EP0652703A1 - Methodes d'immunoselection positive de cellules souches - Google Patents

Methodes d'immunoselection positive de cellules souches

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Publication number
EP0652703A1
EP0652703A1 EP93919834A EP93919834A EP0652703A1 EP 0652703 A1 EP0652703 A1 EP 0652703A1 EP 93919834 A EP93919834 A EP 93919834A EP 93919834 A EP93919834 A EP 93919834A EP 0652703 A1 EP0652703 A1 EP 0652703A1
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European Patent Office
Prior art keywords
cells
antibody
cell
hapten
reagent
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EP93919834A
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German (de)
English (en)
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EP0652703A4 (fr
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Steven Kessler
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Individual
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Individual
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Publication of EP0652703A1 publication Critical patent/EP0652703A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells

Definitions

  • the invention relates to i "proved methods for positive immunoselection of target cells from a . -lerogeneous cell composition.
  • the invention also relates to immunological kit$ for effecting said immunoselection of cells.
  • the invention also relates to improved methods for assaying the purity of the immunoselected cells using indicator cells, which may be autologous or heterologous, as well as methods for subjecting the recovered cells to further immunoselection procedures.
  • the invention in particular relates to improved methods for the positive immunoselection of stem and progenitor cells, and a specific immunological reagent (monoclonal antibody) for effecting said positive immunoselection of stem and progenitor cells.
  • the invention also relates to improved methods for assaying the purity of said immunopurified stem and progenitor cells using indicator cells (e.g., autologous) as well as methods for subjecting the immunoselected cells to further immunoselection.
  • indicator cells e.g., autologous
  • the invention relates to purified stem and progenitor cells resulting from these methods, and methods of using these purified stem cells diagnostically and therapeutically.
  • immunoselection techniques broadly embrace the isolation of a desired moiety from a heterogeneous composition on the basis of its specific reactivity with its immunological binding partner. Essentially, these techniques exploit the specific reactivity of the desired moiety contained in a heterogeneous system, e.g., a particular antigen, bacteria, virus, cell, hapten, antibody, etc. with its immunological counterpart, an immunological reagent which is generally an antibody, antibody-like molecule, or an antigen so as to effect the desired separation.
  • Immunoselection techniques have in particular been utilized for the separation of desired cells from heterogeneous compositions comprising these cells.
  • This application of immunoselection for the purification of specific cell types has been the focus of much recent research given the significant potential applications of purified cell compositions as diagnostics and therapeutics, as well as for biomedical research.
  • the selective recovery of specific highly purified cell types will better enable biological researchers to study the differentiation of these specific cell types, will facilitate the identification of the specific lineage or function-specific antigens comprised on these cell types and will enable researchers to identify the specific discrete functions and bioproducts produced by these cells.
  • BMT bone marrow transplantation
  • BMT bone marrow cells into a recipient in need thereof.
  • a more specific objective of BMT is the infusion of healthy stem cells and progenitor cells into a recipient in need thereof. This is essentially because these cells are the "master cells" from which all the cells of the immune and hematopoietic system develop. Accordingly, an effective method for the infusion of sufficient quantities of healthy stem and progenitor cells into a recipient in need thereof could result in the entire reconstitution of the immune and hematopoietic system.
  • BMT is classifiable into two general types.
  • Autologous BMT comprises the reinfusion of healthy bone marrow or stem cells into the same recipient from which these cells have been derived.
  • heterologous or allogeneic BMT involves the infusion of healthy bone marrow or stem cells into a recipient other than from whom the cells were derived.
  • These two distinct forms of BMT have different applications and comprise different constraints.
  • Autologous BMT is utilized in particular in cancer patients being treated by chemotherapy or radiation. While these treatment protocols are designed to eradicate tumor cells, unfortunately, these methods may also damage normal cells, in particular cells of the hematopoietic and immune system.
  • the bone marrow or stem cells are derived from a heterologous healthy donor.
  • Allogeneic BMT is particularly useful in patients exhibiting congenital defects which result in a defective immune or hematopoietic system.
  • the cells should not be contaminated by malignant cells. Instead, these cells contain allogeneic or heterologous normal T-lymphocytes, which may give rise to a condition known as graft-versus-host disease. Therefore, in the case of allogeneic or heterologous BMT, immunoselection has been utilized to selectively deplete or purge these normal T-lymphocytes from the donor bone marrow.
  • immunoselection comprises particular application in BMT, and more particularly in the selective recovery of healthy stem and progenitor cells.
  • cellular immunoselection comprises general applicability in the selective recovery of any desired cell wherein suitable immunological reagents are available, in particular, antibodies or antibody-like molecules which selectively bind the desired cell or the cells which are to be selectively depleted.
  • Cellular immunoselection may be generally classified into two types. Positive immunoselection implies the direct selection and recovery of cells bearing a given specificity from among a heterogenous population. Depending upon factors including the binding specificity and affinity of the particular immunoreagent (antibody or antibody-like molecule), labeling, and recovery methods these methods may give rise to cell compositions which are highly pure or merely somewhat enriched.
  • the major limitation of positive cellular immunoselection has been the difficulty of physically labeling the cells so that they may be isolated without damaging the cells.
  • a positive cellular immunoselection technique In order for a positive cellular immunoselection technique to be useful it should be (i) gentle, it should result in viable functional cells; ( ⁇ ) rapid - the different operations should be performed with sufficient speed to allow recovery of viable cells; (iii) high capacity - a useful technique should at the minimum permit an input of 10* cells for laboratory-scale immunoselections, and ideally at least 10' - 10 10 cells to be clinically relevant; and (iv) high-resolution - it should discriminate between the desired cells and the other cells in the sample with a high degree of reliability.
  • conventional positive immunoselection methods have not satisfied all these factors.
  • negative cellular immunoselection involves the specific elimination of undesired cells from a heterogenous population. This technique may obtain the desired cell type in highly concentrated form, however, it does not generally give rise to a pure cell sample. This is considered to be the most significant disadvantage of this technique. (For a general review of the subject of positive and negative immunoselection, see Basch et al. (1983), L Immunol. Methods. 56:269.)
  • Negative immunoselection techniques have, however, been utilized most often. These methods have generally involved treatment of the donor bone marrow with anti-tumor or anti-T-cell antibodies which have been incubated with complement, conjugated with toxins or radioisotopes, attached by various linkers or spacer systems to red blood cells or beads for rosetting and density fractionation, and similarly, coupled to plates or to fiber or beads of variable chemical composition which are packed in columns. (These techniques are, e.g., reviewed in Janossy (1984), Br. Med. Bull..
  • Antibodies have also been coupled to magnetic particles or fluids of varying chemical composition or magnetic properties (reviewed extensively in Kemshead (1991), J. Hematotherapy. 1:35). These approaches to negative immunoselection share a number of significant limitations.
  • One limitation is that considerable antigenic heterogeneity exists among tumor cells, even from among those of the same type and patient origin. To overcome this, attempts have been made to assemble cocktails of different monoclonal antibodies specific for each antigenic variant cell type in order to ensure detection of all tumor cells (see e.g., Treleaven et al. (1984), anssi, i:70; Reynolds et al.
  • the agents used can also kill normal hematopoietic progenitors as well as delay engraftment (see e.g., Rowley et al. (1987), Blood. 70:271; Shpall et al. (1990), J. Clin. Oncol.. 9:85).
  • Table 1 depicts the possible number of tumor cells among normal bone marrow cells that could be reinfused in a transplant recipient. Assuming, e.g., a population of 1% tumor cells (7 x 10 7 ) in a bone marrow harvest of minimally 7 x 10 9 nucleated cells for an adult patient, a 4 log purge would theoretically result in 7 x 10 3 tumor cells remaining in the graft. This level of tumor cell contamination (1 tumor cell per 10 6 normal marrow cells) is not detectable by any current technology (Kemshead (1992), J. Hematotherapy. 1:35).
  • a major disadvantage of currently available positive cell immunoselections is that the binding of antibodies to the targeted cell often adversely affects the functional characteristics of the cells and further makes removal of the cells from the immunological substrate difficult.
  • Pilling et al (1989), J. Immunological Methods 122:235 note the difficulties in removing positively selected cells from magnetic Dynabeads, and further acknowledge that such methods may be limited wherein the removal of beads is necessary (as would, of course, be necessary if the cells are to be used in therapy).
  • Another disadvantage of currently available positive immunoselection methods is that the purified cells are contaminated by non-target cells, attributable, e.g., to non-specific binding of non-target cells to the immunological substrate, e.g., antibody bound magnetic beads.
  • the immunological substrate e.g., antibody bound magnetic beads.
  • Janssen et al (1989), J. Immunological Methods 121:289 disclose that while antibody-coated magnetic particles are efficient for purging neoplastic or immune competent cells from bone marrow prior to transplantation, they are unsuitable for positive immunoselection methods wherein the goal is to obtain a homogeneous population of cells.
  • stem cells In order to develop an effective positive immunoselection of stem cells, or in particular, human plunpotent lympho-hematopoietic stem cells and progenitor cells, it is of course necessary to produce antibodies or antibody-like molecules which selectively bind stem and progenitor cells, and which do not bind mature cells.
  • Potentially useful antibodies for this purpose include the anti-MY-10 antibody which reacts with the CD34 antigen, an antigen is expressed on the surface of stem and progenitor cells and which is not expressed on mature lymphoid or myeloid cells. (See, e.g., Civin eLaL (1984), J. Immunol.. 133:157; U.S. Patent Nos.
  • Stem cells or CD34 + ce ⁇ ls typically comprise 1-4% or less of bone marrow mononuclear cells, and a much lower fraction of normal blood mononuclear cells.
  • Various other monoclonal antibodies with similar antigenic specificities have also been developed (Tindle et al. (1984), Leuk es.. 9: 1; Andrews et al. (1986), Blood. 67:842; Watt et al. (1987), Leukemia. 1:417; Kessler (1987), Fed. Proc.. 46: 1363).
  • U.S. Patent Nos. 4,714,680, 4,965,204 and 5,039,994 further contemplate using the disclosed monoclonal antibody for the positive immunoselection of stem cells.
