells comp ⁇ se: (a) contacting a biological sample containing hematopoietic stem cells with a binding partner that binds to a sialylated carbohydrate chain comp ⁇ sing the structure NeuAc ⁇ 2-3Gal ⁇ l-4 to form a stem cell-bindmg partner complex; and (b) separating the stem cell-binding partner complex from unbound biological sample.
  • the binding partner may be attached to a solid support before or after complex formation.
  • Suitable binding partners include antibodies and antigen-binding fragments thereof and lectins.
  • the sialylated carbohydrate comprises the structure NeuAc ⁇ 2-3(Gal ⁇ l- 4GlcNAc ⁇ l-3) n , wherein n is an integer ranging from 1 to 100, and more preferably from 2 to 50.
  • the present invention provides methods for targeting an agent to a hematopoietic stem cell, comprising contacting a biological sample containing hematopoietic stem cells with a binding partner that binds to a sialylated carbohydrate chain comprising the structure NeuAc ⁇ 2-3Gal ⁇ l-4, wherein the binding partner is associated with an agent.
  • Suitable binding partners include antibodies and antigen-binding fragments thereof and lectins.
  • the sialylated carbohydrate chain comprises the structure NeuAc 2- 3(Gal ⁇ l-4GlcNAc ⁇ l-3) n , wherein n is an integer ranging from 1 to 100, and more preferably from 2 to 50.
  • Suitable agents include toxic compounds, therapeutic compounds and detectable markers.
  • kits for use in the above methods comprises: (a) a binding partner for stem cells, wherein the binding partner binds to a sialylated carbohydrate chain comprising the structure: NeuAc ⁇ 2-3Gal ⁇ l-4; and (b) a wash solution, wherein the binding partner and wash solution are provided in separate compartments or containers.
  • the binding partner may, but need not, be attached to a support such as a tissue culture dish.
  • a kit for use in separation and/or purification of stem cells comprising: (a) a binding partner for stem cells, wherein the binding partner binds to a sialylated carbohydrate chain comprising the structure: NeuAc 2-3Gal ⁇ l-4; and (b) a solid support.
  • kits for use in labeling or sorting hematopoietic stem cells comprising (a) a binding partner for stem cells associated with a detectable marker, wherein the binding partner binds to a sialylated carbohydrate chain comprising the structure: NeuAc ⁇ 2-3Gal ⁇ l-4; and (b) a detection reagent, wherein the binding partner and detection reagent are provided in separate compartments or containers.
  • the present invention provides pharmaceutical compositions, comp ⁇ sing: (a) an agent associated with a binding partner for stem cells, wherein the binding partner binds to a sialylated carbohydrate chain comp ⁇ sing the structure: NeuAc ⁇ 2-3Gal ⁇ l-4; and (b) a pharmaceutically acceptable earner.
  • compositions of the present invention are also provided for use as a medicament, and for use for the manufacture of a medicament for the treatment of a disease, e.g., by gene therapy.
  • Figure 1 is a graph illustrating the binding of monoclonal antibody NUH2 to neoglycoprotems within an ELISA
  • BSA bovine serum albumin
  • APEA 2-(4-am ⁇ nophenyl)ethylam ⁇ ne
  • APEA aminophenylene diamine
  • the fourth is M6 linked directly to BSA.
  • the results are presented as OD 5 o as a function of neoglycoprotein concentration.
  • Figure 2 is a histogram illustrating the immobilization of hematopoietic stem cells using representative binding partners. The results are presented as fluorescence units for each binding partner, as indicated.
  • Figure 3 is a histogram illustrating the immobilization of hematopoietic stem cells using representative binding partners The results are presented as fluorescence units for each binding partner, as indicated.
  • Figure 4 presents the results of two-color FACS analysis compa ⁇ ng the binding of the representative binding partner NUH2 to surface markers present on human cord blood cells to the binding observed for the anti-CD34 antibody HPCA2-PE.
  • the data are presented as contour plots which represent the bivariate distribution of green and red fluorescence. Contour lines known as isopleths are drawn to express a cell count at a particular set number.
