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WO2011154615A1 - Procédé d'isolation de cellules souches hématopoïétiques cd34+, de cellules tueuses naturelles et de lymphocytes t régulateurs à partir d'un échantillon de sang de cordon ombilical humain à l'aide d'un anticorps anti-gd3, et compositions les contenant - Google Patents

Procédé d'isolation de cellules souches hématopoïétiques cd34+, de cellules tueuses naturelles et de lymphocytes t régulateurs à partir d'un échantillon de sang de cordon ombilical humain à l'aide d'un anticorps anti-gd3, et compositions les contenant Download PDF

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Publication number
WO2011154615A1
WO2011154615A1 PCT/FI2011/050552 FI2011050552W WO2011154615A1 WO 2011154615 A1 WO2011154615 A1 WO 2011154615A1 FI 2011050552 W FI2011050552 W FI 2011050552W WO 2011154615 A1 WO2011154615 A1 WO 2011154615A1
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Prior art keywords
cells
antibody
cord blood
positive cells
sample
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English (en)
Inventor
Annika Kotovuori
Suvi Natunen
Leena Valmu
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Suomen Punainen Risti Veripalvelu
Glykos Finland Ltd
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Suomen Punainen Risti Veripalvelu
Glykos Finland Ltd
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Priority claimed from FI20105660A external-priority patent/FI20105660A0/fi
Priority claimed from FI20106213A external-priority patent/FI20106213A0/fi
Application filed by Suomen Punainen Risti Veripalvelu, Glykos Finland Ltd filed Critical Suomen Punainen Risti Veripalvelu
Publication of WO2011154615A1 publication Critical patent/WO2011154615A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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

Definitions

  • the present invention relates to a composition
  • a composition comprising an isolated cell population that can be used for cellular therapy.
  • the cell population can be isolated and defined by an antibody
  • the isolated cell population has a characteristic content of cells:
  • haematopoietic stem cells enriched with natural killer (NK) cells and regulatory T
  • T reg lymphocytes
  • T he i pe r T cyt otoxic and B lymphocytes known to be potentially harmful in
  • the present invention also relates to the method for
  • HSC Haematopoietic stem cells
  • haematopoietic stem cells has traditionally been bone marrow but recently cord blood and
  • peripheral blood-derived HSCs are more common.
  • the graft can be even a non- processed sample of bone marrow or cord blood unit.
  • some cell populations in particular CD4 positive T-helper lymphocytes and CD8 positive T-cytotoxic lymphocytes can be partially depleted to decrease the probability or magnitude of so called graft- versus-host disease, the major drawback of HSC transplantation.
  • Umbilical cord blood is a valuable source of HSC, although the small size of a cord blood unit is often a limiting factor in these therapies.
  • the cell population given to patients in cord blood transplantation is most often unspecified fraction of mononuclear cells, although possibility to enrich HSC using either anti-CD34 and/or anti-CD 133 antibodies exists.
  • the purified cord blood HSC population has not, however, proven as efficient as it was expected.
  • some supporting cell population can be assumed to be beneficial in the therapy process.
  • the nature of this supporting cell type is not known. It would be of great value to be able to identify an effective cell population or composition.
  • NK cells Natural killer cells and regulatory T lymphocytes (Treg).
  • Treg regulatory T lymphocytes
  • NK cells have been shown to be potentially beneficial in partially HLA-identical ("haploidentical") HSC transplantation in certain clinical protocols. In particular they may kill those effector cells of the recipient that are essential to graft-versus-host disease (GVHD), decreasing the risk for relapse. Also, NK cells may improve immune reconstitution of the donor (Symons and Fuchs. Bone Marrow Transplant 2008; 42: 365-377).
  • Treg cells are central inhibitors of the immune response and they induce immunological tolerance.
  • GVHD is mediated by the immune system: the cells or tissues of the host are recognised as immunologically foreign by the cells in the transplant and the immune system is targeted to kill these cells, leading to severe destruction of tissues.