  • the described methods for the separation of antibody-bound stem cells from unbound cells include mechanical cell sorting (e.g., with a fluorescence activated cell sorter) and immobilization on supports such as agarose or polystyrene bead columns, hollow fiber membrane and petri dishes.
  • These patents further contemplate two means for separating the bound cells from the column, specifically, vigorous agitation or enzymatic "nicking" or digestion of an enzyme cleavable spacer which intervenes the solid phase and the antibody.
  • the avidin-biotin complex has been used as a tool in molecular biology in methods including: isolation of biotin-derivatized material by affinity chromatography; affinity labeling and identification studies; affinity cytochemical labeling for localization studies in fluorescence and electro- microscopy; inhibition of bacteriophage; and study of cell surface molecular interactions.
  • biotin and avidin complexes in the negative immunoselection of cells is also known in the art.
  • model negative immunoselection systems have been reported using biotinylated anti-T cell antibodies to deplete splenic T cells on avidin coated petri plates (1984, J. Exp.. 159:463 and similarly in U.S. patent No. 4,298,685).
  • biotinylated antibodies have been used with avidin-coupled sheep red blood cells to rosette antigen-positive cells and separate them on density gradients (Wormmeester et al. (1983), J. Immunol. Methods. 67:389; Furfang and Thierfelder (1987), J. Immunol. Methods. 91:123).
  • Antigen negative as well as antigen positive cells could be recovered following disruption of the red cells; however, unfortunately these cells are coated with red membrane "ghosts" which is undesirable if the cells are to be used as a therapeutic.
  • the avidin-biotin complex is widely used for immunoassays and immunoseparations and has many attributes, one significant disadvantage is the fact that the avidin-biotin complex is essentially irreversible. For this reason, it is not easily utilized for positive cellular immunoselections. Therefore, in order to extend the versatility of the biotin-avidin system to positive as well as the negative immunoselections discussed above, several modifications have been described to obviate the irreversibility of this interaction. For example, one reference suggested derivatizing antibodies with biotin analogs exhibiting a reduced affinity for avidin, so that the cells immobilized on avidin coated matrices could be recovered by competition with free biotin (Basch fiLaL (1983), J. Immunol.
  • one object of the present invention is to provide improved methods for the positive immunoselection of desired cells from heterogenous compositions containing these cells, which methods attain levels of cell purity which substantially surpass the levels of cell purity obtained by conventional methods and which further do not disturb the cell surface membrane or membrane antigens.
  • Another object of the invention is to provide immunological kits which are suitable for said positive immunoselection of desired cells from heterogenous compositions containing the desired cells.
  • a further object of the invention is to provide methods of quantifying the purity of the resultant positively immunoselected cells which methods do not require the addition of foreign or unrelated cells to the pre- immunoselection mixture.
  • a further object of the invention is to provide methods for subjecting the resultant positively immunoselected cells to further multiple concurrent or sequential positive or negative immunoselections. These methods may be performed rapidly since the present methods do not disturb the cell surface membranes or membrane antigens.
  • a further object of the invention is to obtain methods for positive immunoselection of cells which use substantially less reagents and are therefore less expensive than conventional cellular immunoselections.
  • Another object of the present invention is to provide a means for assessing the purity of the purified immunoselected cells by further reacting these cells with an immune reagent which binds a distinct antigenic epitope from the immune reagent used for positive immunoselection.
  • a more specific object of this invention is to provide a method for positive immunoselection of stem cells from heterogeneous compositions containing such cells, which results in stem cells virtually devoid of contaminating malignant or tumor cells or mature T-lymphocytes.
  • Another object of the invention is to provide highly pure stem cell compositions which are essentially devoid of contaminating malignant cells or mature T-lymphocytes and which consequently may be used therapeutically in BMT or for biomedical research.
  • Still another object of the present invention is to provide an immunological reagent (monoclonal antibody) which specifically binds lympho-hematopoietic stem cells and a hybridoma cell line which produces this monoclonal antibody.
  • another object of the present invention is to provide immunological kits for selectively recovering stem cells from heterogeneous compositions containing these cells.
  • the present invention provides a significant advance in the art of positive immunoselection of cells, and in particular in the immunoselection of lympho-hematopoietic stem and progenitor cells.
  • PE ⁇ N ⁇ QNS As used herein, the following terms will have the definitions given below:
  • Hapten are those moieties which specifically react with antibodies specific thereto but which moieties themselves are not capable of causing the production of antibodies.
  • Known haptens include, e.g., biotin, arsenilic-acid, dinitrophenol, fluorescein, oligopeptides and glycopeptides.
  • Antibody or “Antibody-like Molecule” are substances capable of specifically binding an antigen.
  • Antibodies or antibody-like molecules in the context of this invention will include monoclonal and polyclonal antibodies, chimeric antibodies, and fragments thereof capable of specifically binding an antigen.
  • Soluble hapten is intended to mean a hapten which is sufficiently soluble to permit a solution containing said hapten to be capable of selectively dissociating target cells bound to a solid phase either directly or indirectly via a hapten-anti-hapten antibody complex.
  • “Monoclonal antibody” is an antibody produced by a clone or hybrid cell line. Methods of producing monoclonal antibodies are well known in the art.
  • Immunonoselection means the selective recovery or removal of a moiety from a heterogeneous composition of the basis of its specific reactivity with an immunological binding partner, or immunological reagent, preferably an antibody or antibody-like molecule.
  • Cellular Immunoselection means the selective recovery of a target cell on the basis of the target cells 'or non-target cells' specific reactivity with an immunological binding partner, preferably an antibody or antibody-like molecule which is directed to an antigen contained on the surface of said target cell type or non-target cells.
  • an immunological binding partner preferably an antibody or antibody-like molecule which is directed to an antigen contained on the surface of said target cell type or non-target cells.
  • “Positive Cellular Immunoselection” means the direct recovery of a target cell type from a heterogeneous cell composition on the basis of its specific reactivity with an antibody or antibody-like molecule which has been produced to an antigen on the surface of said target cell.
  • Negative Cellular Immunoselection or “Negative Cellular Immunodepletion” means the selective depletion of non-target cells from a heterogenous composition containing the target cells on the basis of the non- target cells' specific reactivity with one or more antibodies or antibody-like molecules directed to antigens contained on the surface of these non-target cells.
  • Heterogeneous cell composition means a composition containing the target cells which are to be recovered in admixture with non- target cells.
  • Target Cells are those cells which are to be selectively recovered on the basis of the inventive immunoselection procedure, e.g., stem cells and progenitor cells.
  • Figure 1 depicts an immunoblot analysis involving the CD34 antigen purified by immunoprecipitation which compares the immuno- reactivity of the K6.1 antibody, the anti-MY-10 antibody, and UPC-10 (a negative control myeloma protein of the same isotype as K6.1).
  • Figure 2 depicts an indirect immunofluorescence study involving FTTC-avidin and flow cytometry comparing the effects of pre- binding either the K6.1 or anti-MY-10 antibody to the CD34 antigen or KG- la cells relative to the subsequent binding of the other antibody.
  • Figure 3 depicts a flow cytometric analysis of CD34 antigen expression of cells positively immunoselected according to the invention.
  • Figure 4 depicts the results of immunofluorescence staining and flow cytometry analysis of immunoselected rhesus CD34 + cells.
  • Figure 5 depicts engraftment as determined by peripheral blood neutrophil counts and platelet counts over time.
  • the present invention in its broadest embodiment comprises the immobilization of target cells contained in a heterogeneous composition to an insoluble solid phase matrix or support via an immune complex which contains a primary immune reagent capable of specifically binding the target cells, and an immune complex comprising a soluble hapten and an antibody which specifically binds the soluble hapten.
  • the target cells are immobilized in such a manner that they may be readily dissociated by the addition of a solution containing the soluble hapten.
  • the invention is not limited thereby, and other more conventional methods of cell dissociation including agitation, high temperatures, and addition of enzymes may be used as well. It should be emphasized, however, that dissociation by the addition of the soluble hapten containing solution is preferred.
  • the soluble hapten will comprise biotin or a chemically derivatized biotin.
  • the aforementioned means of cell dissociation involving the addition of a solution of the soluble hapten essentially exploits a phenomenon known in the field of immunology as hapten competition. Although this general phenomenon is known in the art, it is novel in the context of the present invention relating to positive immunoselection of cells. While hapten competition has been utilized in methods of positively immunoselecting cells, it has been limited to cells which are inherently hapten-specific (e.g., hapten-specific lymphocytes).
  • the target cells are not inherently hapten specific, but rather they are complexed with an immune reagent (as antibody) which is either directly or indirectly bound to the particular soluble hapten.
  • an immune reagent as antibody
  • previous methods of immunoselection which have utilized hapten competition have also utilized other cell dissociation methods (e.g., agitation or elevated temperature). This is unlike the present invention wherein the immobilized target cells are dissociable using only hapten competition.
  • haptens which are physiologically non-toxic at the concentrations required for cell dissociation.
  • a higher hapten concentration in solution e.g., in the millimolar range
  • Biotin is a particularly suitable hapten since it is a naturally occurring vitamin. This is an important consideration if the immunopurified cells are to be used as a therapeutic.
  • more conventional haptens such as arsenilic acid (Clark et al. (1982), J. Immunol. Methods.
  • haptens for use in the present invention include oligopeptides, glycopeptides, carbohydrates, etc.
  • the target cells which are immunopurified will comprise human pluripotent lympho-hematopoietic stem cells and progenitor cells derived from human bone marrow or blood since these cells have a known applicability for BMT.
  • the present invention is not intended to be limited to these target cells, but rather er ' -aces the immunopurification of any target cells, cellular organelles or other components, or microorganisms to which immune reagents capable of specifically binding to a membrane antigen on the surface thereof are available.
  • the preferred primary immune reagent capable of specifically binding these cells will be the K6.1 monoclonal antibody produced by the hybridoma cell line K6.1.
  • This monoclonal antibody binds to an epitope on the CD34 antigen comprised on the surface of these cells.
  • This monoclonal antibody was developed by the present inventor (see Kessler (1987), Fed. Proc.. 46:136). This monoclonal antibody binds a distinct CD34 antigenic epitope from the MY- 10 monoclonal antibody described, e.g., in U.S. Patent No. 4,714,680.