  • Figure 5 presents the results of two-color FACS analysis comparing the binding of the control IgM antibody to surface markers present on human cord blood cells to the binding observed for the anti-CD34 antibody HPCA2-PE.
  • the data are presented as contour plots which represent the bivariate distribution of green and red fluorescence. Contour lines known as isopleths are drawn to express a cell count at a particular set number.
  • the present invention is directed to methods and compositions for binding hematopoietic stem cells.
  • the methods described herein employ a binding partner that binds to a sialylated lactosamine structure on the surface of such stem cells to form a binding partner-stem cell complex.
  • the formation of such complexes facilitates a variety of manipulations, such as, for example, immobilization, purification, identification and targeting of hematopoietic stem cells.
  • Compositions according to the present invention generally comprise a binding partner, which may be free, attached to a support material or linked to a label or therapeutic agent, depending on the intended use.
  • binding refers to a noncovalent association between two separate molecules (each of which may be free (i.e., in solution) or present on the surface of a cell or a solid support), such that a "complex" is formed.
  • Such a complex may be free or immobilized (either covalently or noncovalently) on a support material.
  • the ability of one molecule to bind to another molecule may generally be evaluated by determining a binding constant for the formation of the complex.
  • the binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations.
  • two compounds are said to "bind” in the context of the present invention when the binding constant for complex formation exceeds about 10 2 L/mol.
  • a binding constant may be determined using methods well known to those of ordinary skill in the art.
  • the binding constant for the formation of a complex between a relatively small carbohydrate and a macromolecule binding partner may be determined using equilibrium dialysis. Briefly, two chambers of known volume are separated by a dialysis membrane that allows transfer of the small molecular weight carbohydrate, but not the macromolecule binding partner (e.g., an antibody).
  • the carbohydrate is labeled with a reporter group, such as tritium, and is added to the solution in chamber 1, while the binding partner is placed in the solution in chamber 2.
  • the carbohydrate molecules are then allowed to diffuse into chamber 2 until transfer of the carbohydrate reaches equilibrium (generally about 1 to 3 days).
  • the binding constant may then be determined by measuring the amount of carbohydrate in each chamber (which may be readily determined using the reporter group) and using equation I:
  • [C] is the concentration of uncomplexed carbohydrate
  • [B] is the concentration of uncomplexed binding partner
  • [CB] is the concentration of complex. Since [C] is the concentration of carbohydrate in chamber 1 (i.e., [CJi ); [B] is difference between the original concentration of binding partner (i.e., [B 0 ]) and [CB]; and [CB] is the difference between the concentration of carbohydrate in chamber 2 and the concentration of carbohydrate in chamber 1 (i.e., [C 2 ]-[C.]), equation I may be rewritten in measurable terms as equation II:
  • sialylated lactosamine structures are expressed selectively on hematopoietic stem cells.
  • sialylated lactosamine structures are present on the surface of a hematopoietic stem cell at higher concentrations, or in a conformation that favors binding to a binding partner, relative to T cells.
  • a "binding partner,” in the context of this invention, is any agent, such as a compound or a cell, that binds to a sialylated carbohydrate chain comp ⁇ sing the structure NeuAc ⁇ 2-3Gal ⁇ l-4R and/or to a sialylated linear or branched lactosamine comp ⁇ sing the structure NeuAc ⁇ 2-3(Gal ⁇ l- 4GlcNAc ⁇ l-3) n -R, wherein n is an integer ranging from 1 to 100, preferably from 1 to 50, and more preferably from 2 to 50, and wherein R represents the remainder of the molecule, if any
  • R may be a saccha ⁇ de or non-saccha ⁇ de moiety, such as a protein or lipid.
  • Such moieties may be included, for example, to aid in pu ⁇ fication, detection or immobilization
  • R is a protein
  • any agent e.g., a cell or a molecule
  • a binding partner comp ⁇ ses an antibody, or a binding fragment thereof, raised against a suitable immunogen.
  • suitable immunogens include ganghosides adsorbed and coated on acid- treated Salmonella minnesotae, human fetal erythrocytes from cord blood or human colonic adenocarcmoma cells.