  • adding Treg cells in the graft or administering them after the transplantion may reduce the risk of graft-versus-host disease (Di Ianni et al. Blood 2011, Feb 3. published online: doi: 10.1182/blood-2010-10-311894; and Riley et al Immunity 30, May 22, 2009; published online doi 10.1016/j.immuni.2009.04.006) and may induce long-term immunological tolerance.
  • the present invention shows optimized methods which provides effective enrichment of the useful cell populations of cord blood, especially a combination containing both regulatory T cell and NK cells, together with CD34 or CD133 positive hematopoietic stem cells.
  • Cell population can be identified by their surface antigens.
  • a useful tool for detecting and enriching specific population is an antibody specific for the desired antigen.
  • Antibody S2-566 is known to detect the di-sialic acid containing epitope Neu5Ac-alpha-2-8Neu5Ac-alpha-2- 3Gal, linked to lipid and protein carriers (Sato C, et al. J. Biol. Chem. 2000; 275: 15422- 15431). Binding to protein glycan linked structures is characteristic for the antibody, the present invention show specific labelling and cell enrichment by the antibody while a glycolipid GD3 specific antibody is practically non-reactive.
  • the epitope is known to be enriched in HSC and T cell leukemia cell lines (Miyazaki H, Shiku H, Furukawa K. Int. J. Oncol. 1996; 9: 241-245), but nothing is known about other cell type. Due to cell-type and species specificity of glycan structures or epitopes, no predictions can be done beforehand or based on literature (Cagneux and Varki, Glycobiology 1999; 9:747-755). It is realized that glycosylation of cell populations has been reported to vary also according to their activation status and activation- or disease- specific glycosylations can be found. Therefore any glycosylation observed in any one cell fraction will not specifically predict glycosylation of another one, e.g. cells of cord blood origin. Cells of cord blood are special due to their specific niche and developmental phase. The immune cells of cord blood can be assumed as non-challenged, not exposed to most environmental antigens or pathogens.
  • WO 2008 107522 is directed to disialic acid epitopes, including Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal, with different variants on glycolipid structures on ganglioseries ganglioside GD3 and on proteins where it occurs with N-acetyllactosamine. These structures were found to be chemically different and to characterize cells in different manners.
  • the invention in WO2008107522 is directed to the use of antibodies against disialic acid epitopes in context of stem cells but does not teach anything about other cell types.
  • a related glycan structure the GD3 ganglioside, Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal- beta-l-4Glc-Cer, is a minor ganglioside in most normal tissues except placenta and thymus. Its expression increases in tumors of neuroectodermal origin, such as melanomas (Malisan F, Testi R. Biochem. Biophys. Acta 2002; 1585: 179-187).
  • the GD3 ganglioside as detected by the monoclonal antibody R24, is expressed by a subset of T-cells (Welte K, et al. J. Immunol. 1987; 139: 1763-1771.
  • Figure 1 illustrates proportions of different cell populations within umbilical cord blood derived mononuclear cells.
  • Figure 2 illustrates the percentage of cells stained by the two antibodies S2-566 and VIN-IS- 56, within each cell population derived from cord blood.
  • the present invention is based on the finding that by using a single monoclonal antibody specific to glycan epitope Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal, such as S2-566, a novel composition or population of human cells were detected and could be isolated from cord blood samples.
  • composition could be detected using the S2-566 antibody, known to recognize the disialic acid glycan epitope linked to both protein and lipid carrier, but not using the VIN-IS-56 antibody, known to recognize only the lipid-linked epitope, it was obvious that it is the glycan epitope Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal-l- beta4/3GlcNAc that is shared by the cell populations rather than the lipid-linked epitope.