  • the immunopurified human lympho-hematopoietic stem and progenitor cells may be immunoreacted with the MY-10 monoclonal antibody or other CD34 monoclonal antibodies which bind to CD34 epitopes distinct from the one to which the K6.1 antibody binds after the initial immunopurification using the K6.1 monoclonal antibody.
  • the cells may be subjected to further immunoanalysis or additional immunopurification using the MY- 10 monoclonal antibody or other CD34 monoclonal antibodies which bind to CD34 epitopes distinct from the epitope to which the K6.1 antibody binds.
  • the present invention broadly relates to the selective immobilization of target cells contained in a heterogeneous cell composition to an insoluble solid phase matrix or support via an immune complex which contains a primary immune reagent which specifically binds the target cell, a soluble hapten, and an antibody specific to the soluble hapten.
  • This immobilization may in particular be accomplished by the following steps;
  • step (b) reacting the immune complex containing heterogeneous composition produced by step (a) with one or more secondary reagents at least one of which is capable of specifically binding the primary immune reagent and at least one of which is conjugated to the soluble hapten if the primary immune reagent has not been conjugated to the soluble hapten to produce a complex consisting of the target cell-primary immune reagent- secondary reagents; and
  • step (c) reacting the complex containing composition produced by step (b) with a solid phase matrix or support to which has been directly or indirectly bound an immune reagent capable of specifically binding the soluble hapten.
  • the heterogeneous cell composition will comprise cells obtained from blood, bone marrow, spleen, or other tissues which contain the target cells which are to be -nunopurified.
  • the invention broadly contemplates any heterogeneous cell composition containing target cells which are to be im- ⁇ unopurified.
  • the primary immune reagent used in step (a) will preferably be an antibody or antibody-like molecule capable of specifically binding an antigen comprised on the surface of the target cells. Both monoclonal and polyclonal antibodies may be utilized. As noted, this primary immune reagent may be conjugated to a soluble hapten, e.g., biotin. However, it may be preferable to conjugate one of the secondary immune reagents with the hapten since this will enable more of the soluble hapten to be conjugated to the target cells on a per cell basis. After the primary immune reagent has been complexed with the target cells, the unbound or excess antibody may be removed from the cells, e.g., by washing. However, this step is not critical to the invention and may be omitted if so desired.
  • the secondary reagents used in step (b) include, as stated above, at least one immune reagent wi h specifically binds the primary immune reagent used in step (a).
  • this will be an antibody or antibody-like molecule which specifically binds the primary immune reagent, e.g., immunoglobins which are species-specific (e.g., anti-mouse, anti-goat, anti-rabbit, etc.), class or isotype specific (e.g., anti-IgG, IgA, or IgM) or subclass-specific (e.g., anti-IgG 1, IgG2a, IgG2b, IgG3).
  • species-specific e.g., anti-mouse, anti-goat, anti-rabbit, etc.
  • isotype specific e.g., anti-IgG, IgA, or IgM
  • subclass-specific e.g., anti-IgG 1, IgG2a, IgG2b
  • either the primary immune reagent or at least one of the secondary immune reagents is conjugated to the hapten, and preferably the secondary immune reagent.
  • This conjugation may be effected by well known methods for conjugating haptens to antibodies. If the hapten is biotin, there are many commercially available biotin-derivatized antibodies, or they can be prepared by conjugation methods well known to those skilled in the art.
  • a preferred biotin derivative is biotin-e-aminocaproic acid -N-hydroxysuccinimide ester (biotin-X-NHS) which is available from Calbiochem in La Jolla, CA and other commercial sources.
  • the amount of hapten which is conjugated to the immune reagent may be varied so as to enable a greater or lesser amount to be bound per target cell. This enables control of the total amount of crosslinlting between the immobilized target cell and the solid phase matrix or support and the avidity of the target cells to the solid phase.
  • biotin in particular is utilized as the hapten, a preferred ratio has been found to be 6 to 8 biotin molecules bound per immune reagent (e.g., antibody). This avoids possible inactivation of the antibody which would prevent it from binding the anti-hapten antibody.
  • the secondary immune reagents include an antibody directed against the primary immune reagent which is conjugated to the hapten. This results in the cells being immobilized via an indirect or "sandwich” technique. This is advantageous since it
  • step (iii) stabilizes the binding of a low avidity primary immune agent to the target cells; and (iv) enables the positively immunoselected target cells to be subjected to further immunoselections (e.g., by selection of a secondary antibody with a restricted immunoglobin class or subclass binding specificity) using other primary immune reagents of alternative binding specificity.
  • the anti-hapten antibody may be directly or indirectly bound to the solid phase matrix or support. If the hapten is biotin, then of course anti-biotin antibodies will be bound.
  • Anti-hapten monoclonal or polyclonal antibodies are commercially available or may be prepared by methods well known to those skilled in the art.
  • Anti-biotin antibodies are in particular available from Vector Laboratories, Burlingame, California, and Bio-Technology Limited, Oxford.
  • the solid phase matrix or support used to immobilize the anti- hapten antibody may, e.g., be beads, fibers, mesh or tubing.
  • the solid phase support will comprise paramagnetic beads or particles in suspension.
  • Various types of magnetic beads suitable for immunoselection of cells are known in the art (see, e.g., Molday and Mackenzie (1982), J. Immunol. Methods. 52:353; Lea S ⁇ (1985), Scand. J. Immunol.. 22:207) and are available commercially, e.g., by Dynal, A.S. ("Dynabeads," Oslo, Norway through Dynal, Inc., Ft. Lee, NJ), Advanced Magnetics, Inc. ("BioMag” beads, Cambridge, MA), Immunotech Intl./AMAC, Inc.
  • Magnetic beads (“IObeads”), and Baxter/Fenwal (Deerfield, IL).
  • the desirable characteristics of such magnetic beads includes high chemical stability for conjugation, high uniformity in size, shape, magnetic and surface properties. This list of suitable solid supports is by no means intended to be limitative or exhaustive.
  • the direct or indirect binding of the anti-hapten antibodies to the support may be accomplished, e.g., by direct covalent attachment or by adsorption.
  • the anti-hapten antibody may be attached via another antibody directly bound to the support.
  • an immunoaffinity purified F(ab') 2 anti-goat IgG antibody may be covalently bound to the surface of the magnetic beads which beads are tha reacted with an immunoaffihity purified goat anti-biotin antibody.
  • an anti-mouse Ig antibody may be covalently bound to the surface of the magnetic beads which beads are then reacted with a mouse monoclonal anti- biotin antibody.
  • the monoclonal anti-biotin antibody is attached covalently or by adsorption to the support.
  • step (c) When immobilizing the target cells to the anti-hapten antibody containing solid phase in step (c) it is necessary to contact the solid phase and the target cell complexes under conditions which permit the formation of the hapten-anti-hapten linkage.
  • the cell suspension containing the hapten-containing target cell complexes may be passed through or otherwise incubated with the anti-hapten antibody immobilized on the solid support which is in turn comprised in a suitable chamber.
  • Optimal conditions for effecting this complexation will vary depending upon factors including the particular target cells, the concentration of the target antigen on the surface of said target cells, the amount of hapten molecules per antibody molecule, the target cell concentration, the avidity of the hapten for the anti- hapten antibody, and the amount of anti-hapten antibody bound to the solid support.
  • parameters such as cell concentration, density, mass or surface area of the solid support, or the time or temperature so as to obtain optimal complexation of the target cells and the solid support.
  • Also embodied in the subject invention are alternative types of linkages involving a target cell and the solid phase matrix or support containing a hapten-anti-hapten antibody linkage.
  • hapten e.g., biotin
  • This linkage of the hapten to the solid support may, e.g., be effected using a non-immunological spacer molecule.
  • genetically engineered or chimeric antibodies derived from the fusion of two hybridomas i.e., hybrid-hybridomas or tetradomas; Morrison, et al. (1988), Semin. Hematol. 25 (Suppl. 3):23; Chervonsky fiLaL (1988), Mol. Immunol.
  • 25:913 may be used which exhibit binding specificities for both the hapten (e.g., biotin) and also for the cell-specific antigen on the target cells or for immunoglobulin species, class, or subclass antigens.
  • hapten e.g., biotin
  • this support may then be separated from the heterogeneous cell composition. Depending upon the type of solid support this may be accomplished, e.g., by washing or flushing the chambers so as to selectively remove unbound cells.
  • these beads may be drawn to and temporarily fixed to the walls of the container by juxtaposing a magnetic field such as that imposed by a rare-earth (e.g., samarium-cobalt) magnet (See, e.g., Trdeaven c aL (1984), Tanr»» 1:70), or a plurality of magnets, or an electromagnet, and then removing the non-target cells which remain free in suspension.
  • a rare-earth magnet e.g., Trdeaven c aL (1984), Tanr»» 1:70
  • the purity of the immobilized target cells may further be enhanced by repetitive washing of the magnetic beads by removing the magnetic field, resuspending the beads with cells affixed thereto, and reimposing the magnetic field.
  • the target cells will be to introduce a solution containing an amount of the soluble hapten, e.g., biotin, which is sufficient to dissociate the linkage between the hapten and the anti-hapten antibody attached to the support.
  • a solution containing an amount of the soluble hapten e.g., biotin
  • biotin e.g., glucose
  • This hapten concentration may be varied as necessary.
  • an isotonic solution containing biotin in the range of 1-3 mM has been found to be especially suitable.
  • the dissociation temperature should be ambient or cooler than ambient (i.e., 0-4° rather than higher temperatures which favor cell detachment by antigen "capping" or shedding rather than via hapten competition.
  • the time required for cell detachment will depend upon the aggregate affinity between the individual cells and the solid phase, which is affected, e.g., by the amount of crosslinlring between the hapten and the anti-hapten antibody and the avidity of this complex. This dissociation time may be as short as several minutes to as long as 1-2 hours. However, it is contemplated that more rapid dissociation may be achieved with suitable monoclonal rather than polyclonal anti-hapten antibodies because of monoclonal antibodies' more uniform avidities.
  • the target cells may be separated from the solid support by a washing or flushing procedure, which may be repeated as necessary. If the solid support comprises magnetic beads then of course the magnetic field should be r ⁇ mposed so as to remove the beads.