  • Antibodies may be prepared by any of a va ⁇ ety of techniques known to those of ordinary skill in the art See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Sp ⁇ ng Harbor Laboratory, 1988
  • an immunogen is initially injected into any of a wide va ⁇ ety of mammals (e.g , mice, rats, rabbits, sheep or goats). Following one or more injections, the animals are bled pe ⁇ odically.
  • Polyclonal antibodies specific for the immunogen may then be pu ⁇ fied from such antisera by, for example, affinity chromatography using the immunogen coupled to a suitable solid support.
  • Monoclonal antibodies specific for the carbohydrate structure of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J Immunol. 6:511-519, 1976, and improvements thereto B ⁇ efly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity from spleen cells obtained from an animal immunized as desc ⁇ bed above.
  • the spleen cells are immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal.
  • Single colonies are selected and their culture supematants tested for binding activity against a sialylated carbohydrate as desc ⁇ bed herein.
  • Hyb ⁇ domas secreting antibodies having high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supematants of growing hyb ⁇ doma colonies, with or without the use of va ⁇ ous techniques known in the art to enhance the yield. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • Antibodies having the desired activity may generally be identified based on their ability to bind to one or more approp ⁇ ate neoglycoproteins in an lmmunoassay. Suitable neoglycoproteins include those containing one of the following structures:
  • glycoproteins are conjugates of carbohydrate structures to human or bovine serum albumin (HSA or BSA, respectively) Since albumin is non-glycosylated, the resulting neoglycoprotein contains only the carbohydrate structures chemically linked to the molecule.
  • the first neoglycoprotein recited above is commercially available from several sources and both molecules can be synthesized by those skilled in the art.
  • the carbohydrate is linked by the spacer acetylphenylenediamine (APD) by reductive amination of the reducing end of the carbohydrate chain. In these cases, the glucose is reduced, the ⁇ ng opens and creates an ammoalditol The glucose unit is therefore displayed in parenthesis.
  • An antibody may be further characte ⁇ zed by subjecting human blood from the umbilical cord to two color fluorescence activated cell sorter analysis (FACS analysis).
  • FACS analysis Two color fluorescence activated cell sorter analysis
  • One antibody may directed against the hematopoietic stem cell marker CD34.
  • Such an antibody is commercially available labeled with the red fluorescent marker, phycoeryth ⁇ n from Becton Dickinson, San Jose, CA (HPCA-2PE).
  • the test antibody may be labeled with a green fluorescent marker (fluorescein) either directly (using, for example, marker obtained from Molecular Probes, Inc., Eugene, OR) or indirectly (e.g., using fluorescem-labeled goat anti-mouse IgM, available from KPL, Gaithersburg, MD). Antibodies that result in the labeling of all red fluorescent cells with green fluorescence are preferred.
  • antibody NUH2 One suitable binding partner is the antibody NUH2 (see U.S. Patent No. 5,227,160) This antibody is an IgM and was developed from a hyb ⁇ doma produced from mice immunized with disialoganghosides from human colonic adenocarcmoma cells coated on acid-treated, Salmonella minnesotae (see Nudelman, J. Biol. Chem. 264: 18119-25, 1989).
  • Antibody NUH2 has been found to bind the two neoglycoproteins recited above and also to branched disialogangliosides containing sialylated lactosamine structures. In two color FACS analysis, antibody NUH2 also labels all CD34 + cells identified by the commercially-available antibody HPCA-2-PE.
  • the binding partner comprises a lectin, or a derivative such as an altered or truncated form that retains binding properties, and combinations of lectins and/or derivatives.
  • a lectin or a derivative such as an altered or truncated form that retains binding properties, and combinations of lectins and/or derivatives.
  • One suitable truncated form is a complementarity-determining region (CDR) domain (see Saragovic et al., Science 253:792-95, 1991).
  • Suitable lectin binding partners include, but are not limited to, plant lectins such as Tomato lectin, Maackia amurensis lectin, Sambucus nigra lectin, Triticum vulgaris lectin and Erytheina cristagalli lectin; mammalian lectins such as sialoadhesins (see Kelm et al., Glycoconjugate J. 75:913-26, 1996) and galectins; and bacterial lectins such as the carbohydrate receptors of Helicobacter pylori (see Teneberg et al., J. Biol. Chem. 272:19067-71, 1997).