  • the population so isolated still contained a high number of CD34 (and/or CD 133) positive haematopoietic stem cells, but surprisingly, the population additionally was enriched or contained high numbers of both human natural killer (NK) cells, that is cells positive for CD56 antigen, and regulatory T lymphocytes (Treg), that is cells positive for CD4 and CD25 antigens.
  • NK human natural killer
  • Treg regulatory T lymphocytes
  • the cell population obtained by antibody S2-566 has a unique composition of cellular subpopulations, which can be more useful and effective as a cell therapy graft than the current transplants.
  • a major advantage of the present invention is the fact that according to the present invention a mixture of different cell subpopulations, i.e. CD34 (and/or CD133), CD56 and CD4+CD25 positives cell population, can be first detected and then enriched or isolated using a single detecting antibody.
  • use of a mixture of different antibodies, one specific for CD34, one for CD56 and two for CD4+CD25 positive populations, has been essential.
  • the present invention provides a means to readily isolate or enrich a particular cell population using only a single antibody, an obvious advantage for cell isolation procedures, kits or devices for therapeutic cell preparations.
  • the use of only a single antibody minimize the number of steps and reagents needed and hence, simplifies the overall procedure and improves the safety issues.
  • There are a number of procedures, kits and devices described in the art related to handling and purification or isolation of cell preparation (R.I. Freshnay. Culture of Animal Cells, 4 th edition, pp 577, 2000. Wiley-Liss).
  • Typical techniques include use of magnetic beads covered by an antibody or an fluoresence-activated cell sorter (FACS) device, that detects the cells bound to a labelled antibody and sorts them out of other non-labelled cells.
  • FACS fluoresence-activated cell sorter
  • Each cell population can be regarded as unique and has a glycosylation pattern that is specific to the cell type and usually also to its status.
  • the S2-565 antibody has not been known for the characterization or enrichment of a specific T-cell or NK cell population, or their combination with hematopoietic stem cell population.
  • the invention further revealed clear difference to glycan epitope linked to glycolipid.
  • the cell population obtained can be characterized by containing:
  • CD34 positive (HSC) cells at least 50%, preferably over 60% of the CD34 positive cells in the original cord blood sample;
  • CD56 positive NK cells at least 10%, preferably over 20% of the CD56 positive cells in the original cord blood sample;
  • CD4 CD25 positive Treg cells at least 30%, preferably over 40% of the CD4 CD25 positive cells in the original cord blood sample;
  • CD4 positive T helper cells less than 20%, preferably less than 15% of the CD4 positive cells in the original cord blood sample;
  • CD20 positive B lymphocytes less than 20%, preferably less than 15% of the CD20 positive cells in the original cord blood sample;
  • CD8 cytotoxic T lymphocytes less than 20%, preferably less than 15% of the CD8 positive cells in the original cord blood sample;
  • the cell population is depleted from the potentially harmful CD8 T lymphocytes by using anti-CD8 antibodies or from CD20 positive B lymphocytes by using anti-CD20 antibodies, both well known in the art. This step can be done either before or after the step using the S2-566 or equivalent antibody.
  • the cells bound by the anti-CD8 or anti CD20 antibodies are removed from the graft or are destroyed.
  • Champlin et al 2000 Bood 95(12) :3996-4003
  • Table 4 antibodies commonly used in the art are summarized in Table 4.
  • the present invention is directed to a method for the isolation of a cell population from a sample of human umbilical cord blood comprising steps of contacting said sample with an antibody binding to di-sialic acid containing disialylated epitope Neu5Ac- alpha-2-8Neu5Ac-alpha-2-3Gal, such as S2-566, and isolating the cells bound to said antibody.
  • the isolated cells have therapeutic use.
  • the cells can be used as a graft for hematopoietic stem cell transplantation, and/or a cell support product after hematopoietic stem cell transplantation.
  • the cells can also be used as a cellular support therapy after hematopoietic stem cell transplantation to augment the curative process.