  • the purity and integrity of purified target cells recovered by the preferred method, hapten competition are significantly improved relative to cells recovered by conventional methods. (See, e.g., Basch sLfl (1983), J. Immunol. Methods. 56:269). This occurs in particular because this method detachment is specific, i.e., only cells immobilized by the hapten- anti-hapten antibody linkage are dissociated.
  • non-target cells which are non-specifically bound or "trapped" on the solid support are not released by the hapten solution.
  • This is in distinction to the conventional non-specific cell detachment methods such as, e.g., agitation, or enzyme digestion which may also release non-specifically bound cells.
  • this method of target cell detachment is further advantageous since it preserves the integrity of all the cell surface membrane antigens, receptors, and other molecules contained on the target cell, including the target cell antigen which binds the primary immune reagent.
  • nonspecific recovery methods such as agitation, proteolytic digestion, or exposure to chemical reducing agents (e.g., to cleave disulfide or mercury-sulfur bridges, as described in Bonnafous et al. (1983), J. Immunol. Methods. 58:93), oxidizing agents, or acidic or basic conditions may destroy membrane molecules or result in cells with the target antigen "ripped out" of the membrane.
  • cells may be obtained which comprise uniform or non-uniform configurations of antibodies, including various combination of primary, se- idary, or even tertiary layers of antibody linking the cells to the soL ⁇ phase.
  • the inventive immunoselection results in target cells of higher purity and membrane integrity than had been obtained by conventional cellular immunoselection methods.
  • the present invention further relates to a method for testing the purity of the recovered cells and the integrity thereof. This method exploits the fact that "hapten- competition" cell detachment preserves the antigens and other molecules contained on the surface of the target cells.
  • the target cells after the initial immunoselection method, the target cells will be complexed with another antibody which binds a distinct antigenic epitope of the target antigen from that bound by the primary immune reagent used for the initial immunoselection.
  • This other antibody may be utilized in a variety of andard immunoselections, e.g., direct immunostaining techniques.
  • this other antibody may be detected by indirect or sandwich type assays provided that the antibody used does not cross-react with the primary immunosdecting antibody used in the initial immunoselection. This may be accomplished, e.g., by selecting two primary antibodies which are of different immunoglobulin species, class, or subclass.
  • Another and more sensitive method to analyze cell unity after the immunoselection has been effected is to include before immunoselection a subpopulation of labeled "indicator” cells. These may, e.g., be transformed or tumor cell lines, as described in Reynolds et al. (1986), Cancer Res.. 46:5878. . However, there are some instances wherein the addition of such conventional "indicator” cells may be unsuitable. This is particularly so if the cells are to be used as a therapeutic wherein the introduction of transformed cells could have disastrous consequences. Therefore, the present invention further provides improved "indicator" cells relative to those utilized in conventional methods.
  • these improved indicator cells may be prepared by removing a small portion of the heterogeneous cell population from which the target cells are to be immunoselected (e.g., on the order of 5%) and labelling these removed cells with a known label. Methods of labelling cells are well known to those skilled in the art. : However, it should be noted that labels which are physiologically safe in vivo are preferred.
  • the indicator cells (which will be referred to as autologous indicator cells) may be labelled, e.g., with the fluorescent DNA dye Bisbenzimide H 33342 (e.g., as described in Reynolds, et al. (1985), Transplantation Proc.. XVm (1):434 and Reynolds fiUL (1986), £a ⁇ c Res.. 46:5878).
  • These labelled cells will then be reacted with an unhaptenlated form of the same primary immune reagent or primary plus secondary immune reagents used for immunoselection. This "neutralizes" or prevents these autologous indicator cells from being able to bind the anti- hapten antibody bound to the solid support.
  • autologous indicator cells will then be combined with the remainder of the heterogeneous cell composition prior to their incubation with the anti-hapten antibody containing solid support. After immunoselection the purity of the recovered immunopurified cells may be determined by measuring to what extent these autologous indicator cells have been eliminated from the target cell composition.
  • the invention further contemplates subjecting the recovered immunoselected cells to additional immunoselections, which may be performed either sequentially or simultaneously. These methods are possible essentially because of the fact that the embodied cell dissociation method via hapten competition retains the integrity of all or virtually all the membrane antigens comprised on the surface of the target cells.
  • the immunoselected cells may be reacted with one or more antibodies which are specific to the target cells or alternatively be reacted with antibodies specific to non-target cells. These procedures may be effected either concurrently or sequentially from the first immunoselection.
  • the cells After the cells have been reacted with these particular antibodies the cells will preferably be washed to remove excess antibody, and then reacted with solid phase supports which bind only these additional antibodies.
  • This can be accomplished, e.g:, by designing solid phase supports to which are directly or indirectly bound antibodies which bind these other antibodies by virtue, e.g., of their binding distinct haptenic moieties, or immunoglobin species, classes or subclasses.
  • An illustrative example will be to subject the initial heterogeneous cell composition to a positive immunoselection using a biotinylated mouse IgG2a monoclonal antibody, followed by a negative immunoselection of the recovered cells using a mouse IgGl monoclonal antibody which binds a non-target cell using a solid support containing anti- mouse IgGl antibodies.
  • kits for the embodied methods of immunoselection of target cells from heterogeneous cell compositions will typically comprise one or more of the following components: (i) primary immune reagents which bind the target cells, which may or may not be conjugated to a soluble hapten;
  • kits may further include immune reagents directed to additional primary immune reagents specific to the target cells so that these kits may provide for further positive or negative immunoselections.
  • kits may further include conventional indicator cells (transformed or tumor cell lines) or the described autologous indicator cells.
  • kits may be formulated as comprehensive systems for practicing the immunoselections described herein, or they may be formulated as separate but integrated components such as modules for performing positive immunoselections, for testing cell purity of the recovered target cells, or for performing further concurrent or sequential positive or negative immunoselections.
  • kits may incorporate pre-assembled sterilized units of different capacities, such as columns or cartridges.
  • the solid phase in such Irit comprises magnetic beads
  • these beads may be provided in flexible containers resembling plastic freezing or blood-type bags, along with supports or holders containing magnets or a plurality of magnets in the appropriate orientation, and pumping systems to facilitate cell transfer and washing.
  • kits will include a hapten in a form facilitating conjugation to an antibody, a hapten containing solution for cell dissociation, reagents for preparing the described autologous indicator cells, appropriately contained solid supports, anti-hapten antibodies or anti-hapten immunoglobin species-, class-, or subclass-specific antibodies, and the reagents for attaching them.
  • kits may further include the primary antibodies for selecting specific cell populations. Given the described advantages of the inventive immunoselection techniques, these kits should afford improvements in speed, simplicity, cost, and reproducability over conventional cellular immunoselection systems.
  • the target cells which are positively immunoselected will comprise human lympho-hematopoietic stem and progenitor cells from humane bone marrow or blood.
  • Conventional methods for positive immunoselection of these cells employ monoclonal antibodies specific for the MY-10/CD34 antigen comprised on the surface of cells. This monoclonal antibody is described, e.g., in U.S. Patent No. 4,714,680. Additionally, a number of other monoclonal antibodies which specifically bind the CD34 antigen have been described in the literature. (See, e.g., Tindle et al. (1984), Leukemia Res.. 9:1; Andrews s a (1986), Blfifid, 67:842; Watt fij aL (1987), Lfiukemia, 1:417).
  • the present invention provides another monoclonal antibody for this use, which has been called the K6.1 monoclonal antibody and is produced by the K6.1 hybridoma cell line (Kessler (1987), Fed. Proc.. 46: 1363).
  • the K6.1 hybridoma cell line has been deposited at the American Type Tissue Collection on July 20, 1992 according to the Budapest Treaty and is available under ATCC No. HB 11085.
  • the K6.1 antibody is of the mouse IgG2a subclass, and therefore may easily be purified by immunoaffinity chromatography using, e.g., staphylococcal protein A. Unlike the MY- 10 antibody, this antibody is highly stable in solution in both the native and biotin-derivatized forms.
  • this antibody binds the CD34 antigen with higher avidity than the MY- 10 antibody. Moreover, the epitope which is recognized by the K6.1 antibody is separate and distinct from the MY-10 antibody. Therefore, when the K6.1 antibody is bound to the CD34 antigen it does not prevent the binding of the MY-10 antibody thereto.
  • the present invention provides an improved immunoreagent for recovery of stem and progenitor cells relative to CD34 binding immunoreagents which have been available, in particular the anti-MY-10 antibody.
  • the following descriptions are provided to illustrate how the present invention provides an improved method for immunopurification of CD34 expressing cells and lympho-hematopoietic stem and progenitor cells in particular from bone marrow or blood.
  • the present invention embraces the use of all anti- CD34 antibodies, and also antibodies which specifically bind stem cells or lymphatic or myeloid cells. Additionally, the invention embraces the use of any hapten which permits immobilization of the target cells to a solid phase via a hapten-anti-haptcn antibody complex and which permits cell dissociation by hapten competition. Thus, the following description is intended to be only illustrative and in no way limitative of the present invention.
  • the heterogeneous cell composition containing lympho-hematopoietic stem and progenitor cells will be obtained by harvesting bone marrow from an allogeneic or autologous donor (e.g., by aspiration) by standard chemical protocols or from a cadaveric source such as vertebral bodies.
  • This cell composition may be used for immunoselection directly or after cryopreservation and thawing.
  • blood may be withdrawn directly from the circulatory system or from the umbilical cord of a donor by connection through a catheter or shunt to a conventional cell centrifuge used for leukapheresis or hemapheresis. This collection may be performed continuously or stopped once a certain volume has been collected.
  • differential centrifugation may be performed in order to separate the nucleated cells (i.e., the leukocytes) from other constituents, including red blood cells, platelets, and plasma.
  • downstream processing e.g., by centrifugation, may be effected to concentrate the mononuclear cell fraction which contains the desired stem cells and progenitor cells. These separation procedures are generally performed in chemical t heresis units.
  • the immunoselection steps- will generally be carried out further downstream in a closed-in system.
  • this immunoselection will be conducted using the K6.1 monoclonal antibody as the primary immunoreagent and biotin as the soluble hapten.