  • plant lectins such as Tomato lectin, Maackia amurensis lectin, Sambucus nigra lectin, Triticum vulgaris lectin and Erytheina cristagalli lectin
  • mammalian lectins such as si
  • Binding partners may generally be identified by evaluating the ability of a putative binding partner to bind a test sialylated carbohydrate chain comprising the NeuAc ⁇ 2-3Gal ⁇ l-4R structure, as described above. Any of a number of well known binding assays may be employed for screening putative binding partners, including immunoassays. Binding constants for the interaction between binding partner and test carbohydrate may be measured as described above.
  • Test sialylated carbohydrate chains may generally be prepared from commercially available materials, using methods that will be apparent to those of ordinary skill in the art.
  • lactosamine chains of varying lengths may generally be prepared according to the method of Srivastava et al., J. Carbohydrate Chem. 10:921-933 (1991).
  • Such chains may be sialylated using sialyltransferases or transsialidase enzymes.
  • Sialyltransferases enzymatically transfer sialic acid (NeuAc) from the sugar nucleotide CMP-NeuAc to the acceptor carbohydrate chain.
  • Transialidases transfer sialic acid between sialylated and non-sialylated carbohydrate chains.
  • a biological sample refers to any tissue sample or preparation of cells that contains hematopoietic stem cells, including blood, bone marrow, buffy coat cells, cord blood and cells that are grown and/or expanded in vitro. Such preparations may, but need not, be treated and/or fractionated prior to use within the methods of the present invention.
  • a biological sample may be a blood preparation, with or without cytokine treatment.
  • a binding partner can be used to immobilize stem cells.
  • immobilization is used herein to refer to a noncovalent interaction between a stem cell and a binding partner, resulting in the formation of a stem cell-binding partner complex, wherein the binding partner or the complex is attached to a solid support.
  • the binding partner binds to the stem cell prior to immobilization of the complex.
  • the binding partner is attached to the solid support before contact with the stem cells.
  • attachment or “attachment,” as used herein, refers to a covalent or noncovalent interaction between a binding partner or stem cell-binding partner complex and a solid support, such that the binding constant of the interaction is at least 10 3 L/mol.
  • Attachment may generally be achieved through noncovalent association, such as adsorption or affinity, or via covalent attachment.
  • Attachment of a binding partner or complex by adsorption may be achieved by contact, in a suitable buffer, with a solid support for a suitable amount of time. The contact time varies with temperature, but is generally between about 5 seconds and 1 day, and typically between about 10 seconds and 2 hours.
  • Attachment by affinity is generally achieved using a support that contains a compound that binds to the binding partner and/or complex (e.g., a support to which avidin is attached may be used to attach a binding partner associated with biotin). Such a compound may itself be attached by adsorption or covalently.