  • Another cell therapy use is prevention of graft- versus-host-disease when given before transplantation or inhibition of on-going graft-versus-host disease after stem cell
  • the original graft and possible supportive cell therapy product can be from the same individual or from a third party. It is well established in the art that both fresh cord blood units or cells and freezed, banked units can be applied in therapeutic purposes. It is further possible to apply cord blood or a fraction thereof, that has been in vitro cultivated in conditions supporting the growth or survival of at least CD34 and/or CD 133 positive hematopoietic stem cells and regulatory T cells and/or NK cells.
  • the present invention is further directed to a composition
  • a composition comprising human cells isolated from umbilical cord blood, wherein all said cells contain a glycan epitope Neu5Ac-alpha-2- 8Neu5Ac-alpha-2-3Gal. Consequently, these cells or this cell population can be isolated by the use of a single antibody binding to epitope Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal; one such antibody is S2-566.
  • the present invention is also directed to composition, wherein said cells are bound to said antibody.
  • the proportion of CD34+ cells in the composition of the present invention can be as high as 9.7 %.
  • the present invention is directed to a composition comprising at least 5 %, preferably at least 6, 7, 8 or 9 %, of CD34+ cells. Accordingly, in one specific embodiment of the invention, the composition contains (i) at least 9% of the CD34 positive cells; (ii) about 36% of the CD56 positive cells; (iii) about 3% of the CD20 positive cells; (iv) about 16% of the CD14 positive cells; and (v) about 35% of the CD3 positive cells.
  • the aim of this invention is to provide a composition that contains at least (i) CD34 positive cells; (ii) CD56 positive cells; and (iii) CD4 CD25 positive cells, since these are supposed to be advantageous in the prevention of graft-versus-host-disease.
  • the cell population is further depleted from the remaining potentially harmful CD8 T lymphocytes by using anti-CD8 antibodies or from the remaining CD20 positive B cells by using anti-CD20 antibodies, or by other means well known in the art.
  • This step can be done either before or after the step using S2-566 antibody.
  • the cells bound by the antibodies are removed from the graft or are destroyed.
  • Antibodies commonly used for cell depletions are summarized in Table 4.
  • the graft is treated with humanized anti-CD20 antibody that detects B -lymphocytes; there are many commercial preparations of anti-CD20 monoclonal antibodies available, for example, rituximab, Ibritumomab tiuxetan, or tositumomab.
  • the rationale in the cell depletion is further removal of harmful effector lymphocytes, the cells known to mediate graft-versus-host disease and whose removal reduce the severity and incidence of graft-versus-host disease, as discussed e.g. by Champlin et al. (Blood 95(12): 3996-4003). It is obvious that also other cell types may be removed if required, using similar approaches. It is obvious the removal step can done by using each single antibody separately or by using a combination of different antibodies in a single step. Further, the invention also relates to the use of an antibody binding to glycan epitope
  • Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal such as S2-566
  • the isolation or enrichment can be done using methods known by a person skilled in the art.
  • the antibody can be linked to magnetic beads, with which the starting cell material or cord blood sample is incubated followed by washing with a buffer.
  • the cells detected by the antibody can be collected using magnetic device.
  • the cells can be enriched using cell sorting apparatus (FACS device) wherein cells labelled by a fluorescent- linked antibody can be "sorted” or separated from all other cells in the sample.
  • FACS device cell sorting apparatus
  • the cord blood samples were incubated with 1.5 or 2 ⁇ g of the antibodies per 200 000 cord blood mononuclear cells. This corresponds to 7.5 - 10 micrograms of antibody, e.g. IgM or equivalent antibody, per million cells or per about 100 000 target cells.
  • Target cells correspond about one tenth fraction of the cells in a sample. In case the sample is already depleted from some cell populations, the number or fraction of the target cells may accordingly vary.