  • This separation procedure may further include re-infusion of the separated blood or marrow constituents depending upon the need of the particular donor.
  • These steps may be effected using commercially available devices for delivering cells to columns or receptacles by, e.g., pumping, 'gravity-feeding or centrifugation.
  • CD34+ cells are purified according to the present invention that it is possible to obtain CD34+ cell compositions of significantly higher cell purities than had been obtainable using conventional methods.
  • Tr tact, CD24+ cell compositions purified according to the methods taught herein comprise cell purities in excess of 99.95%. This exceeds the levels of cell purities obtainable by prior methods by two orders of magnitude or more. Additionally, if the object of immuROpurificatior -vas to deplete or purge malignant cells from bone marrow (so that it may X ⁇ used for autologous BMT), it has been found that the factor of depletion may equal or significantly exceed what is obtainable by specific negative immir ""lection techniques.
  • This factor is a p r oduct of the intrinsic purity of the 34+ cells and the degree of enrichment of these marrow repopulating cells (e.g., a 50-fold enrichment of these cells should on average allow 50-fold fewer total cells/kg to be returned to the patient). It is believed that these significantly enhanced cell purities are obtained by virtue of the increased specificity of the use of hapten for cell dissociation. However, increased cell purities may further be attributed to the use of magnetic beads as the solid phase and repeated washing cycles. Secondly, it has been found that when CD34+ cells are prepared according to the present invention that the purity thereof may be evaluated simply and rapidly.
  • cell purity may be easily measured by assaying for CD34+ expression.
  • This can be done, e.g., by labeling the immunopurified cells with commercially available CD34 specific antibodies, such as, e.g., the anti-MY-10/CD34 antibody (e.g., mouse IgGl anti-HPCA-1, Becton-Dicl ⁇ nson), followed by staining with fluorescent anti- IgGl antibody, which cells are then analyzed by microscopy or flow microfluorometry.
  • CD34 specific antibodies such as, e.g., the anti-MY-10/CD34 antibody (e.g., mouse IgGl anti-HPCA-1, Becton-Dicl ⁇ nson), followed by staining with fluorescent anti- IgGl antibody, which cells are then analyzed by microscopy or flow microfluorometry.
  • a third advantage of the present invention is attributable to the manner by which the cells are immobilized to the solid support. Specifically, because immobilization occurs via a linkage which recognizes only the hapten (e.g., biotin) conjugated form of the K6.1 antibody, it provides a means for selectively preventing immobilization of a portion of CD34+ cells, e.g., by preincubation with non-biotinylated K6.1 antibody. This allows a portion of the same or similar bone marrow cells which are used for immunoselection to be converted to "autologous indicator cells". Accordingly, this provides an alternative method for assaying CD34+ cell purity.
  • hapten e.g., biotin
  • a fourth advantage of the present immunoselection is the fact that the recovered CD34+ cells should be less fragile than those CD34+ cells obtained by conventional methods. Accordingly, these cells should be capable of better withstanding further manipulations, such as cryopreservation and/or additional immunoselections. With respect to bone marrow transplantation, this is frequently performed following cryopreservation of cells in a fixed concentration of dimethylsulfoxide, and the thawed cells are transplanted without washing. Therefore, the ability to obtain purified stem cells in highly concentrated form is advantageous in that smaller volumes could be administered than with purged or less purified cell preparations, thereby causing less discomfort or toxicity to the recipient.
  • a fifth advantage of the present immunoselection is attributable to the fact that it preserves the expression of all cell surface antigens, and not just CD34. Accordingly, the purity of the CD34+ cells may be increased further, or any subset within this population may be enriched or depleted by effecting further immunoselections with another antibody or plurality of antibodies. This may result in cell purities approaching 100% or even absolute purity ' with respect to antigen expression.
  • residual contamination of CD34+ cells by malignant cell expression e.g., of a known differentiation or tumor associated antigen "X” may be eliminated by labeling with anti-"X" antibody and then immobilizing on a solid phase containing an antibody that binds anti-"X” antibody.
  • the anti-"X" antibody may be of different immunoglobulin isotype or subclass from the CD34 antibody used for the initial immunoselection or may be conjugated with a different hapten. Therefore, this will provide the capability of removing malignant cells which are CD34 antigen positive, as in the case of hematologic malignancies such as certain leukemias and lymphomas. This would extend the range of capability of malignancies treatable by autologous BMT. However, in these situations, there may still be a risk of co-purifying malignant cells, particularly given the antigenic variation of malignant cells even from the same donor and cdl type. Accordingly, BMT in such situations may still be preferred.
  • This method should also provide a means for the purification of cell subsets within the CD34+ population, such as, e.g., the totipotent stem cell, the oligopotent lymphoid, or mydoid progenitor cell, or the bone ma ⁇ ow repopulating stem cell using suitable primary immune reagents with specificity to these CD34+ subsets. This should be possible because of the fact that this method does not disturb the cell surface antigens expressed on the surface of the immunopurified CD34 cells.
  • a sixth advantage of the present immunoselection involves economics.
  • the quantity of reagents used are significantly less than required in conventional methods.
  • reagent savings using magnetic beads may exceed tenfold.
  • An seventh advantage obtained by the present invention is attributable to the K6.1 antibody.
  • this antibody binds a CD34 epitope which is conserved in other primates, it should be possible to utilize this antibody for the immunopurification of CD34+ cells from non-human primates. This would result in alternative sources of CD34+ for research and for therapeutic investigative analysis, e.g., the optimization of genetic therapy procedures which may not be desirable in humans.
  • any method which relies upon the use of highly purified cell populations containing lympho-hematopoietic stem and progenitor cells will be significantly enhanced.
  • One noteworthy example involves the production of monodonal antibodies which recognize specific antigens on lympho- hematopoietic stem and progenitor cells.
  • Another example involves the devdopment and use of various in vitro bioassays to identify and test the actions of growth factors and other drugs (e.g., anti-neoplastic agents, immune modulators) directly on normal lympho-hematopoietic cells.
  • Prior art isolation methods have been limited to 1-2 week clonal assays, rather than large population sampling assays because of the problem of ruling out indirect or secondary effects caused by contaminating cells. Results with large population sampling, however, may be obtained more quickly than with clonal assays. Accordingly, because of the highly pure stem and progenitor cells provided by the present invention, it should be possible to screen the effects of such drugs and growth factors on stem and progenitor cells much more rapidly than had been possible heretofore.
  • Another example involves the purification of these stem and progenitor cells with the object being expanding their number or directing their maturation in vitro with the aid of specific growth factors or suitable agents.
  • the purified stem and progenitor cells obtained by the present invention will facilitate the design of tissue culture media and culturing methods which promote their amplification and/or maturation.
  • these methods could provide larger quantities of purified stem and progenitor cells for reinfusion into BMT patients, an obvious concern of conventional bone marrow treatment protocols.
  • the isolation and expansion of totipotent stem cells may reduce the amount of bone marrow or blood collected for autologous BMT, and may permit their use as "universal donors" for allogendc BMT.
  • Another example involves the use of d e present invention to obtain other cdl types to accompany purified stem and progenitor cells in BMT, in particular for alleviating the severity of the immunodeficient state which accompanies this procedure.
  • This immunodeficiency occurs in both autologous and allogendc BMT when the donor marrow is purged of T- cells. Only a small number of donor T-cdls can sustain engraftment, possibly attributable to the suppression of residual host immunity or the inhibition of the production of hematopoietic growth factors.
  • Conventional methods of obtaining CD34+ cells, in particular stem and progenitor cells have been variably contaminated by tither T-cdls or tumorigenic cells.
  • this disclosure provides improved composition for BMT which are virtually devoid of T-cells or tumorigenic cells. Therefore, this invention provides methods for the positive immunosdection of cells other than CD34+ selected cells (e.g., T-cell subsets such as CD4+ helper cells) from donor blood or marrow using commonly available monoclonal antibodies. Accordingly, it should be possible to infuse these cells along with the stem or progenitor cells or CD34+ cells in BMT in order to provide immunocompetent cells earlier during recovery. This is particularly attractive since the patients undergoing BMT (e.g., cancer patients) tend to be immunocompromised. Thus, a more immediate means of compensating for this immunocompromised state should -be beneficial.
  • CD34+ selected cells e.g., T-cell subsets such as CD4+ helper cells
  • TIL cells tumor-infiltrating lymphocytes
  • cytotoxic lymphocytes cytotoxic lymphocytes
  • CMV cytomegalovirus
  • HAV human immunodeficiency virus
  • hapten-anti-hapten antibody linkage system are not limited to the use of cell specific antibodies.
  • lymphocyte populations binding to virus particles, other microorganisms, or parasitic organisms labelled with a soluble hapten e.g., biotin
  • a soluble hapten e.g., biotin
  • transformation vehicles and methods may be utilized to introduce heterologous genetic material (e.g., genes for repair of inherited metabolic defects, or resistance to drugs or viral infections) into stem cells in vitro.
  • heterologous genetic material e.g., genes for repair of inherited metabolic defects, or resistance to drugs or viral infections
  • the cells purified by the present invention will be particularly suitable for this purpose since membrane structural integrity, receptor expression, and cell viability are important for efficient gene transfer.
  • the present techniques will enable directing or targeting the carrier vehicles used for genetic transformation to the membrane bound hapten (e.g., biotin) on positivdy immunosdected cells.
  • hapten e.g., biotin
  • methods for malting monoclonal antibodies could be improved by using a hapten (biotin) labeled antigen to presdect antigen binding lymphocytes prior to fusion with immortal cells to produce hybridomas. This would significantly reduce or possibly eliminate the complexity of screening large numbers of hybridomas to identify ant;, sn specific clones.
  • positive immunoselection could replace the subcloning procedures which are necessary to preserve the specificity of hybridoma lines.
  • EXAMPLE 1 Development of K6.1 anti-CD34 monoclonal antibody.