  • Covalent attachment of a compound to a solid support may be a direct linkage between a binding partner or complex and functional groups on the support, or may be a linkage by way of a cross-linking agent. Attachment using a cross-linking agent may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or ammo group, on the compound
  • a binding partner may be bound to a support having an approp ⁇ ate polymer coating using benzoquinone, by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e g , Pierce Immunotechnology Catalog and Handbook (1991) at A12-A13) or by condensation of an amino group on the support with a carboxyhc acid on the binding partner
  • an agent may be associated with a binding partner such that the agent can be targeted to hematopoietic stem cells
  • An “agent” may be a compound or a cell, and may be a therapeutic compound, a cytotoxic compound or a label or detectable marker suitable for labeling hematopoietic stem cells (e g , for cell sorting or in vivo imaging)
  • Such association may be noncovalent (such as by way of hydrophobic interactions or incorporation into a hposome), but is preferably covalent and may be achieved by standard recombinant or chemical means "Targeting," withm the present invention, refers to specifically directing an agent to hematopoietic stem cells in vitro or in vivo, such that the agent is delivered to hematopoietic stem cells more efficiently than to other cell types
  • a binding agent associated with a detectable marker may be used to identify hematopoietic stem cells within a mixture of cells using standard techniques (
  • methods are provided for immobilizing hematopoietic stem cells
  • a biological sample containing such stem cells is initially contacted with a binding partner
  • the binding partner may be attached to a solid support, or such attachment can be achieved after complex formation
  • Preferred solid supports for expansion and gene transduction include tissue culture dishes, hollow fiber bioreactors and bags, such as blood- and cell-collection bags For pu ⁇ fication purposes, chromatography mat ⁇ ces are preferred supports
  • an excess of binding partner over the number of stem cells is employed, to facilitate the formation of stem cell-binding partner complexes
  • Suitable conditions for contacting the binding partner with the biological sample are generally conditions that favor complex formation and maintain cell viability Such conditions may be readily determined by one of ordinary skill in the art by evaluating the binding constant for complex formation at a senes of different conditions Suitable conditions include physiological conditions, such as pH 7 4 lsotomc saline (e g , 0 15M NaCl) at room temperature It will be evident to those of ordinary skill in the art that the amount of binding partner necessary to achieve adequate complexation of the stem cells in a given biological sample will depend upon the concentration of stem cells within the sample, the binding constant for the given binding partner and the other mate ⁇ als present in the biological sample In general a concentration of binding partner ranging from about 0 1 ⁇ g/mL to 10 mg/mL, and typically from about 1 ⁇ g/mL to 1 mg/mL, is sufficient After complex formation, the stem cell-binding partner complex may be attached to a solid support if necessary The remainder of the biological sample may then be removed from the immobilized stem cells by any of
  • Hematopoietic stem cells immobilized as descnbed herein may generally be used for a va ⁇ ety of in vitro manipulations, including expansion and gene transduction
  • the present immobilization procedure generally has the advantage of permitting expansion of stem cells in the dedifferentiated state.
  • the present invention provides methods for pu ⁇ fying hematopoietic stem cells
  • "Punfication” refers to a separation of the stem cells from at least a portion of the components of the biological sample, and pu ⁇ fied stem cells may be present withm a stem cell-binding partner complex
  • a biological sample is contacted with a binding partner as descnbed above If the binding partner was not attached to a support p ⁇ or to contact with the biological sample, the complex is attached after such contact and separated from the unbound portion of the biological sample Cells may then be released from the binding partner using any suitable technique Preferably, release is achieved by incubation with high concentrations of carbohydrate hapten to compete for binding to the binding partner.
  • cells may be eluted by incubation under conditions that dimmish or eliminate the activity of the binding partner (e.g., a change in pH and/or cation concentration) Under appropnate circumstances, cells may be eluted from immobilized binding partners by mechanical agitation.
  • further pu ⁇ fication steps may be performed using other affinity mate ⁇ als.
  • a binding partner as descnbed herein may be used to target an agent to hematopoietic stem cells in vitro or in vivo.
  • any of a va ⁇ ety of agents associated with a binding partner may be directed specifically to hematopoietic stem cells.
  • an agent may be a detectable marker suitable for cell identification and sorting in vitro or imaging m vivo. Such markers are well known in the art and include radionuchdes, luminescent groups, fluorescent groups, enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, constant immunoglobulin domains and biotm.
  • an agent may be a therapeutic or cytotoxic compound.
  • Other agents include polynucleotides encoding a detectable marker, therapeutic agent or cytotoxic compound.
  • a biological sample is contacted, as desc ⁇ bed above, with the agent associated with the binding partner. Unbound binding partner is then removed by standard techniques, such as filtration, centnfugation, precipitation, elution or other appropnate means. For detectable markers, the agent may then be detected by any appropnate method known to those of ordinary skill in the art, the selection of which will depend in part upon the agent used. For example, a fluorescent agent, such as fluoresce , associated with a binding partner may be used for FACS analyses, using well known techniques. For in vivo use, an agent associated with a binding partner is typically administered to a subject in the form of a pharmaceutical composition.
  • a pharmaceutical composition may be a ste ⁇ le aqueous or non-aqueous suspension or emulsion, which additionally compnses a pharmaceutically acceptable earner (i.e., a non-toxic matenal that does not interfere with the activity of the active ingredient).