  • the invention is directed in one embodiment to the use of a large amount of antibodies: at least 0.1 micrograms per million cord blood mononuclear cells (or 0.1 million target cells) in a sample. It is obvious that the amount of antibody can vary. In other embodiments the amount may be at least 0.5 micrograms, at least 1.0 microgram, at least 2.5 microgram, at least 5.0 microgram, at least 6.0 microgram, at least 7.5 microgram, at least 10 microgram, at least 12.5 microgram, at least 15 microgram, or at least 20 microgram per million sample cells or 0.1 million target cells.
  • a high excess of antibody is used, an amount by far more than necessary for a stem cell population preparation, and a cell population comprising NK and Treg cell populations is thus efficiently provided. It is realized that the high excess of antibody may reveal a group of less strongly labelled cells in competition of labelling of subpopulations. It is further realized that very high amounts of antibodies may not be useful or increase the effect. In an embodiment the amount of antibody is between 0.1- 100 micrograms, between 0.5-50 micrograms or between 1- 20 micrograms or between 2.5 and 15 micrograms per million sample cells or 0.1 million target cells. It is realized that IgM has ten antigen binding sites and it binds oligovalently to cell surface.
  • IgM Equivalently to IgM, also other antibodies with suitable affinity may be used, such as monovalent antibody fragments such as single chain antibodies or divalent antibodies such as IgG or equivalents or multivalent antibodies such as oligomeric nanobodies or oligomerically conjugated FAb or FAb2 fragments.
  • monovalent antibody fragments such as single chain antibodies or divalent antibodies such as IgG or equivalents
  • multivalent antibodies such as oligomeric nanobodies or oligomerically conjugated FAb or FAb2 fragments.
  • the cord blood cells populations are produced as described in examples by using preincubation of S2-566 with the cord blood cells for times of at least 1 min, in some embodiments at least 5 min, at least 10 min, at least 15min, at least 20 min, at least 25 min, at least about 30 min, and the in an embodiment less than 120 min, and temperature between for the incubation between 0-2, 0-5 or 0-10 degrees of Celsius, in an embodiment on ice between about 0-3 degrees of Celsius.
  • the disialylated N-acetyllactosamine epitope is in a preferred embodiment disialic epitope comprising preferably NeuAcccNeuAccc3Gaip4GlcNAc or NeuAcccNeuAccc3Gaip3GlcNAc, in an embodiment NeuAca8NeuAccc3Gaip4GlcNAc.
  • the invention also revealed that the structure can be recognized by specific binder molecules recognizing terminal disialylated epitopes especially, when the structure is recognized on N- acetyllactos amine such as NeuAccc8NeuAccc3GaipGlcNAc, preferably
  • NeuAccc8NeuAccc3Gaip3/4GlcNAc preferably NeuAccc8NeuAccc3Gaip4GlcNAc, preferably on a protein.
  • the preferred binders include antibody S2-566 (Seikagaku), especially when the structrure is recognized on N-acetyllactosamine such as NeuAccc8NeuAccc3GaipGlcNAc or NeuAc 8NeuAc 3Gaip3/4GlcNAc, preferably NeuAccc8NeuAccc3Gaip4GlcNAc, preferably on a protein.
  • the S2-566 antibody recognizes GTla or GQlb ganglioside as indicated in the background and/or the protein linked disialic acid epitope but not GD3 on the target cells.
  • the disialic acid target epitope is recognized on a protein of 45 kD.
  • the invention is directed to use of combination of specific disialic acid recognizing antibodies wherein the first antibody can recognize the protein and/or lactosamine linked epitope NeuAccc8NeuAccc3Gal( GlcNAc), preferably
  • NeuXcc8NeuXcc3Gal-on glycolipids preferably on GD3, but not the protein and/or N- acetyllactos amine linked epitope.
  • the invention is directed to exclusively "ganglio disialic acid” specific binder, wherein the binder, such as an antibody, binds to "ganglio disialic acid” also referred here as “glycolipid” epitope, but does not recognize the protein or N- acetyllactos amine linked epitope.