  • the monoclonal antibody K6.1 was produced by fusing SP- 2/0-AG14 plasmacytoma cells (American Type Culture Collection, Rockville, MD) with splenocytes from a BALB/cByJ mouse (Jackson Laboratory, Bar Harbor, ME) which had been hyperimmunized with viable KG-la cells (ATCC). Injections containing 10 to 20 million KG-la cells washed in saline were performed approximately monthly for a period of 6 months; the first and last immunizations were intravenous and the other immunizations were intraperitoneal. The last injection was performed 3 days prior to fusion. Cell hybridization and selection in HAT medium were performed according to the techniques of Kohler and Milstein (1975), Nature. 256:495, as modified by Fazekas de St. Groth and Scheidegger (1980), J. Immunol. Methods. 35:1, and Lane fiLaL (1984), J. Immunol. Methods. 72:71.
  • pll5/MY-10 (1984; L Immunol.. 133:157, U.S. Patent No. 4,714,680).
  • pools of about 10 growth positive hybridoma wells were screened, and individual wells of antibody positive pools were then screened.
  • Antibody positive wells were subcloned by limiting dilution (Oi and Herzenberg, in Selected Methods in Cellular Immunology (1980), Mishell and Shigii, eds., pp. 351-72), and clones were screened the same way.
  • the hybridoma was expanded in roller bottles in Iscove's Modified Dulbecco's Medium (IMDM, MA Bioproducts, Walkersville, MD) containing fetal calf serum (Hyclone). After supernatant harvesting, the K6.1 antibody was purified by hydroxylapatite chromatography (Stanker ej aL (1985), J. Immunol. Methods. 76: 157), followed by pH-gradient elution from protein A-Sepharose (Ey et al. (1978), Immunochemistrv. 15:429). The yield of antibody was 40-45 mg/liter of supernatant.
  • Immunochemical identity between the antigens recognized by K6.1 and anti-MY-10 antibodies was established by showing that anti-MY-10 reacted in immunoblot analysis with antigen purified by immunoprecipitation with K6.1 antibody.
  • About 50 million KG- la cells prewashed in saline were solubilized in 1 ml of lysis buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.5% Triton X-100 detergent and 2 mM PMSF.
  • the Sepharose was washed 5 times by centrifugation in washing buffer containing 0.05% Triton, and the pellet was then divided into 3 equal parts.
  • part a the pellet was suspended in 100 ul of Laemmli sample buffer containing 2% SDS and 25 mM DTT, and incubated for 60 min at 22°C to elute the CD34 antigen.
  • part b the pellet was suspended in buffer containing the enzyme endoglycosidase F (Genzyme Corp., Cambridge, MA) to digest off asparagine-linked oligosaccharides and incubated according to published procedures references supplied by the company.
  • Part c was the incubation control for part b and lacked the enzyme.
  • Sections of the nitrocellulose membrane containing the antigen immunoprecipitated by K6.1 antibody were incubated with the following monoclonal antibodies or immunoglobulins at a concentration of 5 ug/ml: K6.1 antibody (positive control for antigen retention), anti-MY-10 antibody (purchased from Becton Dickinson Immunocytometry Systems, Mtn. View/San Jose, CA, under the trade name HPCA-1), UPC- 10 (negative control myeloma protein of the same isotype as K6.1, purified by protein A- Sepharose chromatography of ascites from BALB/c mice bearing the tumor). Detection with alkaline phosphatase-goat anti-mouse IgG and BCIP/NBT was as described above.
  • FIGURE 1 depicts the results of this analysis. This shows that the patterns of reactivity of K6.1 and MY-10 antibodies to the untreated (lane “a”), enzymatically treated (lane “b"), or control antigen (lane “c") immunoprecipitated by K6.1 antibody were the same.
  • the intense band coinciding with the pi 15 marker in the K6.1 and anti-MY-10 sections is the CD34/MY-10 antigen.
  • the lower minor band coinciding with the ⁇ -symbol is the reduced 7-chain of the K6.1 antibody originally involved in the immunoprecipitation, which is also detected by the alkaline phosphatase-anti- IgG.
  • the more intense staining with K6.1 indicates that this antibody reacts with the antigen more strongly than anti-MY-10.
  • EXAMPLE 3 Reactivity of K6.1 and anti-MY-10 antibodies with distinct and non-overlapping epitopes on CD34 antigen.
  • the design of this analysis was to determine if pre-binding of either K6.1 or anti-MY-10 antibody to CD34 antigen on KG-la cells would interfere with or block the subsequent binding of the other antibody. Absence of inhibition or blocking is interpreted as indicating that the respective epitopes recognized by the antibodies are distinct and non- overlapping.
  • the antibody to be tested after the preincubation step was pre-conjugated with biotin, and was detected by indirect immunofluorescence with FTTC-avidin (Becton Dickinson) and flow cytometry with a Becton Dickinson FACS ⁇ .
  • the antibodies were conjugated with biotin as follows: Purified K6.1 antibody from Example 1 was mixed with NaHCC ⁇ to give a solution containing 3 mg antibody/ml in 0.1 M NaHCO 3 . Biotin-N- hydroxysuccinimide ester (Calbiochem, La Jolla, CA) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 12 mg/ml, and 5.0 ul of this was added to each ml of antibody solution. After 1 hr at room temperature, NH 4 HCO 3 was added to 50 mM final concentration to stop the reaction.
  • DMSO dimethylsulfoxide
  • the mixture was then passed through a Sephadex PD-10 column (Pharmacia) equilibrated in phosphate-buffered saline (PBS, 6.7 mM Na phosphate, pH 7.2, 137 mM NaCl) to desalt and exchange the buffer.
  • PBS phosphate-buffered saline
  • Anti-MY-10 purchased from Becton-Dickinson as HPCA-1 was dialyzed into normal saline. 100 ug of this antibody was mixed with 900 ug of carrier MOPC-21 myeloma protein and NaHCO,, to give a 1 mg/ml solution of IgG in 0.1 M N--HCO 3 .
  • This myeloma protein has no known binding specificity, and was previously purified by DEAE-cellulose chromatography from ascites of BALB/c mice bearing the MOPC-21 plasmacytoma (gift of Dr. M.
  • each of the 4 reagents was titered by indirect immunofluorescence staining of KG-la cells, using FITC-avidin or FITC- anti-mouse IgG antibody (Becton-Dickinson), to determine the minimum amount of primary antibody needed to give saturation staining of CD34.
  • FITC-avidin or FITC- anti-mouse IgG antibody Becton-Dickinson
  • KG-la cells were preincubated with 10-20 times the saturating amount of either non-biotinylated antibody. Next, the minimum saturating amount of either biotinylated antibody was added to each sample (4 combinations), and incubation was continued.
  • FIGURE 2 The results of this analysis are depicted in the histograms in FIGURE 2, in which cell number on the y-axis is plotted against linearly increasing channels of fluorescence intensity on the x-axis. Binding of biotinylated-K6.1 antibody (left panels) was blocked by preincubation with excess non-biotinylated K6.1 (positive blocking control; lower panel), as expected, but was unaffected by preincubation with anti-MY-10 (upper panel).
  • EXAMPLE 4 Positive immunoselection of CD34+ cells from bone marrow.
  • Biotinylated K6.1 antibody stock Purified K6.1 antibody from
  • Example 1 was mixed with NaHC to give a solution containing 5 mg antibody/ml in 0.1 M NaHCOj.
  • Biotinyl-e-aminocaproyl-N- hydroxysuccinimide ester (B-X-NHS; Calbiochem, La Jolla, CA) was dissolved in dimethylsulfoxide at a concentration of 16 mg/ml, and 10.0 ul of this was added per ml of antibody solution. After 1 hr at room temperature, N ⁇ 4 HCO 3 was added to 50 mM to stop the reaction, and the mixture was passed through a Sephadex PD-10 column equilibrated in sterile PBS for desalting and buffer exchange.
  • the beads were washed back into water by reversal of the above sequence. Finally, the beads were suspended in 20 ml 0.1 M NaHCO 3 solution containing 1.5 to 2.0 mg affinity purified rabbit F(ab') 2 anti-goat IgG antibody, and the suspension was tumbled (approx. 30 rpm) for 16-20 hr at 22°C. At the end of this period, 5 mg of clinical reagent grade bovine serum albumin was added (Bovuminar, Armour Pharmaceutical Co., Kankakee, IL; note: it was assumed that pharmaceutical grade human serum albumin also could be substituted for use in BMT) and the beads were tumbled for another 3-4 hr.
  • bovine serum albumin was added (Bovuminar, Armour Pharmaceutical Co., Kankakee, IL; note: it was assumed that pharmaceutical grade human serum albumin also could be substituted for use in BMT) and the beads were tumbled for another 3-4 hr.
  • Tween 20 detergent was added (from 10% stock) to 0.1% final concentration, along with ethanolamine-HCl (from 5 M stock, pH9) to 0.1 M final concentration, and the beads were mixed for another 2 hr. Finally, the beads were washed twice in sterile-filtered Tris-buffered saline (20 mM Tris-HCl, pH7.4, 0.137 M NaCl) containing 0.1% Tween-20 and 0.01 % merthiolate, and thrice washed in Tris-buffered saline containing merthiolate. The beads were then stored at 4°C.
  • a stock of affinity purified rabbit F(ab') 2 anti-goat IgG antibody was made in preparation for the above coupling reaction. This was done by immunizing rabbits with DEAE-cellulose-purified goat IgG, and subjecting the serum to (NH 4 ) 2 SO 4 precipitation, pepsin digestion, passage over protein A-Sepharose, and affinity chromatography on goat IgG- Sepharose (with MgCl 2 as eluant) according to published methods (Stanworth and Turner, in Handbook of Experimental Immunology. Vol 1 (1978), ed. Weir, pp.12.1-46; Goding (1978), J. Immunol. Methods. 20:241; and Kessler et al. (1978), J. Immunol.. 127:1674).
  • the cells were suspended and washed in a citrate-phosphate buffered saline dextrose solution, and then washed and incubated in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal calf serum and DNAse (usually 1000 units/ml).
  • IMDM Iscove's Modified Dulbecco's Medium
  • fetal calf serum 10% fetal calf serum
  • DNAse usually 1000 units/ml
  • smaller amounts of iliac crest bone marrow were obtained by aspiration from volunteer donors. Bone marrow debulking and mononuclear cell enrichment.