  • a pharmaceutically acceptable earner i.e., a non-toxic matenal that does not interfere with the activity of the active ingredient.
  • Any suitable earner known to those of ordinary skill in the art may be employed m the pharmaceutical compositions of the present invention.
  • Representative earners include physiological saline solutions, gelatin, water, alcohols, natural or synthetic oils, saccharide solutions, glycols, injectable organic esters such as ethyl oleate or a combination of such materials.
  • a pharmaceutical composition may additionally contain preservatives and/or other additives such as, for example, antimicrobial agents, anti-oxidants, chelating agents and/or inert gases, and/or other active ingredients.
  • preservatives and/or other additives such as, for example, antimicrobial agents, anti-oxidants, chelating agents and/or inert gases, and/or other active ingredients.
  • the binding partner binds to the hematopoietic stem cells and can be detected using conventional imaging techniques, such as x-ray technologies.
  • kits for use in any of the methods described herein are provided. Kits for use in immobilizing hematopoietic stem cells generally contain a binding partner, which may be attached to a tissue culture dish.
  • kits may contain a binding partner in combination with a filter or chromatographic support.
  • Kits for use in labeling, cell sorting or in vivo imaging typically contain a binding partner associated with a detectable marker.
  • a kit may contain binding partner associated with a therapeutic agent.
  • one or more additional compartments or containers of a kit generally enclose elements, such as reagents, buffers and/or wash solutions, to be used in the method. The following Examples are offered by way of illustration and not by way of limitation.
  • This Example demonstrates use of antibody specific for CD34+ cells to identify sialylated lactosamine structures on the surface of hematopoietic stem cells.
  • Monoclonal antibody NUH2 (ATCC Accession Number HB 9762) was employed within enzyme-linked immunoassays (ELISAs) for binding to carbohydrate structures.
  • Monoclonal antibody NUH2 is an IgM isotype that was developed by immunizing mice with disialogangliosides isolated from colon adenocarcinoma and adsorbed on acid-treated Salmonella minnesotae (see U.S. Patent No. 5,227,160).
  • neoglycoprotein library To generate a neoglycoprotein library, purified oligosaccharides were chemically coupled to human serum albumin. Each neoglycoprotein molecule contained about 10 to 15 chains of one specific purified oligosaccharide.
  • the neoglycoprotein library was coated onto the surface of the wells in a microtiter plate by incubating the wells filled (lOO ⁇ l) with neoglycoproteins diluted in phosphate- buffered saline, pH 7.4 (PBS) overnight at 4°C. Wells were then blocked by adding 2% bovine serum albumin in PBS and incubating for 2 hours at room temperature.
  • This Example illustrates the use of an antibody binding partner to immobilize hematopoietic stem cells.
  • Immobilization of CD34+ cells to surfaces coated with antibodies and lectins was tested by a static cell adhesion assay.
  • Antibodies and lectins diluted in a buffer containing lOmM Tris, 0.15M NaCl, lmM CaCl 2 at pH 7.4 were added to wells of a 96 well microtiter plate and incubated overnight at 4°C.
  • Bovine serum albumin (2% in TrisCa buffer) was added to each well and incubated at room temperature for at least 2 hours in order to reduce nonspecific binding.
  • CD34+ cells (10 6 cells/ml), purchased from Poetic Technologies Inc.
  • calcein AN Molecular Probes, Eugene OR
  • DPBS Dulbecco's PBS
  • 100 ⁇ l of cells were added to each well.
  • the plate was incubated in a stationary position for 25 minutes at room temperature. After incubation, the plate was inverted in a wash chamber (GlycoTech Corp., Rockville MD), for exactly 6 minutes.
  • Example 3 IDENTIFICATION OF HEMATOPOIETIC STEM CELLS USING AN ANTI-CARBOHYDRATE ANTIBODY
  • FACS fluorescence activated cell sorting
  • the cells were washed and incubated with FTTC-labeled goat anti-mouse IgM and FTTC-labeled streptavidin (l ⁇ g/ml) respectively (KPL Labs, Gaithersburg, MD). After incubation at 4°C for 1 hour, the cells were washed twice with DPBS + azide and the commercial PE-labeled (red fluorescence) anti-CD34 antibody, HPCA2-PE (Becton Dickinson, San Jose, CA) was added to each tube.