  • the invention is directed to exclusively "protein/LacNAc disialic acid” specific binder, wherein the binder, such as an antibody, binds to "protein/lacNAc disialic acid", but does not recognize the "ganglio disisalic acid” epitope.
  • the invention is further directed to dual specificity antibody, wherein the antibody can recognize both the "ganglio disialic acid” epitope and "protein/N-acetyllactosamine disialic acid” epitope.
  • the invention is in an embodiment directed to specific specific recognition oof the "protein/N-acetyllactosamine disialic acid" epitope It is realized that with high specificity binders selectively and specifically recognizing the epitope(s), less binders may be needed e.g. these would be preferably used as combinations of at least two reagents, more preferably at least three epitopes, even more preferably at least four, even more preferably at least five, most preferably at least six antibodies.
  • the high specificity binders selectively and specifically recognizing the "protein/N-acetyllactosamine disialic acid" epitopes binds one of the elongated epitopes at least inorder of increasing preference, 5, 10, 20, 50, or 100 fold affinity, methods for measuring the antibody binding affinities are well known in the art.
  • the invention is also directed to the use of lower specificity antibodies capable of effective recognition of one elongated epitope but also at least one, preferably only one additional elongated epitope with same terminal structure
  • the invention is directed to combined isolation of both the S2-566 cells and CD34+ and/or CD133+ by using antibodies against CD34+ and/or CD133+ with S2-566.
  • the preferred disialic acid cell preparations are depleted with GD3 glycolipid comprising cells, preferably by GD3 antibody in examples.
  • Glycosidic linkages are shown partly in shorter and partly in longer nomenclature, the linkages of the Neu5 Ac -residues cc3 and cc6 mean the same as cc2-3 and 2-6, respectively, and with other monosaccharide residues ccl-3, ⁇ 1-3, ⁇ 1-4, and ⁇ 1-6 can be shortened as cc3, ⁇ 3, ⁇ 4, and ⁇ 6, respectively.
  • Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal can be written as Neu5Ac-alpha-(2-8)-Neu5Ac-alpha-(2-3)-Gal or by IUPAC shortened nomenclature as Neu5 Accc8Neu5 Ac 3Gal and Neu5 Ac-alpha-2-8Neu5 Ac-alpha-2-3Gal-beta- 1 -4/3GlcNAc is Neu5Aca8Neu5Aco 3Gai 3/ GlcNAc. 3/4 means either 3 or 4.
  • UMB umbilical cord blood
  • MNCs mononuclear cells
  • UCB mononuclear cells were washed with buffer (PBS - 0,3 % BSA - 2mM EDTA) before antibody labelling.
  • buffer PBS - 0,3 % BSA - 2mM EDTA
  • Primary antibodies VPU005 or VPU031 were incubated (1,5 or 2 ⁇ g / 100 ⁇ cell suspension / 200 000 cells) for 30 minutes on ice and washed once with buffer. Cells were then labelled with a secondary antibody, Alexa Fluor 488 conjugated goat anti- mouse (1:2000) for 30 minutes on ice in the dark.
  • tertiary antibodies (1 or 4 ⁇ of each cell marker/isotype control) were added and incubated for 30 minutes on ice in the dark. After washing the cells were fixed with stabilizing fixative (BD biosciences). As a negative control cells were incubated without primary antibody and otherwise treated similar to labelled cells. Cells were analysed by FACSAria and
  • EXAMPLE 1 Characterization of different cell populations within mononuclear cells of umbilical cord blood unit
  • the relative amount of different cell populations within cord blood derived mononuclear cells were calculated.
  • the cell populations were stained with marker antibodies and the amount of cells expressing these marker epitopes were then calculated.
  • EXAMPLE 2 Cord blood derived cell populations expressing disialylated epitope recognized by antibody S2-566
  • Example 2 the expression of disialylated glycan epitope in different cell types present within cord blood derived mononuclear cell population was investigated.