  • red cells were removed and mononuclear cells were enriched prior to positive immunoselection. This was accomplished by any of the following methods: a. Density gradient centrifugation.
  • Diluted bone manow cells were centrifuged over Percoll (d. 1.077, Pharmacia) or Ficoll-Hypaque or equivalent at d. 1.077, e.g., Lymphocyte Separation Medium (LSM, Litton Bionetics, Washington, SC) or Ficoll-Paque (Pharmacia), and "light
  • ⁇ I ⁇ ⁇ U cells. Bone marrow cells were first depleted of red cells by NH 4 CI lysis using ACK Lysis Buffer (MA Bioproducts). BioMag anti-human IgG immunobeads (M4320, Advanced Magnetics, Inc., Cambridge MA) were preincubated with heat-inactivated pooled human AB serum (#34004-1, Pel- Freez Clinical Systems, Brown Deer, WI) to generate immobilized magnetic human immune complexes.
  • immobilized magnetic mouse immune complexes were made by incubating BioMag anti-mouse IgG immunobeads (M4400) with ascites fluid from BALB/c mice previously inoculated with the IgG2a hybridoma D3-2H2-9-21 (anti-Dengue complex, ATCC HB 114) or the plasmacytoma PI.17 (ATCC TIB 10). Neither the human serum immunoglobulins nor the two mouse IgG2a proteins had antibody specificity for human cells, but could bind to Fc receptor-bearing cells.
  • each of the washed magnetic bead complexes was mixed at a ratio of 10-20 beads/cell (assuming 1.3 x 10 9 beads/mg) for 60 min at 4°C on a rotator (approx. 30 rpm). Cells reacting with the magnetic beads were removed by holding the mixture next to a samarium-cobalt magnet (Treleaven et al. (1984), Lancet 1:70) for several minutes and collecting the cells remaining in suspension.
  • BioMag anti-mouse IgG beads were preincubated with ascites fluid from BALB/c mice inoculated with the plasmacytoma OKM1 (ATCC CRL 8026) producing anti-human CD lib monoclonal antibody. They were then incubated with a suspension of human bo ⁇ &marrow cells as described in method b above. n. £;. Positive immunoselection of CD34+ cells.
  • Bone marrow mononuclear cells were washed and adjusted to a concentration of 50 x lOVml.
  • Biotinylated-K6.1 antibody prepared as described above, was added at a ratio of 6-10 ug/ml of cell suspension and incubated with occasional mixing for 30 min at 4°C. The cells were then washed by centrifugation 3-4 times, and set to a concentration of 25 x lOVml.
  • Dynabeads M-450 coated with anti-goat IgG antibody as described above were activated with goat anti-biotin antibody.
  • the number of Dynabeads used was proportional to the total number bone marrow mononuclear cells, using a ratio of 1 bead/ 10 cells (the underlying objective was to provide at least 3 beads target cell contained in this population, but not to provide an overabundance of beads). It is to be noted that manufacturer's specifications and prior art use of these beads for negative immunosdections of tumor cells from bone marrow commonly employ a ratio of 10-20 beads/total number of cells. Thus, positive immunoselection as described here reflects a 100-fold or greater savings in bead consumption.
  • Anti-goat IgG Dynabeads were washed magnetically 4-5 times with a rare- earth magnet. The beads were then suspended at a concentration of 1 x lOVml in washing buffer containing 2.5 ug/ml affinity purified goat anti- mouse biotin antibody (#SP-3000, Vector Laboratories, Burlingame, CA), mixed vigorously for 30 min at room temperature, and then washed magnetically twice, and resuspended to 1 x 18 ⁇ /ml. This concentration of anti-biotin was optimized in preliminary titration studies using as an endpoint the kinetics of cell dissociation from the magnetic beads (described below).
  • the binding of anti-goat IgG to goat anti-biotin does not reach equilibrium under these conditions; varying the anti-biotin provided a convenient way to compensate indirectly for different cell surface antigen densities, by controlling the amount of biotin-anti-biotin crosslinking.
  • the bone manow mononuclear cells containing biotinylated- K6.1 antibody coated target cells, were incubated with the anti-biotin Dynabeads for 30 min on a rotator (approx. 30 rpm).
  • the magnetic CD34+ cells were then selected by attraction to a samarium-cobalt magnet, and removal of non-target cells free in suspension. These cells were purified by a further 4-5 washing cycles in which the magnet was removed, the cells resuspended, and the magnet reopposed, with removal of non-target cells free in suspension each time.
  • the magnetic CD34+ cells were suspended in medium (e.g., IMDM) containing 2.5 mg ml biotin, put on a rotator for 1-2 hr, and free CD34+ cells were recovered from magnetically immobilized Dynabeads.
  • medium e.g., IMDM
  • free CD34+ cells were recovered from magnetically immobilized Dynabeads.
  • CD34+ immunoselection experiments performed this way, these recovery conditions were observed to result in the complete detachment of beads from over 90% (usually well over 95%) of the immunoselected cells.
  • EXAMPLE 5 Yields of CD34+ cells from human cadaveric bone marrow.
  • An important test of the practicality of an immunoselection method is whether it can be scaled up to the cell numbers normally encountered in clinical settings, such as in BMT.
  • the data provided in TABLE 2 address this point by providing cell yields from positive immunoselections of CD34+ cells from 4 cadaveric bone marrows.
  • the total number of white blood cells from 5 vertebral bodies approximates the number obtained from 0.5 to 1 liter of aspirated marrow from BMT donors.
  • the debulking procedures described in Example 4 to enrich for mononuclear cells (examples shown in this table employed OKMl anti-CD lib depletion) typically remove 60-75% of the cells.
  • CD34+ cells are well into the theoretical useful range for BMT, since it is believed that 10* CD34+ cells may have the marrow repopulating capacity of 10* unfractionated bone manow cells (normally provided at 10* cells kg body wt) based on their normal frequency in manow.
  • 10* CD34+ cells may have the marrow repopulating capacity of 10* unfractionated bone manow cells (normally provided at 10* cells kg body wt) based on their normal frequency in manow.
  • Considerable variation occurs in yields of CD34+ cells from cadaveric marrows due to differences in donor age, medications received before patient death, and the amount of time passed between death and marrow harvesting.
  • EXAMPLE 6 Measurement of CD34+ cell purity by analysis of CD34 antigen expression.
  • Example 4 The retention of CD34 antigen expression of cells which are positively immunoselected as described in Example 4 provides a unique capability for measuring cell purity as soon as the cells are recovered, rather than after prolonged cell culture (e.g., overnight) during which the antigen may be reexpressed. To accomplish this, the recovered cells are stained for flow cytometry analysis with another monoclonal antibody that recognizes a separate epitope on the CD34 molecule. This second CD34 antibody is distinguishable from the K6.1 antibody that resides on the cells by virtue of belonging to a different immunoglobulin isotype or class, or being directly conjugated with a fluorochrome or other hapten.
  • Example 3 showed that anti-MY-10 fulfills this property.
  • CD34+ cells purified from bone manow as in Example 4 were stained with anti-MY-10 (anti-HPCA-1, Becton-Dickinson) or with isotype-matched MOPC-21 IgGl myeloma protein as a negative control. After washing, they were then stained with phycoerythrin-conjugated goat anti-mouse IgGl antibody (Fisher Biotech Antibodies, Fisher Scientific) and then analyzed by flow cytometry with an Ortho Cytofluorograf (Ortho Diagnostic Systems, Inc., Westwood, MA).
  • FIGURE 3a The results are depicted in FIGURE 3a, in which cell number is plotted on the y-axis against channels of logarithmically increasing fluorescence intensity on the x-axis.
  • Analyses of several other commercially available CD34 mouse monoclonal antibodies on purified CD34+ cells revealed at least 2 that fulfilled the above criteria.
  • cells were stained with TUK3 mouse monoclonal antibody (Civin et al.. in Leucocyte Typing IV. White Cell Differentiation Antigens (1989), ed. Knapp sLaL, PP- 818-25, available from DAKO Corp., Carpenteria, CA), or the negative control myeloma protein J606 belonging to the IgG3 isotype.
  • the cells were then stained with phycoerythrin-conjugated goat anti-mouse IgG3 antibody (Fisher Biotech). Other cells were stained with directly phycoerythrin-conjugated anti-HPCA-2 (Landsdorp et al. in Leucocyte Typing IV. White Cell Differentiation Antigens (1989), ed. Knapp et al. pp. 826-27, available from Becton-Dickinson), or with a phycoerythrin- conjugated IgGl isotype control. The cells were then analyzed with a Coulter Epics Elite flow cytometer.
  • FIGURE 3b (TUK3) and 3C (anti-HPCA-2) with the same channel parameters as above.
  • Staining with the specific antibodies is represented by the solid lines, and staining with the negative control immunoglobulins is depicted by the broken lines.
  • the discrimination (i.e., peak separation) between the CD34+ and negative control histograms is a reflection of the avidities of the different antibodies. Nevertheless, this method shows the cells are minimally 98-99% CD34+, which is at the upper limit of detection by this approach. To the knowledge of the present inventor, except for flow cytometry sorting, this exceeds the levels of CD34+ cell purity reported by others using other separation methods.
  • EXAMPLE 7 Measurement of CD34+ cell purity bv ""rare event analysis" with normal labeled or “autologous indicator” cells.
  • H342+ PRESEL is the actual percentage of Bisbenzimide H-33342 stained indicator cells in the bone manow suspension before positive immunoselection (a minimum of 500 total cells were counted).
  • #H342+/TOTAL (X10 5 ) is the number of stained indicator cells per number of CD34+ cells counted. In BM1993, for example, there were 2 fluorescent cells per 470,000 CD34+ cells.
  • PURGE FACTOR is the magnitude of indicator cell depletion, obtained by dividing the percentage of fluorescent cells before selection by the percentage after selection. In BM1993, for example, there was a 4277-fold depletion of indicator cells.
  • %CD34+ is the per centum purity of recovered CD34+ cells, obtained directly from the "purge factor.” In BM1993, for example, 4276 of every 4277 cells were CD34 + .
  • BMT EQUIVALENT LOG PURGE is the clinically equivalent “purge factor” that would be realized if these cells were to be used for bone manow transplantation. It is 100-fold higher than the "log purge,” because it assumes that 1 CD34+ cell has the manow repopulating capacity of 100 unfractionated bone marrow cells.