  • FTTC-labeled goat anti-mouse IgM and FTTC-labeled streptavidin l ⁇ g/ml
  • FIGS. 4 and 5 display data obtained from light scattered by lymphocytes in human blood from umbilical cords.
  • the data are presented as contour plots which represent the bivariate distribution of green and red fluorescence. Contour lines known as isopleths are drawn to express a cell count at a particular set number.
  • the X-axis is a logarithmic scale of green fluorescence labeled on cell surfaces, whereas the Y-axis is a logarithmic scale of red fluorescence labeled on cell surfaces.
  • Quadrant 2 contains data that statistically demonstrate the presence of cells that express both surface markers, each detected by either the red or green fluorescent labeled antibody.
  • This Example illustrates the use of antibodies to detect cell surface structures on a vanety of cultured cell lines by immunofluorescent staining followed by fluorescence activated cell sortings (FACS).
  • FACS fluorescence activated cell sortings
  • the expression of markers on the surface of cultured cell lines was analyzed by indirect immunofluorescent staining of the cell lines by specific monoclonal antibodies followed by fluorescence activated cell sorting.
  • the following cell lines were obtained from the ATCC (Ame ⁇ can Type Culture Collection, Manassas, VA).
  • the ATCC designation follows the cell type abbreviation: KGla, CCL246.1; HL60, CCL240; NCI-H446, HTB171; NCI-H82, HTB175; MCF7, HTB22 and Molt4, CRL1582.
  • Adherent cell lines were removed from tissue culture surfaces and disaggregated by pipetting in IX Versene (PBS-EDTA solution, Gibco, Life Technologies, Grand Island, NY). Cells were washed in phosphate-buffered saline (PBS) and resuspended in Dulbecco's PBS (DPBS) containing 0.1% sodium azide and 1% bovine serum albumin (BSA) The cells were then divided into 3 separate tubes.
  • PBS-EDTA solution Gibco, Life Technologies, Grand Island, NY
  • DPBS Dulbecco's PBS
  • BSA bovine serum albumin
  • Monoclonal antibodies NUH2, ant ⁇ -Le ⁇ and a control mouse IgM were each added to separate tubes at final concentration of lO ⁇ g/ml After incubation at 4°C for 1 hour, the cells in each tube were washed with DPBS + azide and incubated with FTTC-labeled goat anti -mouse IgM (KPL Labs, Gaithersburg, MD) at a final concentration of 1 ⁇ g/ml. After incubation for 45 minutes at 4°C, the cells were washed twice with DPBS + azide and resuspended in DPBS + azide and 1% BSA. Labeled cells were maintained at 4°C and analyzed immediately by fluorescence activated cell sorting gated for fluorescem.
  • Cells were directly immunostained for antigens, CD34 and Thy-1 using fluorescence-labeled antibodies ant ⁇ -HPCA2-FTTC and ant ⁇ -Thy-1-PF, respectively.
  • cells prepared as descnbed above were incubated at 4°C for 1 hour in DPBS containing 0.1% sodium azide, 1% BSA and 10 ⁇ g/ml of staining antibody for either CD34 (ant ⁇ -HPCA2-FTTC, Becton-Dickenson, San Jose, CA) or Thy-1 (ant ⁇ -CDw90-PE, Serotec, U.K.).
  • the cells were washed twice with DPBS + azide and resuspended in DPBS + azide and 1% BSA. Labeled cells were maintained at 4°C and analyzed immediately by fluorescence activated cell sorting gated for either fluorescem or phycoerythnn, respectively.

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Abstract

L'invention concerne des procédés et des compositions utilisés pour lier des cellules souches hématopoïétiques. Les procédés de l'invention consistent généralement à utiliser un partenaire de liaison qui forme un complexe avec une structure à base de lactosamine syalylée présente sur la surface des cellules souches. La formation desdits complexes facilite, par exemple, l'immobilisation, la purification, l'identification et le ciblage de cellules souches hématopoïétiques. Lesdites compositions de l'invention comprennent généralement un partenaire de liaison qui peut être libre, fixé à un matériau de support ou lié à un agent marqueur ou thérapeutique.