  • the cell populations depicted in Example 1 were analyzed using flow cytometry for the cell surface expression of diSA glycan epitope using (i) S2-566 antibody, that recognizes diSA linked to both protein and lipid carrier, and (ii) VIN-IS-56 antibody, which recognizes only the lipid-linked diSA epitope.
  • S2-566 antibody that recognizes diSA linked to both protein and lipid carrier
  • VIN-IS-56 antibody which recognizes only the lipid-linked diSA epitope.
  • the percentage of S2-566 epitope positive cells within each cell population is visualized in Figure 2. As much as 60% of CD34 positive HSCs carried this epitope and approximately 50% of CD 133 positive HSCs did the same.
  • the antibody investigated in Example 2 can be used to purify cell populations expressing these epitopes on their surface.
  • the antibody can either be coupled to a solid carrier, for example to a magnetic bead, of sorting by flow cytometry can be utilized.
  • a solid carrier for example to a magnetic bead
  • flow cytometry can be utilized.
  • the advantage of using glycan binders in these type of isolation protocols is the possibility, due to relatively low affinity of anti-glycan antibodies, to elute or detach the cells from the antibody and its carrier using epitope-like glycan structures, here disialylated structures.
  • the characteristic cell population derived from umbilical cord blood mononuclear cells using antibody S2-566 was based on the data obtained in the above mentioned Examples.
  • the distinct cell populations stained by tertiary marker antibodies (Table 1) were gated and the number of cells was calculated (Table 3, left column).
  • the expression of disialic acid epitope, (stained by S2-566 antibody) was investigated. According to this data, the simulated cell numbers were derived (Table 3, right column).
  • EXAMPLE 4 Isolation of a cell population from UCB-MNC by antibody S2-566 using fluoresence activated cell sorter (FACS) or magnetic beads
  • UCB mononuclear cells recognized by antibody S2-566 can be isolated by cell sorting using FACS flow cytometry. UCB mononuclear cells are filtered using a 30 ⁇ cell filter and 1 -5 x 10 cells are suspended in cold buffer (standard Phosphate Buffered Saline (PBS), 0,3 % Bovine Serum Albumin, 2mM EDTA) at 10 cells/ml. Appropriate amount of S2-566 antibody, typically approximately 2-10 ⁇ g of antibody per 10 cells, the optimal amount has to be titrated, is added.
  • PBS standard Phosphate Buffered Saline
  • 2mM EDTA standard Phosphate Buffered Saline
  • Appropriate amount of S2-566 antibody typically approximately 2-10 ⁇ g of antibody per 10 cells, the optimal amount has to be titrated, is added.
  • the cells After a 30-minute incubation on ice the cells are washed once with the buffer and labelled with a secondary antibody, Alexa Fluor 488 conjugated goat anti-mouse (1:2000) for 30 minutes on ice in the dark. The cells are washed twice. After filtering (30 ⁇ cell filter) the cells are suspended in cold buffer and the volume is adjusted to approximately 3 x 10 cells/ml. The cell sorting can be performed by FACS Aria (Becton Dickinson) using standard protocols.
  • UCB mononuclear cells recognized by antibody S2-566 can be isolated using anti-mouse Ig MicroBeads (Miltenyi Biotec) according to the manufacture's instructions. Briefly, UCB mononuclear cells (up to 10 cells) are suspended in buffer (PBS - 0,3 % BSA - 2mM
  • HSC hematopoietic stem cell
  • CD34 positive and negative cells as well as CD133 positive and negative cells from human umbilical cord blood are isolated using magnetic affinity cell sorting and double selection (Miltenyi Biotec, Germany) as described in Kekarainen et al (2006, BMC Cell Biol 7:30), the cells negative with regard to the markers are further reacted with beads comprising S2-566, and bound the cells are combined to get maximum HSC and useful Treg and NK cell population.