  • the efficiency of cell depletion (of non-CD34+ cells) obtained by positive immunoselection of CD34+ cells equals or exceeds the capabilities of any negative immunoselection system (e.g., for bone marrow tumor cells or T cells). In addition, this substantially exceeds the capabilities of any known positive immunoselection system (including fluorescence activated cell sorting, which has a much lower cell throughput).
  • EXAMPLE 8 Expression of other surfac-: membrane antigens on purified CD34+ cells.
  • Example 4 The preservation of CD34+ cell surface membrane integrity by immunoselection as described in Example 4 enabled these cells to be analyzed for expression of other membrane antigens. Analysis was further facilitated by the ability to recover CD34+ cells in large quantity (Example
  • Monoclonal antibodies with the specificities indicated were obtained from several commercial suppliers, including Becton-Dickinson (Mountain View, CA), Coulter (Hialeah, FL), Hybritech (La Jolla, CA), Ortho Diagnostic Systems (Raritan, NJ) and Sera-Lab (Accurate Chemical & Scientific Corp., Westbury, NY), and were used according to the suppliers' directions.
  • Antibodies MHM24 and H52 were a gift of Dr. J. Hildreth (Johns Hopkins Univ., Baltimore, MD).
  • antibodies that were available as FITC or phycoerythrin conjugates were used for direct immunostaining, along with negative control immunoglobulins matched wherever possible for isotype, conjugation method, concentration, and supplier.
  • Antibodies that were available as unconjugated proteins were used in indirect immunostaining with FITC or phycoerythrin conjugated, immunoglobulin isotype or subclass-specific secondary antibodies (Fisher Biotech), along with isotype or subclass-, concentration-, and source-matched negative control immunoglobulins.
  • EXAMPLE 9 Sequential immunoselections: Positive immunoselection of CD34+ cells followed by negative immunoselection of tumor cells. The capability to subject purified CD34+ cells to further immunoselection procedures was desirable. These could be used, e.g., to further increase CD34+ cell purity, or to enrich or deplete any cell subset or lineage within this population. Studies using "rare event analysis” were performed according to the protocol described in Example 7, but with the following modifications: "Indicator” cells were from the human T cell leukemia line Jurkat Clone E6-1 (catalog no. 177, obtained from the AIDS Research and Reference Reagents Program, NIAID, NM), instead of normal light density mononuclear cells.
  • a suspension of Dynabeads M-450 coated with sheep anti-mouse IgGl (Dynal product no. 11004) was then added at a ratio of 1 bead/2 cells (approx. 1.25 x lOVml), and the mixture was rotated (approx. 30 rpm) for 30 min.
  • the magnetic Jurkat cells were then removed with a samarium- cobalt magnet, and the CD34+ cells free in suspension were collected.
  • CD34+ cell preparations and against which antibodies to tumor-associated antigens are available or may be obtained for this purpose include breast cancer, neuroblastoma, multiple myeloma, lymphoma, and leukemias.
  • uncommitted CD34+ progenitor cells which are CD38- and lack lineage-specific antigens such as CD5, CD10, CD33, and CD71, could be separated from CD34+ cells that are lineage- committed and express the CD38 antigen in high density (Terstappen et al. (1991) Blood 77:1218).
  • EXAMPLE 10 In vitro growth of recovered CD34+ cells. The growth capabilities of CD34+ cells immunoselected as described in Example 4 were analyzed in a variety of cell culture systems.
  • hematopoietic, clonogenic, lineage-restricted (granulocyte-macrophage colony forming units, CFU-GM; erythroid burst- forming units, BFU-E) and multipotential (mixed colony forming units, CGU-Mix) progenitors were assayed in methylcellulose cultures using an adaptation of standard methods (Iscove et al. (1974), J. Cell. Physiol.. 83:309 and Lu et al. (1986), Cancer Res.. 46:4357).
  • Cells were plated in 35 mm diameter gridded tissue culture plates (Nunc Inc., Naperville, IL) in IMDM supplemented with 1.1% methylcellulose (4000 CP; Fluka, Buchs, Switzerland), 30% (vol/vol) FBS, 2.5 U/ml final concentration human recombinant erythropoietin (Amgen, Thousand Oaks, CA), and 10% (vol vol) giant-cell tumor conditioned media (GCT, Gibco). Cultures were incubated at 37°C with 5% CO 2 and 100% humidity, and colonies were counted using an inverted phase microscope and 40x magnification at 14-21 days.
  • IMDM IMDM supplemented with 1.1% methylcellulose (4000 CP; Fluka, Buchs, Switzerland), 30% (vol/vol) FBS, 2.5 U/ml final concentration human recombinant erythropoietin (Amgen, Thousand Oaks, CA), and 10% (vol vol) giant-cell tumor conditioned media (GCT, Gibco). Cultures were in
  • CFU-GM CFU-GM
  • hemoglobinized BFU-E CFU-Mix
  • CFU-Mix mixed erythroid-nonerythroid colonies
  • progenitors constituted 15-20% more of the CD34+ cells, and were enriched in progenitors from over 20-fold to over 70-fold compared to unseparated manow (depending on the source of the cells and particularly the percentage of CD34+ cells before immunoselection).
  • CD34+ cells depleted of cells which coexpress the CD33 antigen have been obtained by treating them with an anti-CD33 immunotoxin (anti-CD33 blocked ricin; LaRussa et al.. ibid.).
  • CD33 is expressed by most normal clonogenic myeloid progenitors (CFU-GM, BFU-E, CFU-Mix), as well as most acute myelogenous leukemia cells, but not on stem cells or more primitive progenitors (Andrews et al. (1989), J. Exp. Med..
  • a distinguishing surface membrane marker on these cells e.g., the expression of receptors for IgE
  • a haptenylated ligand e.g., biotinylated IgE
  • EXAMPLE 11 Immunoselection and analysis of CD34 + cells from nonhuman primates.
  • Monoclonal antibody 12.8 (described in Example 3) also crossreacts with nonhuman primate CD34, but because the binding of 12.8 antibody is blocked by K6.1 antibody (Example 3) this cannot be used for reanalysis of cells immunoselected with K6.1.
  • this population may be more prominent than the published report with human cells (Baum et al. (1992), ibid.) because prior to marrow harvest the donor was injected with a bolus of 5-fluorouracil to kill cycling cells and increase the frequency of stem cells and immature progenitors (Wieder et al. (1991), ibid.).
  • EXAMPLE 12 Transplantation of CD34+ cells.

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Abstract

L'invention se rapporte à des méthodes améliorées et à des kits destinés à l'immunosélection positive de cellules souches, ces cellules étant immobilisées sur une matrice solide par l'intermédiaire d'un complexe immun contenant un haptène soluble et un anticorps anti-haptène, et pouvant être dissociées par compétition de haptènes. L'invention se rapporte en particulier à une immunosélection positive améliorée, permettant de purifier des cellules précurseurs et des cellules souches lympho-hématopoïétiques dans des compositions de cellules hétérogènes, ainsi qu'à un nouveau réactif immun permettant d'effectuer cette purification.
EP93919834A 1992-07-28 1993-07-27 Methodes d'immunoselection positive de cellules souches. Withdrawn EP0652703A4 (fr)

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AU2593192A (en) 1992-09-14 1994-04-12 Oystein Fodstad Detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations
NO180658C (no) 1994-03-10 1997-05-21 Oeystein Fodstad Fremgangsmåte og anordning for deteksjon av spesifikke målceller i spesialiserte eller blandede cellepopulasjoner og opplösninger som inneholder blandede cellepopulasjoner
US6017719A (en) * 1994-06-14 2000-01-25 Nexell Therapeutics, Inc. Positive and positive/negative cell selection mediated by peptide release
US5972627A (en) * 1994-06-15 1999-10-26 Systemix, Inc. Method of purifying a population of cells enriched for dendritic and/or lymphoid progenitors and populations of cells obtained thereby
US5985660A (en) * 1994-06-15 1999-11-16 Systemix, Inc. Method of identifying biological response modifiers involved in dendritic and/or lymphoid progenitor cell proliferation and/or differentiation
CA2211380A1 (fr) * 1995-02-02 1996-08-08 Stichting Centraal Laboratorium Van De Bloedtransfusiedienst Van Het Ned Erlandse Rode Kruis Enrichissement de cellules souches hematopoietiques provenant du sang ou de la moelle osseuse
ATE340254T1 (de) * 1995-04-06 2006-10-15 Miltenyi Biotec Inc Verfahren zur freisetzung von magnetischen teilchen, die an zelloberflächen gebunden sind
US6015554A (en) * 1995-06-07 2000-01-18 Systemix, Inc. Method of reconstituting human lymphoid and dendritic cells
NO961031D0 (no) * 1996-03-13 1996-03-13 Det Norske Radiumshospital Tum Fremgangsmåte til å drepe uönskede målceller
US5928638A (en) * 1996-06-17 1999-07-27 Systemix, Inc. Methods for gene transfer
US6096540A (en) * 1996-09-30 2000-08-01 Becton Dickinson And Company Differentiation of granulocytic and monocytic progenitor cells
AU5432798A (en) 1996-11-08 1998-05-29 Morphogenesis, Inc. Materials and procedures for the purification of cells
CA2285918C (fr) * 1997-04-04 2007-07-03 Biosite Diagnostics Methodes mettant en oeuvre des particules magnetiques pour concentrer des ligands
AU7493101A (en) * 2000-05-23 2001-12-03 Nexell Therapeutics Inc Reagents for cell selection and methods of use
ITUD20020092A1 (it) * 2002-04-30 2002-07-30 Univ Degli Studi Udine Metodo per l'identificazione e l'isolamento di cellule staminali di un tessuto umano
DE10256042B4 (de) * 2002-11-30 2006-06-08 Universität Potsdam Verfahren zur Selektion von Zellen, die spezifisch bindende Moleküle produzieren
EP2039348A1 (fr) * 2007-09-21 2009-03-25 Jürgen Schliefelbein Préparation cosmétique et procédé pour obtenir une préparation de cellule souche somatique

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