EP98948540A 1997-09-25 1998-09-24 Procedes et compositions utilises pour lier des cellules souches hematopoietiques Withdrawn EP1017790A1 (fr)

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US5997297P 1997-09-25 1997-09-25
US59972P 1997-09-25
PCT/US1998/020063 WO1999015628A1 (fr) 1997-09-25 1998-09-24 Procedes et compositions utilises pour lier des cellules souches hematopoietiques

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CA2368423C (fr) 1999-03-30 2011-06-28 Netech Inc. Procede de separation selective de cellules sanguines au moyen de lectine
AU2001277635A1 (en) * 2000-03-14 2001-10-08 Exiqon A/S New methods for separation and isolation of subpopulations of cells in biological samples
WO2002040644A1 (fr) * 2000-11-15 2002-05-23 Yukio Kato Agent conferant une resistance a des stimuli exterieurs
AU2004227205B2 (en) * 2003-04-08 2010-06-10 Yeda Research And Development Co. Ltd Stem cells having increased sensitivity to a chemoattractant and methods of generating and using same
US8691774B2 (en) 2005-07-11 2014-04-08 Glykos Finland Oy Carbohydrate profile compositions from human cells and methods for analysis and modification thereof
WO2007054622A1 (fr) * 2005-11-08 2007-05-18 Suomen Punainen Risti, Veripalvelu Nouvelles compositions a profils glucidiques tirees de cellules humaines et procedes d'analyse et de modification correspondants
AU2007264846A1 (en) * 2006-06-29 2008-01-03 Glykos Finland Oy Novel cellular glycan compositions
KR20080037883A (ko) * 2006-10-27 2008-05-02 세원셀론텍(주) 메디컬 키트 및 이의 사용방법
US20100068806A1 (en) * 2007-01-18 2010-03-18 Suomen Punainen Risti, Veripalvelu Novel methods and reagents directed to production of cells
JP2010516240A (ja) * 2007-01-18 2010-05-20 スオメン プナイネン リスティ，ヴェリパルベル ヒト細胞からの新規の炭水化物、ならびにその分析および修飾のための方法
JP6182135B2 (ja) * 2011-06-06 2017-08-16 レゲネシス ベーフェーベーアー 中空繊維バイオリアクター中での幹細胞の増幅
WO2013060283A1 (fr) * 2011-10-28 2013-05-02 Li Fusheng Procédé d'utilisation de lectines pour reconnaître et purifier des cellules souches, kits de réactifs et procédé d'application associé
CN103087979A (zh) * 2011-10-28 2013-05-08 李福生 用于富集、提纯干细胞的方法、试剂盒及其应用方法
KR20150023297A (ko) * 2012-05-31 2015-03-05 에이전시 포 사이언스, 테크놀로지 앤드 리서치 고상 우레이드에 대한 생물학적 표적의 선택적 결합
JP6057574B2 (ja) * 2012-07-11 2017-01-11 キヤノン株式会社 造血幹細胞識別用プローブ
US20180320136A1 (en) * 2015-11-03 2018-11-08 GlycoMimitics, Inc. Methods and compositions for the production of monoclonal antibodies, hematopoietic stem cells, and methods of using the same
WO2020158943A1 (fr) * 2019-02-01 2020-08-06 住友化学株式会社 Anticorps et fragment fonctionnel de celui-ci

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US5227160A (en) * 1988-07-15 1993-07-13 The Biomembrane Institute Monoclonal antibody nuh2 capable of inactivating motility of human sperm, antigen defined by said monoclonal antibody and methods of using said monoclonal antibody and antigen
IL106255A0 (en) * 1993-04-23 1993-11-15 Baxter Int Method for isolating human blood cells

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JP2001517427A (ja) 2001-10-09
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AU9508998A (en) 1999-04-12
CN1280611A (zh) 2001-01-17

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