  • the present magnetic beads and HSC binding CD34 and CD 133 binding beads are used together to isolate the optimal preparation.
  • N 450 (52%) CD6 + CD7, CD6 + CD8, CD6 + CD7 + CD8, CD 8 + CD7
  • N 73 (8%) CD2 + CD3 + CD4 + CD8, CD2 + CD3 + CD4 + CD5 + CD6 + CD8 +

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Abstract

Cette invention concerne un procédé d'isolation d'une population cellulaire à partir d'un échantillon de sang de cordon ombilical humain comprenant les étapes de mise en contact dudit échantillon avec un anticorps se liant à l'acide di-sialique contenant l'épitope saccharidique Neu5Ac-alpha-2-8Neu5Ac-alpha-2-3Gal, tel que S2-566, et d'isolation des cellules liées audit anticorps. L'invention concerne également des compositions contenant des cellules humaines isolées à partir de sang de cordon ombilical.
PCT/FI2011/050552 2010-06-10 2011-06-10 Procédé d'isolation de cellules souches hématopoïétiques cd34+, de cellules tueuses naturelles et de lymphocytes t régulateurs à partir d'un échantillon de sang de cordon ombilical humain à l'aide d'un anticorps anti-gd3, et compositions les contenant Ceased WO2011154615A1 (fr)

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FI20105660 2010-06-10
FI20105660A FI20105660A0 (fi) 2010-06-10 2010-06-10 Menetelmä solujen eristämiseksi ja menetelmällä eristetty solupopulaatio
FI20106213A FI20106213A0 (fi) 2010-11-17 2010-11-17 Menetelmä solujen eristämiseksi ja menetelmällä eristetty solupopulaatio
FI20106213 2010-11-17

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002933A2 (fr) * 2006-06-26 2008-01-03 Macrogenics, Inc. COMBINAISON D'ANTICORPS DE FCγRIIB ET D'ANTICORPS SPÉCIFIQUES DE CD20 ET LEURS PROCÉDÉS D'UTILISATION
WO2008107522A1 (fr) * 2007-03-08 2008-09-12 Suomen Punainen Risti, Veripalvelu Nouveaux marqueurs de glycanes acides de cellules humaines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002933A2 (fr) * 2006-06-26 2008-01-03 Macrogenics, Inc. COMBINAISON D'ANTICORPS DE FCγRIIB ET D'ANTICORPS SPÉCIFIQUES DE CD20 ET LEURS PROCÉDÉS D'UTILISATION
WO2008107522A1 (fr) * 2007-03-08 2008-09-12 Suomen Punainen Risti, Veripalvelu Nouveaux marqueurs de glycanes acides de cellules humaines

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALYEA, E.P. ET AL.: "CD8+ cell depletion of donor lymphocyte infusions using cd8 monoclonal antibody-coated high-density microparticles (CD8-HDM) after allogeneic hematopoietic stem cell transplantation: A pilot study", BONE MARROW TRANSPLANTATION, vol. 34, no. 2, 2004, pages 123 - 128 *
MIYAZAKI, H. ET AL.: "Differential effects of a murine monoclonal antibody reactive with the disialylgalactosyl residue on the growth of melanoma cells and T cell activation: Comparison with anti-GD3 antibody R24", INTERNATIONAL JOURNAL OF ONCOLOGY, vol. 9, no. 2, 1996, pages 241 - 245 *
ORTI, G. ET AL.: "Phase I study of high-stringency CD8 depletion of donor leukocyte infusions after allogeneic hematopoietic stem cell transplantation", TRANSPLANTATION, vol. 88, no. 11, 2009, pages 1312 - 1318 *
RATANATHARATHORN, V. ET AL.: "Treatment of chronic graft-versus- host disease with anti-CD20 chimeric monoclonal antibody", BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION, vol. 9, no. 8, 2003, pages 505 - 511 *

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